Physiol Rev 95: 645– 690, 2015
doi:10.1152/physrev.00026.2014
TRANSIENT RECEPTOR POTENTIAL CHANNELS IN
THE VASCULATURE
Scott Earley and Joseph E. Brayden
Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; and Department of
Pharmacology, University of Vermont College of Medicine, Burlington, Vermont
Earley S, Brayden JE. Transient Receptor Potential Channels in the Vasculature.
Physiol Rev 95: 645– 690, 2015; doi:10.1152/physrev.00026.2014.—The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP)
superfamily of cation channels, which exhibit varying degrees of selectivity for different
ionic species. Multiple TRP channels are present in all cells and are involved in diverse
aspects of cellular function, including sensory perception and signal transduction. Notably, TRP
channels are involved in regulating vascular function and pathophysiology, the focus of this review.
TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in
perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they
participate in the regulation of vascular tone. Almost all of these functions are mediated by changes
in global intracellular Ca2⫹ levels or subcellular Ca2⫹ signaling events. In addition to directly
mediating Ca2⫹ entry, TRP channels influence intracellular Ca2⫹ dynamics through membrane
depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including
hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic
inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide
an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial
cells, and perivascular cells under normal and pathophysiological conditions.
L
I.
II.
III.
IV.
V.
INTRODUCTION
A BRIEF REVIEW OF TRP CHANNELS
PHYSIOLOGICAL ROLES OF TRP...
INVOLVEMENT OF TRP CHANNELS...
SUMMARY AND CONCLUSIONS
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653
671
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I. INTRODUCTION
Since the initial discovery and characterization of the transient receptor potential (trp) channel in Drosophila phototransduction mutants, a growing body of work has demonstrated the diversity, tissue distribution, and remarkable
range of functions performed by these cation channels in
mammals. Twenty-eight mammalian TRP homologs, distributed throughout the body, have been described. TRP
channels are critically involved in sensory and signal transduction processes in excitable and nonexcitable cells present in virtually every organ system. A common theme regarding the specific physiological roles of these channels is
their significant contribution to the regulation of intracellular Ca2⫹ concentration ([Ca2⫹]i) and distribution, which
directly influence a multitude of cellular functions. The biophysics, molecular and cellular biology, and pathophysiological involvement of these “truly remarkable proteins”
have recently been reviewed in considerable depth (95,
249).
The importance of TRP channels in the cardiovascular
system has been clearly demonstrated over the past 15
years. TRP channels in the heart are involved in normal
pacemaker function and contractility and contribute to
mechanisms underlying pathologies such as cardiac hypertrophy, fibrotic disease, and arrhythmias. In the circulatory system, TRP channels are present and functional
in smooth muscle cells and endothelial cells constituting
the vascular wall, and are also expressed in perivascular
neurons and astrocytes, which are closely associated with
cerebral parenchymal arterioles. TRP channels contribute to mechanosensation and G protein-coupled receptor
(GPCR)-initiated signaling pathways that modulate vasoconstrictor and vasodilator activity and cellular proliferation. In vascular endothelial cells, Ca2⫹ entry through
TRP channels is an important contributor to endothelium-dependent vasodilation, vascular wall permeability,
and angiogenesis. TRP channels present in perivascular
sensory nerves and astrocytes participate in vasodilator
responses in some tissues. Altered TRP channel function
has been linked to a number of common vascular disorders, including hypertension, vascular occlusive disease,
0031-9333/15 Copyright © 2015 the American Physiological Society
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
neurogenic inflammation, neointimal injury, and pulmonary edema.
This review is organized into three main sections. The first
provides a brief overview of TRP channel structure and
function. The second considers the normal physiological
roles of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells (i.e., neurons and astrocytes). And the last reviews evidence supporting the involvement of TRP channels in vascular disease.
II. A BRIEF REVIEW OF TRP CHANNELS
larger role for multiple trp homologs in humans, concluding
that “it will be interesting to determine the full size of the
human TRP family and whether the human TRPs have
different functional characteristics . . .” Indeed, the discovery of the first human TRPs spurred a decade-long race to
clone all of the TRP genes and characterize the functional
properties of the encoded proteins in heterologous expression systems. All members of the TRP superfamily now
appear to be accounted for, and current efforts are focused
on understanding how TRP channels affect the physiology
and pathophysiology of different organs and tissues. Detailed insight into the early days of TRP channel research is
provided by reviews and commentaries by the pioneers in
this field (130, 223, 229).
A. Discovery
The discovery of the TRP superfamily can be traced to
Cosens and Manning’s seminal study of Drosophila phototransduction mutants that were able to navigate normally
under low light conditions but behaved as if blind under
bright lights (63). These flies also displayed characteristic
abnormalities in electrical responses in the retina during
prolonged light exposure. Minke et al. (224) further characterized the electrophysiological properties of photoreceptors from these mutants and reported that, in contrast to the
steady-state depolarization induced by bright light recorded
from wild-type flies, light-induced depolarization of the
photoreceptor membrane potential in mutant flies was transient in nature. Consequently, the strain was designated
transient receptor potential (trp) (224). Isolation of DNA
encoding a portion of the affected gene by Montell et al.
(230) and subsequent cloning of the full-length trp cDNA
by Montell and Rubin (231) and Wong et al. (394) provided
additional insight into the function of the trp gene product.
Hydropathy plots of the predicted amino acid sequence
suggested that trp encoded a membrane protein with intracellular NH2 and COOH termini and six to eight putative
transmembrane domains. Montell et al. (231) noted that
the membrane topology of the protein encoded by trp was
reminiscent of previously characterized voltage-dependent
ion channel proteins, and predicted that trp also encoded an
ion channel. Vaca et al. (366) validated this hypothesis,
reporting an electrophysiological analysis of trp and the
related gene trp-like (trpl) in a heterologous expression system. When expressed in Sf9 insect cells, trp formed a Ca2⫹permeable, nonselective cation channel that was activated
by the sarco(endo)plasmic ATPase (SERCA) inhibitor thapsigargin. Evidence that trp homologs are present in vertebrates was reported almost simultaneously by Petersen et al.
(268), who found that sequences related to trp are present
in Xenopus oocytes and mouse brain. More comprehensive
studies were performed by Wes et al. (388) and Zhu et al.
(425), who cloned and characterized the first mammalian
trp homologs, and Zhu et al. (426), who reported the first
evidence of a receptor-operated mammalian TRP channel.
The first mammalian TRP to be cloned is now known as
TRPC1. Wes et al. (388) were prescient in predicting a
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B. TRP Superfamily Organization and
Channel Structure
Mammalian genomes encode 28 distinct TRP protein subunits, which together comprise the TRP superfamily (FIGURE 1, TABLE 1). A standard nomenclature was established
(61), grouping these genes and their products into six subfamilies based on amino acid sequence homology. These
subfamilies are designated canonical (TRPC), vanilloid
(TRPV), melastatin (TRPM), ankyrin (TRPA), mucolipin
(TRPML), and polycystin (TRPP) (FIGURE 1, TABLE 1). One
member of the TRPC subfamily, TRPC2, is present in rodents and is important for pheromone sensing (206), but is
a nonexpressed pseudogene in humans (409).
The terminology used to describe the TRPP subfamily requires some clarification. The polycystin-1 protein encoded
by PKD1 is sometimes referred to as TRPP1 (or TRPP4).
This is confusing, as PKD1 encodes a protein with 11 transmembrane domains and a long extracellular NH2 terminus
that does not form an ion-conducting pathway and is structurally unrelated to authentic TRP channels (390). The
name “TRPP4” was also briefly used to describe the product of the PKDREJ (polycystic kidney disease and receptor
for egg jelly related protein) gene in an early report. But
PKDREJ is also unrelated to the mammalian TRP superfamily, and the name “TRPP4” is no longer in use. TRPP2,
TRPP3, and TRPP5 have also been used to describe the products of the PKD2 (polycystic kidney disease 2) gene (gene ID:
5,311; mRNA: NM_000297), PKD2L1 gene (gene ID: 9,033;
mRNA: NM_016112), and PKD2L2 gene (gene ID: 27,039;
mRNA NM_014386), respectively. In this review, we use
TRPP1 for PKD2, TRPP2 for PKD2L1, and TRPP3 for
PKD2L2. The current International Union of Basic and
Clinical Pharmacology (IUPHAR) database (http://www.
guidetopharmacology.org/GRAC/FamilyDisplayForward?
familyId⫽78) currently uses both nomenclatures, adding to
confusion.
All TRP subunits are expressed as polypeptides of 553–
2,022 amino acids (⬃64 –230 kDa) containing six trans-
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
Trpm1
Trpm6
Trpp3
Trpm3
Trpp2
Trpm7
Trpp1
Trpml3
Trpm5
Trpml2
Trpm4
Trpml1
Trpm2
Trpm8
FIGURE 1. Phylogenetic tree of the mouse TRP
superfamily.
Trpc7
Trpv5
Trpc6
Trpv6
Trpc3
Trpv3
Trpc2
Trpv2
Trpv1
Trpc5
Trpc4
Trpv4
Trpa1
Trpc1
membrane domains (S1–S6) and intracellular NH2 and
COOH termini of variable length (FIGURE 2). An ⬃25amino acid conserved element called the TRP domain is
present immediately distal to the S6 domain in members of
the TRPC, TRPM, and TRPV subfamilies. The TRP domain contains two highly conserved, six-residue TRP boxes
bordering a central region that has greater sequence variability. The function of the TRP domain is not completely
understood, but it may be involved in phosphatidylinositol
4,5-bisphosphate (PIP2) binding (287) or subunit assembly
(105). Multiple regulatory elements, interaction sites, and
enzymatic domains are present in the intracellular termini.
Numerous ankyrin repeat domains are present on the NH2
terminus of TRPC, TRPV, and TRPA subunits. These are
particularly prominent in TRPA1 subunits (⬃15–19 repeats), where they contribute to channel regulation. The
COOH terminus of TRPC channels encodes calmodulin/
IP3R-binding (CIRB) domains, Ca2⫹-binding EF hands
and, for TRPC4, a PDZ domain. A domain known as the
TRPM homology region is present in the NH2 terminus of
TRPM subfamily members. Functional serine-threonine kinase domains are present in the COOH terminus of TRPM6
and TRPM7, and an ADP-ribose-binding NUDIX phosphohydrolase domain is present in the COOH-terminal region of TRPM2. TRPP and TRPML channels share homology through the transmembrane region, including a large
extracellular loop between the S1 and S2 domains that is
not present in other subfamilies. Transcriptional splice variants, some with functionally distinct properties, have been
described for almost all TRP subunits (373).
Functional TRP channels arise from the assembly of four
subunits, with the S5 and S6 transmembrane domains forming the ion-conducting pore. All TRP subunits, with the
possible exception of TRPC1 (328), are capable of forming
functional homomeric cation channels. Many cell types express multiple TRP subunits, and heteromultimeric channels composed of two or three related subunits can form
and are present in vivo. Heteromultimeric channels display
conduction properties and regulatory mechanisms that are
distinct from those of homomeric channels. Formation of
heteromultimeric channels is best characterized for TRPC
channel subunits. TRPC1 subunits can form heteromultimeric channels with TRPC3, TRPC4, TRPC5, TRPC6, and
TRPC7 subunits. TRPC4 and TRPC5 can heteromultimerize, as can combinations of TRPC3, TRPC6, and TRPC7
subunits (141). Other arrangements include channels composed of the closely related subunits TRPV5 and TRPV6
(138), TRPM6 and TRPM7 (186), and TRPML1 and
TRPML3 (367). There is less evidence supporting interactions between channels from different subfamilies, although formation of TRPC1/TRPV4 heteromultimeric
channels in the endothelium (207), TRPV1/TRPA1 channels in sensory nerves (93, 290), and other combinations
(263) has been reported.
The most detailed study of TRP channel structure to date
was provided by Liao et al. (192) and Cao et al. (43), who
used cryoelectron microscopy to determine the structure of
TRPV1 channels at high resolution (3.4 Å). These studies
revealed that four TRPV1 subunits are symmetrically ar-
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647
SCOTT EARLEY AND JOSEPH E. BRAYDEN
Table 1. The human transient receptor potential cation channel superfamily
TRP
Gene Name
TRPC1
TRPC1
TRPC2
TRPC2
TRPC3
TRPC3
TRPC4
TRPC4
TRPC5
TRPC5
TRPC6
TRPC6
TRPC7
TRPC7
TRPV1
TRPV1
TRPV2
TRPV2
TRPV3
TRPV3
TRPV4
TRPV4
TRPV5
TRPV5
TRPV6
TRPV6
TRPA1
TRPA1
TRPM1
TRPM1
TRPM2
TRPM2
TRPM3
TRPM3
TRPM4
TRPM4
TRPM5
TRPM5
TRPM6
TRPM6
TRPM7
TRPM7
TRPM8
TRPM8
TRPP1
TRPP2
TRPP3
TRPML1
TRPML2
TRPML3
PKD2
PKD2L1
PKD2L2
MCOLN1
MCOLN2
MCOLN3
Description
Transient receptor potential cation channel, subfamily
member 1
Transient receptor potential cation channel, subfamily
member 2
Transient receptor potential cation channel, subfamily
member 3
Transient receptor potential cation channel, subfamily
member 4
Transient receptor potential cation channel, subfamily
member 5
Transient receptor potential cation channel, subfamily
member 6
Transient receptor potential cation channel, subfamily
member 7
Transient receptor potential cation channel, subfamily
member 1
Transient receptor potential cation channel, subfamily
member 2
Transient receptor potential cation channel, subfamily
member 3
Transient receptor potential cation channel, subfamily
member 4
Transient receptor potential cation channel, subfamily
member 5
Transient receptor potential cation channel, subfamily
member 6
Transient receptor potential cation channel, subfamily
member 1
Transient receptor potential cation channel, subfamily
M, member 1
Transient receptor potential cation channel, subfamily
M, member 2
Transient receptor potential cation channel, subfamily
M, member 3
Transient receptor potential cation channel, subfamily
M, member 4
Transient receptor potential cation channel, subfamily
M, member 5
Transient receptor potential cation channel, subfamily
M, member 6
Transient receptor potential cation channel, subfamily
M, member 7
Transient receptor potential cation channel, subfamily
M, member 8
Polycystic kidney disease 2 (autosomal dominant)
Polycystic kidney disease 2-like 1
Polycystic kidney disease 2-like 2
Mucolipin 1
Mucolipin 2
Mucolipin 3
ranged around a central ion-permeable pore in a configuration reminiscent of previously characterized voltage-gated
ion channels. Six transmembrane ␣-helices (S1–S6) span the
lipid bilayer, with a pore-forming loop between the S5 and
S6 domains. The TRP domain forms an interfacial helix
distal to the S6 domain that is positioned to interact with
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UniGene ID
Chromosome
C,
Hs.250687
3
C,
Hs.131910
17
C,
Hs.150981
4
C,
Hs.262960
13
C,
Hs.657709
X
C,
Hs.159003
1
C,
Hs.591263
5
V,
Hs.579217
17
V,
Hs.279746
17
V,
Hs.446255
17
V,
Hs.506713
12
V,
Hs.283369
7
V,
Hs.302740
7
A,
Hs.667156
8
Hs.155942
15
Hs.369759
21
Hs.47288
9
Hs.467101
19
Hs.272287
11
Hs.272225
9
Hs.512894
15
Hs.366053
2
Hs.181272
Hs.159241
Hs.567453
Hs.631858
Hs.591446
Hs.535239
4
10
5
19
1
1
the region preceding the S1 domain and the linker region
between the S4 and S5 domains. The outer pore of the
assembled channel is very wide. The selectivity filter is short
and narrow in the inactive configuration, but dilates substantially when the channel is activated by capsaicin. This
work confirmed the general structural features of TRP
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
S1 S2 S3 S4 S5
TRPA
S6
TRPM
S1 S2 S3 S4 S5
S6
TRPM
homology region
Kinase
(TRPM6/M7)
Coiled-coil
C
C
N
Nudix
(TRPM2)
N
Ankyrin repeat
S1 S2 S3 S4 S5
TRPV
S6
TRPP
S1 S2 S3 S4 S5
S6
TRP box
PDZ (TRPV3)
C
N
N
C
Ankyrin repeat
TRPC
S1 S2 S3 S4 S5
S6
TRPML
S1 S2 S3 S4 S5
S6
TRP box
Coiled-coil
PDZ (TRPC4, C5)
N
FIGURE 2.
C
N
C
Membrane topology and functional domains of TRP subunits.
channels that had been derived using lower-resolution cryoelectron microscopy methods in studies of TRPV1 (227),
TRPV4 (313), and TRPC3 (225) channels. Not surprisingly, these studies, as well as the few available crystallography analyses of the ankyrin repeat domain of TRPV2
(150, 215), revealed specific structural differences in both
intracellular and extracellular domains that are clearly associated with the unique signaling partners and sensory
modalities served by individual TRP channel family members.
C. Functions in Mammalian Cells
TRP channels influence numerous important biological
processes by controlling the flux of cations across the
plasma membrane. Several TRP family members, including
TRPP2, TRPM8, TRPV1, and TRPA1, are also present and
active on the membranes of intracellular organelles and are
involved in protein trafficking and vesicular ionic homeostasis (see Ref. 71 for review). Cation permeability has substantial biological significance. The distribution of the monovalent cations Na⫹ and K⫹ is particularly important for
the regulation of cellular membrane potential and excitability. The divalent cations Ca2⫹ and Mg2⫹ also function as
intracellular second messengers and as required cofactors
for enzymatic activity. Although TRP channels are often
described as “nonselective cation channels,” this definition
is too broad and rather imprecise. TRPV5 and TRPV6
channels are highly selective for Ca2⫹, with relative permeability of Ca2⫹ versus Na⫹ (PCa:PNa) of ⬃100:1 (253, 368,
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
415). TRPM4 (179, 251) and TRPM5 channels (139, 276)
are selective for monovalent cations (Na⫹ and K⫹) and are
essentially impermeant to Ca2⫹ and other divalent cations.
Ion substitution experiments suggest that the remaining
TRP channels are permeant to both mono- and divalent
cations, but with ionic preference ranging from essentially
nonselective (i.e., TRPC1; Ref. 334) to significantly selective for Ca2⫹ (e.g., TRPV3; PCa:PNa ⬃12:1; Ref. 401) or
preferentially selective for Mg2⫹ under physiological conditions (TRPM6 and TRPM7) (186). Selectivity for particular monovalent species also varies, with certain channels
favoring Na⫹ versus K⫹ (e.g., TRPC3, TRPC6) and others
with the opposite bias (e.g., TRPM4, TRPV4). This property is significant, since the reversal potential of channels
with high K⫹ permeability occurs at negative membrane
potentials in physiological ionic gradients; thus activation
of these channels will have only a small direct effect on
membrane potential depolarization. Membrane depolarization is greater for channels with a higher Na⫹ conductance,
which diminishes the electrochemical driving force for
Ca2⫹ influx in nonexcitable cells. Ultimately, this leads to
channels with greater K⫹ permeability (e.g., TRPV4) conducting mixed currents having a higher fraction of Ca2⫹
compared with channels with higher Na⫹ permeability
(e.g., TRPC6). Among the channels that conduct currents
with a significant (⬎20%) Ca2⫹ fraction under physiological conditions are TRPV1-4 and TRPA1; in contrast, the
fractional contribution of Ca2⫹ to TRPC channel currents
is expected to be very low. Native currents are often not
apparent from patch-clamp records reported in the literature, since these experiments are usually performed using a
symmetrical cationic distribution. Few studies have actually
attempted to record and characterize mixed currents under
physiological ionic conditions.
1. Membrane potential regulation
Asymmetric concentration gradients maintained by cellular
ion transporters and pumps establish the resting membrane
potential. TRP channels contribute to membrane potential
regulation in both excitable cells (i.e., those expressing voltage-gated Na⫹ or Ca2⫹ channels), such as neurons, glia,
myocytes, and endocrine cells, and nonexcitable cells (i.e.,
those lacking voltage-gated ion channels), such as epithelial
and endothelial cells, by regulating the flux of cations across
the plasma membrane.
TRPM4 channels have a well-documented influence on
membrane potential regulation in excitable and nonexcitable cells. These channels have a unitary conductance of
⬃25 pS, are selective for monovalent cations and impermeant to Ca2⫹ ions, and are activated by high levels of
intracellular Ca2⫹ (179). TRPM4 channels are equally permeant to Na⫹ and K⫹, resulting in a reversal potential of
⬃0 mV in physiological solutions. At the hyperpolarized
resting membrane potentials that are typical for most cells,
these channels conduct inward currents that are primarily
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composed of Na⫹ when activated by global or localized
increases in [Ca2⫹]i, resulting in membrane depolarization.
TRPM4-mediated Na⫹ currents influence the membrane
excitability of pancreatic ␤-cells (53), detrusor (258, 321,
322) and cerebral artery (64, 76, 83, 104, 114, 115, 117,
118) smooth muscle cells, atrial cardiomyocytes (317), and
pre-Botzinger neurons (226). In addition, membrane potential regulation in nonexcitable mast (369) and dendritic
cells (26) is disrupted in TRPM4⫺/⫺ mice. Membrane hyperpolarization associated with TRPM4 knockout and loss
of depolarizing Na⫹ currents increases the driving force for
Ca2⫹ influx, resulting in Ca2⫹ overload and impaired
chemokine-dependent migration of dendritic cells (26).
2. Ca2⫹ signaling
Changes in the intracellular concentration of free Ca2⫹ influence muscle contraction, hormone secretion, gene expression, cell proliferation, and many other critical processes (32). The cytosolic free [Ca2⫹] is normally maintained at low levels (⬃100 –300 nM) by stationary and
mobile Ca2⫹-buffering proteins and by the activity of Ca2⫹pumping ATPases located on the plasma membrane and
other cellular compartments. Changes in intracellular Ca2⫹
occur as a result of the influx of Ca2⫹ from the extracellular
space and/or through the release of Ca2⫹ from intracellular
structures. The endoplasmic/sarcoplasmic reticulum (ER/
SR) is the best characterized intracellular Ca2⫹ storage organelle, but the membranes of mitochondria, nuclei, and
other structures also delimit regions with high [Ca2⫹]. Ca2⫹
signals can be global, occurring throughout the cytosol, or
can be localized, creating subcellular domains where [Ca2⫹]
is transiently elevated. Almost all TRP channels are permeable to Ca2⫹, and several have a strong influence on Ca2⫹dependent signaling pathways (62). TRP channels have
been described as direct (i.e., “ionotropic”) Ca2⫹ influx
pathways, metabotropic (receptor-operated) Ca2⫹ influx
channels, or store-operated Ca2⫹ channels, although this
latter property is disputed.
A) TRP CHANNELS AS IONOTROPIC CA2⫹ INFLUX PATHWAYS: TRP
Elementary Ca2⫹ signals representing Ca2⫹ influx through single TRPV4 channels, called TRPV4 sparklets, have been recorded from airway (423) and vascular
smooth muscle cells (221), endothelial cells (337), and the
intact endothelium (324) (for review, see Ref. 336). Recordings of TRPA1 sparklets have also been reported for cerebral artery endothelial cells (338). Theoretically, all TRP
channels with sufficient unitary conductance and Ca2⫹ permeability (i.e., TRPV1-6; TRPA1) should produce detectable sparklets. TRPV4 and TRPA1 sparklets are optically
recorded from cells expressing Ca2⫹ biosensors or loaded
with Ca2⫹-indicator dyes using total internal reflection fluorescent (TIRF) or confocal microscopy (FIGURE 3). Individual TRPV4 sparklets are very large Ca2⫹ influx events,
with the amount of Ca2⫹ entering estimated to be ⬃100
times that of a single L-type Ca2⫹ channel sparklet (221).
SPARKLETS.
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
A
2.0
1.5
1.0
0.5
t = 0 ms
t = 30 ms
CPA 30 µM
CPA 30 µM + GSK 10 nM
d
d
t = 90 ms
t = 60 ms
c
t = 150 420 ms
t = 450 ms
1.5
1.0
0.5
a
3
2
b
1
c
0
1
2
3
2
4
4
6
8
10
d
10 s
Time (s)
Control
600
90
Counts
tsA-201 cell
400
10 nM GSK
45
50 nM
Δ [Ca2+]i (nM)
B
0
200
0
0
2s
Control
50
100
60
Counts
100
50
Δ [Ca2+]i (nM)
80
Arterial myocyte
100 nM GSK
40
20
0
50 nM
Δ [Ca2+]i (nM)
b
4
0
0
a
a
t = 480 ms
b
2.0
Channels
Fluorescence
(F)
t = 120 ms
c
1 F/F0
Fluorescence
(F)
C
2s
0
0
50
100
150
Δ [Ca2+]i (nM)
200
FIGURE 3. TRPV4 sparklets in endothelial cells and vascular smooth muscle cells. A: time-lapse image of a
typical TRPV4 sparklet recorded from a primary cerebral artery endothelial cell using TIRF microscopy. The cell
was stimulated with the selective TRPV4 agonist GSK1016790A (GSK; 100 nM). B: TRPV4 sparklets
recorded from a tsA-201 cell transfected with TRPV4 (top) and from a native cerebral artery myocyte (bottom).
The middle and right panels demonstrate the single channel-like behavior of these events. TRPV4 sparklets
were stimulated with GSK. C: TRPV4 sparklets recorded from the intact endothelium of cerebral arteries from
mice expressing the Ca2⫹-indicator protein GCaMP2 exclusively in the endothelium using high-speed, highresolution confocal microscopy before and after stimulation with GSK. Experiments were performed in the
presence of cyclopiazonic acid (CPA) to eliminate interference from ER Ca2⫹-release events. Representative
traces indicating single channel-like events are shown below. [A from Sullivan and Earley (336). B from
Mercado et al. (221). C from Sonkusare et al. (323), with permission from American Association for the
Advancement of Science.]
The unitary amplitude of TRPA1 sparklets (338) is approximately twice that of a TRPV4 sparklet recorded under
identical conditions (337), suggesting that more Ca2⫹ enters during the opening of a single TRPA1 channel than is
the case for a single TRPV4 channel. TRPV4 sparklets generate subcellular microdomains in which local Ca2⫹ levels
are very high, leading to activation of a variety of Ca2⫹dependent signaling cascades, including endothelium-dependent vasodilation (324), negative feedback inhibition of
vasoconstrictor stimuli (221), and NFATc3-dependent
smooth muscle proliferation (423). TRPA1 sparklets are
linked to endothelium-dependent dilation of cerebral arteries (338).
B) TRP CHANNELS AS RECEPTOR-OPERATED CHANNELS.
Activation
of specific GPCRs on the cell surface causes a biphasic
change in global [Ca2⫹]i, with an initial transient increase
followed by a sustained plateau phase (FIGURE 4A) (for review, see Ref. 32). The transient Ca2⫹ signal results from
release of Ca2⫹ from the ER/SR, whereas influx of Ca2⫹
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
Agonist
Ca2+
A
Gq
IP3
IP3R
ROCE
ER/SR
Ca2+
Time
Ca2+ Ca2+
Orai1
Ca2+ release
Plasma membrane
DAG
C
GPCR agonist
TRPC3/C6/C7
PIP2
PLC
B
Intracellular [Ca2+]
GPCR
D
Thapsigargin
TRPC1/C4/C5 (?)
Ca2+ free
STIM1
ER/SR
Ca2+
Intracellular [Ca2+]
Plasma membrane
SOCE
Time
FIGURE 4. Ca2⫹-influx mechanisms mediated by TRP channels. A: receptor-operated Ca2⫹ entry (ROCE).
Agonist binding to GPCRs stimulates the activity of PLC, which cleaves the membrane phospholipid PIP2 into IP3
and DAG. IP3 binds to IP3Rs on the ER/SR to cause release of Ca2⫹ into the cytosol. DAG directly activates
Ca2⫹ influx through TRPC3, TRPC6, or TRPC7 channels on the plasma membrane. B: example trace of
changes in intracellular Ca2⫹ following stimulation of a GPCR, showing the phasic increase resulting from
ER/SR Ca2⫹ release and sustained elevation resulting from ROCE. C: store-operated Ca2⫹ entry (SOCE).
Depletion of Ca2⫹ in the ER/SR causes clustering of STIM1 in the ER/SR membrane proximal to the plasma
membrane. STIM1 interacts with Oria1 at the plasma membrane to promote Ca2⫹ influx. TRPC1, TRPC4,
and/or TRPC5 channels, either independently or in association with STIM1/Oria1, may participate in SOCE.
D: example of a typical SOCE experiment. Under Ca2⫹-free conditions, cells are treated with the SERCA inhibitor
thapsigargin, causing Ca2⫹ to leak from the ER/SR into the cytosol. Reintroduction of Ca2⫹ to the bathing
solution after ER/SR stores are depleted causes SOCE.
through receptor-operated channels (ROCs) is responsible
the sustained phase (FIGURE 4B). This latter mode of Ca2⫹
influx is called “receptor operated Ca2⫹ entry” (ROCE).
TRPC3, TRPC6, and TRPC7 channels have been characterized as ROCs that are activated by GPCRs linked to
phospholipase C (PLC) (144, 199, 266, 323). In response to
receptor stimulation, PLC cleaves the membrane phospholipid PIP2 into the second messenger molecules inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the latter of which activates protein kinase C (PKC). IP3 binds to
IP3 receptors (IP3Rs) present on the ER/SR to cause Ca2⫹
release from intracellular stores. DAG is a direct (i.e., independent of PKC) activator of TRPC3, TRPC6, and TRPC7
channels, as well as heteromultimeric channels that include
652
these subunits (140); ROCE through these channels accounts for sustained elevations in intracellular Ca2⫹.
The PLC substrate PIP2 can directly influence, both positively and negatively, the activity of almost all TRP channels. For example, application of PIP2 to excised membrane
patches has been shown to directly activate the ROCs discussed above (TRPC3, TRPC6, and TRPC7) in HEK cell
expression systems (181). This extensive topic is the subject
of several recent reviews (285, 286, 288).
C) TRP CHANNELS AS STORE-OPERATED CHANNELS. Store-operated
Ca2⫹ entry (SOCE) is defined as Ca2⫹ influx that occurs in
response to depletion of intracellular Ca2⫹ stores, typically
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
accomplished experimentally by prolonged stimulation of
cell surface receptors or blocking SERCA activity with
pharmacological agents such as thapsigargin or cyclopiazonic acid (260, 278) (FIGURE 4). Ca2⫹ directly enters the
ER/SR compartment during SOCE, providing a dedicated
mechanism for refilling depleted intracellular Ca2⫹ stores.
The ion channels that conduct SOCE are called store-operated channels. A number of early studies suggested that
TRPC1, TRPC4, and/or TRPC5 channels are store-operated channels (6, 97, 122, 261, 396). However, the discovery that complexes formed by stromal interacting protein 1
(STIM1) and Orai1 are critically involved in SOCE (for
review, see Ref. 21) cast doubt on an obligate role for TRP
channels in this response. STIM1 is a single-transmembrane-domain protein present on both the plasma membrane and the ER/SR. The NH2 terminus of STIM1 contains an EF hand Ca2⫹-sensing domain that resides in the
ER/SR lumen and two coiled-coil domains in the cytosolic
COOH terminus that mediate homomeric and heteromeric
protein-protein interactions. Orai1 is a Ca2⫹-selective ion
channel present on the plasma membrane (275). Depletion
of intracellular Ca2⫹ stores is sensed by the EF hand domain
located in the ER/SR lumen and leads to rearrangement and
clustering of STIM1 proteins at the surface of the ER/SR
proximal to the plasma membrane. STIM1 clusters interact
with Orai1 on the plasma membrane, thereby initiating
Ca2⫹ entry. Studies have shown that SOCE can occur in the
absence of TRPC1, TRPC4, and TRPC5 channel expression and that TRPC channels can function independently of
STIM1 and Orai1 (68), leading some to conclude that TRP
channels are not involved in SOCE. However, other studies
have reported that TRPC channels are required for SOCE in
certain settings, and some investigators have noted that the
structure of Orai1 is more reminiscent of a regulatory subunit than classically described ion-permeable channels
(393). It has been proposed that Orai1 proteins are a regulatory component of a store-operated channel composed of
TRPC1 subunits (or vice versa) (16), but this issue is currently unresolved. Experimental details are important in
interpreting SOCE experiments. For example, in whole cell
patch-clamp experiments, if cytosolic Ca2⫹ is not fully buffered, SOCE through the STIM1/Oria1 pathway can cause
Ca2⫹-dependent activation of certain TRP channels that is
independent of store depletion. In this context, it has been
proposed that SOCE through Orai1 causes TRPC1 channels to traffic to the plasma membrane where they mediate
a secondary Ca2⫹ influx event (55). The precise functional
relationships between STIM1, Oria1, and TRPC channels
during SOCE remain to be determined.
3. Mg
2⫹
TRPM7 has a unitary conductance of ⬃40 –105 pS, is
abundantly expressed in many types of cells, and is constitutively active in heterologous expression systems (240).
TRPM7 is permeable to both monovalent and divalent cations, although some reports suggest greater permeability of
divalent cations under physiological conditions. The channel is inhibited by intracellular [Mg2⫹] greater than 1 mM.
Under physiological conditions, the channel may act as a
Mg2⫹/Ca2⫹ influx pathway, with intracellular [Mg2⫹] providing negative feedback. The ion permeation and regulatory properties of TRPM6 are similar to those of TRPM7,
but its unitary conductance is greater (⬃80 pS). TRPM6 can
form heteromultimeric channels with TRPM7 with properties distinct from those of the homomeric channels. Significantly, mutations within TRPM6 are associated with familial hypomagnesemia with secondary hypocalcemia
(303), and TRPM7 deletion causes Mg2⫹ deficiency, indicating that TRPM6 and TRPM7 channels are vital for
Mg2⫹ homeostasis. TRPM6 and TRPM7 channels are also
permeable to other divalent cations, including the essential
elements Zn2⫹, Co2⫹, and Mn2⫹, and toxic metals such as
Sr2⫹, Cd2⫹, Ba2⫹, and Ni2⫹, and may be involved in transporting these ions across the plasma membrane (228).
III. PHYSIOLOGICAL ROLES OF TRP
CHANNELS IN THE VASCULATURE
The primary function of the vasculature is to dynamically regulate blood flow and vascular permeability to ensure appropriate matching of oxygen and nutrient supply with the metabolic demands of the tissue. Vascular resistance and blood
flow are primarily regulated by the contractile state of smooth
muscle in the arterial circulation, but can also be chronically
altered by cellular proliferation and vascular remodeling that
occur during vascular development and in association with
vascular diseases. Endothelial cells have a profound influence
on vascular tone and permeability, an influence that can vary
between physiological and pathophysiological conditions.
Perivascular neurons and astrocytes closely apposed to blood
vessels can also influence smooth muscle contractility and
blood flow regulation. A growing body of evidence, reviewed
below, clearly demonstrates the importance of TRP channels
in the initiation, modulation, and integration of regulatory
pathways that govern the contributions of these diverse cell
types within the vascular wall to control of vasomotor tone
and tissue blood flow.
A. TRP Channels in Vascular Smooth Muscle
homeostasis
Mg2⫹ are central to the maintenance of nucleic acid structure, regulation of certain Ca2⫹ and K⫹ channels, and the
biological activity of ATP and hundreds of enzymes (for
review, see Ref. 67). TRPM6 and TRPM7 channels are
important pathways for Mg2⫹ influx (228, 303, 305).
Luminal diameter and hence vascular resistance are determined by the contractile state of vascular smooth muscle
cells, which are arranged circumferentially around the lumen of all blood vessels. Vascular smooth muscle contraction is primarily regulated by the phosphorylation state of a
myosin regulatory light chain (RLC) domain in the smooth
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653
SCOTT EARLEY AND JOSEPH E. BRAYDEN
muscle myosin complex. The net balance of myosin light
chain (MLC) kinase versus MLC phosphatase activity determines the phosphorylation state of the RLC. MLC kinase, a Ca2⫹/calmodulin-dependent enzyme, is activated by
increases in cytosolic Ca2⫹ levels, but its phosphatase activity is regulated by intracellular signaling pathways that
serve to modify the sensitivity of the contractile apparatus
to intracellular Ca2⫹. Smooth muscle [Ca2⫹]i is dynamically regulated by the relative activities of Ca2⫹-entry, -release, and -extrusion mechanisms. The L-type voltage-dependent Ca2⫹ channel (VDCC) is the primary Ca2⫹-influx
pathway in vascular smooth muscle cells. Thus membrane
depolarization and subsequent influx of Ca2⫹ through these
channels is a primary mechanism of vascular smooth muscle cell contraction (FIGURE 5). Increased cation permeability associated with TRP channel activation results in membrane depolarization and Ca2⫹ influx, which are the
primary mechanisms by which these channels cause vasoconstriction. Ca2⫹ influx through TRP channels with significant selectivity for Ca2⫹ could theoretically directly activate the contractile process, but there is little experimental
evidence in support of this possibility in the systemic circulation. However, some data suggest that SOCE through
TRP channels can support a vasoconstrictor response in
pulmonary arteries (see sect. IIIA2).
1. TRP channels and vascular smooth muscle
contractility
Nearly a dozen TRP channels have been detected in vascular smooth muscle using RT-PCR, Western blotting, and
immunolabeling approaches (TABLE 2). Lacking selective
pharmacological blockers or activators, early researchers in
the field relied on antisense nucleotide-mediated knockdown of TRP channel expression or antibodies that bind to
epitopes on extracellular domains of the channels to inhibit
cation currents. In some cases, the specificity of these treatments was not fully demonstrated; thus the conclusions
drawn from the results of these studies should be carefully
Na+, Ca2+
considered. In addition, many studies have attempted to
study smooth muscle cell contractility using cultured cells,
such as the aorta-derived A7r5 transformed cell line. This
approach is questionable, as A7r5 cells (and other transformed cell lines) display little or no contractility and are
phenotypically distinct from native vascular smooth muscle
cells. Primary smooth muscle cells are also problematic
since they rapidly (within a few hours) differentiate to a
proliferative, noncontractile state under standard culture
conditions. Accordingly, conclusions about regulation of
smooth muscle cell contractility derived from data obtained
using cultured cells should be interpreted with caution.
Even now, many studies continue to rely on nonselective
pharmacological agents, such as trivalent cations (Gd3⫹,
La3⫹), 2-aminoethoxydiphenyl borate (2-APB), ruthenium
red, and SKF96365 without consideration of the broad
range of targets affected by these compounds. Although
these experimental tools have their place, knockout animals, small inhibitory RNA (siRNA) approaches, and selective pharmacological agents that have been developed in
recent years are more definitive approaches for studying
contractile responses.
A) TRPC6. Compelling evidence indicates that TRPC6 channels are functionally important in arterial myocytes.
TRPC6 channel mRNA and protein have been identified in
vascular smooth muscle, both arterial and venous, from
many species and in many vascular beds (145). In native
cells, it is often difficult to determine whether the observed
ion channel activity is attributable to a specific TRPC channel isoform, perhaps because many TRPC subunits form
heteromultimeric associations, creating a challenge for
characterizing endogenous TRPC channels. This is in contrast to expression systems, where the composition of channel isoforms is controlled. Furthermore, TRPC channels are
often activated or modulated by diverse cellular signaling
mechanisms, the activity and function of which are often
altered by cell isolation procedures or the specific patchclamp configuration (i.e., on-cell, whole-cell, perforated
Ca2+
TRP
VDCC
ΔEm
Local Ca2+
SR
Ca2+-CaM
?
Global
Ca2+
Relaxation
MLC20
MLCK
MLCP
P-MLC20
Smooth muscle cell
654
Contraction
FIGURE 5. TRP channels contribute to
vascular smooth muscle cell contractility.
Cation influx through TRP channels depolarizes the plasma membrane (⌬Em), initiating
Ca2⫹ influx through VDCCs. Elevated Ca2⫹
levels increase binding of calmodulin-Ca2⫹
(Cd-Ca2⫹) complexes to MLCK, stimulating
phosphorylation of the regulatory myosin
light chain (MLC20). MLC20 is dephosphorylated by myosin light-chain phosphatase
(MLCP). Some studies suggest that Ca2⫹
influx through TRP channels can directly initiate myocyte contraction.
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
Table 2. TRP channels in vascular smooth muscle
TRP
Function
TRPC1
TRPC3
ROCE (ET-1 constriction), SOCE
GPCR-mediated vasoconstriction
TRPC4
TRPC5
TRPC6
TRPV1
SOCE
SOCE
GPCR-mediated vasoconstriction, myogenic tone
Vasoconstriction
TRPV2
TRPV3
TRPV4
TRPA1
TRPM4
TRPM8
TRPP2
Mechanosensitive Ca2⫹ influx
mRNA present in VSM but no known function
Ca2⫹ influx, vasodilation
Vasodilation
Depolarization, myogenic tone
Vasoconstriction
Ca2⫹ entry, vasoconstriction, myogenic tone
Tissue
Brain (124), aorta (344), lung (245–247)
Brain (4, 5, 283, 398), aorta (198), heart (266), kidney (296),
lung (420)
Aorta and mesentery (196, 197)
Brain (403)
Portal vein (146), aorta (323), brain (217, 387)
Aorta, skeletal muscle (158, 201), mesentery (273), cephalic
circulation (46)
Aorta (238)
Aorta and lung (408)
Brain (79, 221), mesentery (81)
Aorta (405)
Brain (64, 82, 83, 104, 114, 115, 117)
Aorta (151)
Mesentery (279, 308), brain (243)
ROCE, receptor-operated Ca2⫹ entry; SOCE, store-operated Ca2⫹ entry; ET-1, endothelin-1; GPCR, G proteincoupled receptor. Reference numbers are given in parentheses.
patch) employed. Nevertheless, single-channel activity that
likely is determined primarily by TRPC6 channels has been
observed in a variety of vascular smooth muscle types, including portal vein (9), mesenteric artery (293), and cultured aortic smooth muscle cells (144). Single-channel conductance values ranging from 33 to 45 pS have been reported in these studies. The presence of functional ion
channels in mesenteric (293) myocytes that are composed,
at least in part, of TRPC6 subunit proteins has been confirmed through the use of current-blocking antibodies that
target TRPC6.
In work growing out of initial observations that TRPC
channel activity in expression systems is linked to activation
of GPCRs (62), Inoue et al. (146) provided the first evidence
that TRPC6 channels are present and functionally active in
native vascular smooth muscle. These seminal studies
clearly demonstrated that a nonselective cation channel
with biophysical and pharmacological properties resembling those of cloned TRPC6 channels expressed in heterologous systems is present in portal vein myocytes. The
activity of this portal vein smooth muscle channel was substantially elevated following ␣1-adrenoceptor activation,
and was greatly reduced or was absent in cells treated with
TRPC6-targeted antisense oligonucleotides. Subsequent
studies have demonstrated that TRPC6 channels in vascular
myocytes are activated by a variety of GPCR ligands. For
example, nonselective cation channels with a TRPC6 biophysical and pharmacological profile (154), or that are suppressed by TRPC6 siRNA (323), are activated by vasopressin in A7r5 aortic smooth muscle-derived cells. Likewise,
vasopressin-induced Ca2⫹ entry (323) and Ca2⫹ oscillations (210) in A7r5 cells are suppressed by treatment with
TRPC6 siRNA. In freshly isolated rabbit mesenteric artery
myocytes, low levels (1 nM) of angiotensin II (ANG II) were
shown to activate a nonselective cation channel that was
inhibited by TRPC6 antibodies (293). Activation of TRPC6
in these studies followed stimulation of a GPCR and subsequent signaling via PLC and generation of DAG (327). The
stimulatory effect of DAG on TRPC6 channel activity appeared to be direct and was independent of PKC activation
(137, 140). Furthermore, there are apparent interactions
between receptor-mediated and other signaling modalities
that result in amplification of vascular TRPC6 channel activity under some conditions. Specifically, transmembrane
Ca2⫹ mobilization through TRPC6 activation is amplified
through combined receptor and mechanical stimulation via
signaling pathways that involve contributions by both PLC
and phospholipase A2 (PLA2) (144). Other examples of
vascular TRPC6 coregulation by various cellular signaling
molecules have been noted. Synergistic stimulatory effects
on vascular TRPC6 channels through coactivation of IP3Rs
and DAG (9, 147, 152) or IP3 and arginine vasopressin
receptors (213) have been described, and it has been shown
that protein kinase A (PKA) activity amplifies the activating
effects of ANG II on TRPC6 channels (255). In contrast to
findings obtained using a HEK cell expression system (181),
PIP2 was shown to inhibit TRPC6 currents in native mesenteric artery smooth muscle cells (13). TRPC6 channels
may also be inhibited by interactions with other TRPC
channels, including TRPC1/C5 heteromultimeric channels
(312).
Functional studies have linked the activation of TRPC6
channels in vascular smooth muscle cells with pressureinduced (myogenic) vasoconstriction. Myogenic constriction, which contributes to the autoregulation of blood flow
in some vascular beds (28), is mediated by depolarization of
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
the arterial myocyte plasma membrane and Ca2⫹ entry
through VDCCs (163). TRPC6 channels in vascular
smooth muscle cells and expression systems are activated by
mechanical forces such as cell swelling (387) (327) or application of suction to the plasma membrane (327). However, the exact mechanism responsible for mechanical activation of TRPC6 is a matter of controversy, with some
studies indicating that TRPC6 channels are directly activated by application of force (327) and others reporting
indirect activation downstream of mechanosensitive signaling pathways (217). Data from some laboratories point to
GPCRs as the primary mechanosensors that activate
TRPC6 channels through downstream signaling involving
the formation of DAG (217, 302). A study by Welsh et al.
(387) demonstrated that swelling-activated cation currents
and intravascular pressure-induced smooth muscle cell depolarization and vasoconstriction were substantially reduced in cerebral arteries treated with antisense oligonucleotides targeting TRPC6, suggesting the critical involvement
of this channel in the myogenic response in this vascular
bed. It should be mentioned that neither agonist-evoked
contraction nor myogenic tone were reduced in arteries
from TRPC6⫺/⫺ mice (70, 302). However, upregulation of
constitutively active (12) TRPC3 channels in TRPC6⫺/⫺
mice may provide a compensating depolarization and vasoconstrictor pathway in these animals.
in vasoconstriction) (FIGURE 6). This model clearly delineates the architecture, interactions, and mechanisms of action of a variety of signaling molecules recognized as critical
players in the development and modulation of arterial tone
in the brain circulation.
TRPC6 channels are also involved in vasoconstrictor responses in the pulmonary arterial circulation (385). Small
pulmonary arteries constrict in response to hypoxic challenge, a response called hypoxic pulmonary vasoconstriction (HPV). HPV improves oxygenation of the blood by
matching perfusion to well-ventilated regions of the lung.
Weissmann et al. (385) found that robust HPV, present in
the lungs of wild-type mice, was absent in TRPC6⫺/⫺ mice.
Hypoxia-induced activation of Ca2⫹ influx as well as
TRPC6-like cation currents were also absent in pulmonary
artery myocytes from TRPC6⫺/⫺ mice. Interestingly, in
contrast to the apparent compensatory upregulation of
TRPC3 expression in nonpulmonary arteries previously
documented in TRPC6⫺/⫺ mice (70), no change in the expression of TRPC3 or other TRPC channels (other than
TRPC6) was reported in this study. A follow-up study by
this group reported the clear involvement of DAG as a
mediator of TRPC6 activation in pulmonary artery myocytes during hypoxia (99). Others have reported a key role
for epoxyeicosatrienoic acid (EET) derivatives as mediators
of enhanced TRPC6 contributions to HPV (272). Much of
the ability of EETs to increase channel activity in pulmonary artery vascular smooth muscle cells is likely attributable to recruitment of TRPC6 channels to caveolae (159).
TRPC6 appears to be an integral part of a force-sensing
complex in cerebral artery smooth muscle cells that regulates myogenic vasoconstriction (118). Gonzales et al. (118)
demonstrated that Ca2⫹ entry through TRPC6 channels,
activated either by PLC-␥1-dependent generation of DAG
or by direct mechanical stimulation, reinforces IP3R-dependent Ca2⫹ release from the SR, which then activates nearby
TRPM4 channels. TRPM4 channels, in turn, mediate
smooth muscle cell depolarization, promoting Ca2⫹ influx
through VDCCs and subsequent vasoconstriction (see below for more details regarding the role of TRPM4 channels
It is now abundantly clear that TRPC6 channels are fundamentally important for the onset, maintenance, and modulation of vascular tone in many different vascular beds.
Additional studies are needed to further define the unique
contributions of these channels in various regions within
the cardiovascular system and to characterize other important modulatory inputs that may influence the activity of
Na+
AT1R
PM
Na+
TRPC6
PLCγ1
Src
Ca2+
(+)
DAG
PtdIns(4,5)P2
IP3
TRPM4
VDCC
ΔVM
?
(+)
Ca2+
IP3R
Ca2+
SR
Contraction
FIGURE 6. A force-sensitive signaling network involving TRPC6 and TRPM4 channels in cerebral artery
smooth muscle cells. A stretch-sensing pathway in cerebral artery myocytes that includes 1) activation of
AT1R, Src tyrosine kinase, and PLC-␥1, leading to the generation of IP3; and 2) Ca2⫹ influx through TRPC6
channels onto IP3Rs, promoting CICR that results in activation of TRPM4 channels, which are responsible for
pressure-induced depolarization of the plasma membrane. PM, plasma membrane; ⌬Vm, change in membrane
potential. [From Gonzales et al. (118).]
656
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
these channels in various physiological and pathological
contexts. For instance, more detail is needed about the regulation of TRPC6 by PLC-derived molecules, the influences
of related TRPC channels that form heteromultimers with
TRPC6, and the contributions of TPRC6-mediated Ca2⫹
entry to local and global cellular signaling processes.
B) TRPC3. Expression of TRPC3 channel mRNA and/or protein has been demonstrated by RT-PCR, Western blotting,
and/or immunohistochemical approaches in vascular
smooth muscle cells in cerebral (256, 283), cephalic (12),
coronary (168, 199), renal (373), uterine (306), and pulmonary arteries (420) and in the aorta (202). However, the
properties of TRPC3 currents in native vascular smooth
muscle cells have been described only rarely. Albert et al.
(12) recorded a constitutively active nonselective cation
channel in vascular myocytes from the rabbit ear artery
with pharmacological properties very similar to cloned
TRPC3 channels. This channel appeared to have three distinct conductance levels (10), although none matched the
single value reported for the cloned TRPC3 channel expressed in HEK cells (427). Heteromultimeric TRPC channel assembly, in particular between TRPC3 and TRPC7
channels, has been documented in coronary artery myocytes, and related diversity in channel properties has been
described (266).
TRPC3 channels in vascular smooth muscle cells are involved in contractile responses, primarily evoked by receptor-ligand interactions. This function has been best described in cerebral and carotid arteries and the aorta.
TRPC3 channels in vascular smooth muscle are activated
by stimulation of a variety of GPCRs, including pyrimidine
(283), endothelin-1 (ET-1) (266), and ANG II (199) receptors. In brain pial arteries, suppression of TRPC3 channel
expression using antisense oligonucleotides was shown to
greatly reduce inward currents, depolarization, and vasoconstriction induced by the purinergic receptor agonist uridine triphosphate (UTP) (283). Interestingly, in this study,
intrinsic myogenic tone was unaltered by TRPC3 downregulation, suggesting a specific role for TRPC3 in receptormediated vasoconstriction and not in pressure-induced vasoconstriction. This concept is supported by the findings of
another laboratory that showed Ca2⫹ entry and cerebral
artery constriction induced by ET-1 was decreased following suppression of TRPC3 expression (398). ET-1 also activated nonselective cation channels in coronary artery
myocytes that have properties conferred, in part, by TRPC3
channel protein (266). Liu et al. (199) reported that ANG II
receptor-mediated Ca2⫹ entry was decreased by suppression of TRPC3 expression and increased by TRPC3 overexpression in cultured aortic vascular smooth muscle cells.
In rat afferent arterioles, a heteromultimeric TRPC channel
structure containing TRPC3 together with TRPC1 and
TRPC6 subunits was shown to participate in a norepinephrine-stimulated, nifedipine-resistant constrictor response
(296). Taken together, these studies indicate that TRPC3
channels, alone or in association with other related TRPC
subunits, are important mediators of agonist-evoked vasoconstriction in many different types of arteries. An interesting aspect of TRPC3 behavior in general, and specifically in
vascular smooth muscle cells, is that these channels are
constitutively active (12). This basal TRPC3 activity suggests possible roles in tonic regulation of membrane potential and arterial tone (70, 306) as well as potential involvement in the enhancement of myogenic tone and/or agonistevoked vasoconstriction (306).
The primary mechanism for activation of TRPC3 downstream of receptor stimulation in expression systems (140)
and in native vascular myocytes (11) was initially thought
to be through the direct actions of DAG on the channel.
However, work from Kiselyov et al. (162) showed that
TRPC3 channels functionally interact with IP3Rs in HEK
cell expression systems, and Boulay et al. (35) showed that
TRPC3 and IP3Rs could be coimmunoprecipitated. Jaggar
and colleagues (3–5, 398) showed that IP3Rs are critical
elements in the activation mechanism for TRPC3 channels
in cerebral artery smooth muscle cells. This activation pathway does not involve IP3-mediated Ca2⫹ release from the
SR, but rather reflects direct coupling between IP3Rs and
TRPC3 channels mediated by IP3. Coupling of TRPC3
channels and IP3Rs in vascular smooth muscle cells leads to
activation of TRPC3-mediated cation currents, followed by
membrane depolarization, Ca2⫹ entry through VDCCs,
and myocyte contraction. Close physical association of
IP3Rs with TRPC3 channels is orchestrated by the integral
membrane protein caveolin-1. Activation of heteromultimeric
TRPC3/TRPC7 channels by ET-1 in coronary artery myocyte
(266) also involves products of phosphoinositide metabolism. Phosphatidylinositol-3-phosphate, formed by the actions of specific phosphatidylinositol 3-kinase isoforms, appears to be a direct activator of this channel (311). Interestingly, in this case IP3 itself inhibited the TRPC3/C7
heteromultimeric channel, demonstrating the potential differential control of homomeric versus heteromultimeric
TRPC channels. In expression systems, TRPC3 trafficking,
membrane localization, and activity are regulated by PIP2,
which is the primary substrate for phosphoinositide metabolism (142, 181). Whether this holds true for native vascular smooth muscle cells has not been determined.
Other kinase systems have substantial roles in the regulation of TRPC3 channels in vascular smooth muscle. In several instances, kinases have inhibitory effects on TRPC3
channel function. For instance, the serine-threonine kinase
WNK4 exerts a tonic inhibitory action on TRPC3 function
in rat aorta (262). Protein kinase G (PKG) also inhibits
TRPC3, an action that is correlated with dilation of isolated
carotid artery segments (49). This is consistent with previous observations that PKG inhibits TRPC3 channels expressed in HEK293 cells (175). In contrast, studies employ-
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657
SCOTT EARLEY AND JOSEPH E. BRAYDEN
ing TRPC3 knockout mice found no apparent link between
TRPC3 function and cGMP- or PKG-induced relaxation of
the aorta or the hindlimb circulation (202), perhaps suggesting species- or tissue-specific differences in this regard.
In expression systems (360) and in arterial myocytes (8),
TRPC3 channels are inhibited by PKC.
The weight of evidence supports the contention that TRPC3
channels in vascular smooth muscle play important roles in
regulating vascular contractility. Constitutive activity of
these channels contributes to basal vascular tone, and activation of TRPC3 channels by vasoconstrictor agonists
causes further membrane depolarization and Ca2⫹ entry
through VDCCs. Future studies will provide needed details
of the mechanisms by which the basal and ligand-activated
states of TRPC3 channels are achieved.
C) TRPM4.
TRPM4 and the closely related TRPM5 are the
only TRP channels that are essentially impermeant to Ca2⫹.
However, because of their relative high Na⫹-versus-K⫹ permeability and significant inward current amplitudes in standard ionic gradients and at physiological membrane potentials, TRPM4 channels have considerable capacity to affect
membrane depolarization and thereby alter cellular function. TRPM4 channels have been reported to influence
Ca2⫹ entry in excitable (125) and nonexcitable cells (178),
primarily through their depolarizing influence. In excitable
cells, depolarization leads to activation of VDCCs and a
global increase in Ca2⫹, whereas in nonexcitable cell depolarization diminishes passive Ca2⫹ influx because it reduces
the electrical driving force.
Molecular and functional studies have shown that TRPM4
channels are present in native smooth muscle cells. TRPM4
mRNA has been reported in smooth muscle cells in the rat
aorta (145, 408), as well as mesenteric (145), pulmonary
(408), and cerebral arteries (83, 145). Immunohistochemical and Western blot analyses have also demonstrated the
presence of TRPM4 protein in cerebral artery myocytes (64,
114). Single-channel and whole cell currents with properties very similar to those recorded from HEK cells overexpressing TRPM4 channels have been detected in cerebral
artery myocytes (82, 83, 234). The activity of TRPM4 channels is primarily regulated by the intracellular [Ca2⫹]
through interactions of the Ca2⫹/calmodulin complex with
defined sites near the COOH terminus (252). Activation of
native and exogenously expressed TRPM4 channels requires a very high (micromolar) [Ca2⫹] at the intracellular
face of the channel (82) (251). However, global intracellular Ca2⫹ levels in vascular smooth muscle cells are typically
in the range of 100 –500 nM, suggesting that TRPM4 activation must occur within subcellular regions with locally
elevated [Ca2⫹]. Such domains are created when Ca2⫹ is
released through ryanodine receptors (234) or IP3Rs (114).
Work by Gonzales et al. demonstrated that TRPM4 channels in native cerebral artery smooth muscle cells are acti-
658
vated by release of Ca2⫹ from IP3Rs (114) and that channel
activation is dependent on intracellular Ca2⫹ buffering
(115).
Ca2⫹-dependent activation of TRPM4 can be fine tuned by
other signaling pathways. For example, Nilius et al. (252)
demonstrated that the innate Ca2⫹ sensitivity of cloned
TRPM4 channels expressed in HEK cells is elevated by
PKC-dependent phosphorylation. Similarly, stimulation of
PKC activity was shown to increase TRPM4 whole cell
currents in cerebral artery myocytes and cause constriction
of isolated cerebral arteries (82). This increase in Ca2⫹ sensitivity was found to result from PKC-␦-dependent translocation of TRPM4 channels to the plasma membrane of
atrial myocytes (64, 104). PIP2 has been clearly implicated
as an important regulator of TRPM4 channel activity in
expression systems (250, 422). These studies demonstrated
that the exogenous administration of PIP2 prevented desensitization of TRPM4 currents in response to high levels of
Ca2⫹ (⬃10 –100 ␮M) at the cytosolic face; however, the
significance of this pathway in native vascular smooth muscle cells remains to be determined.
TRPM4 channels are critically important for cerebral artery
function (76, 116). Suppression of TRPM4 expression in
cerebral arteries was shown to result in a substantial reduction in smooth muscle membrane depolarization and vasoconstriction accompanied by increased intravascular pressure (83). This pioneering study was reinforced by a subsequent report showing that 9-phenanthrol, a highly specific
pharmacological inhibitor of TRPM4 channels (119), inhibited pressure-induced depolarization and myogenic tone
in isolated pial arteries (117). However, interpretation of
these latter data may be called into question by a recent
study suggesting that 9-phenanthrol can activate intermediate-conductance Ca2⫹-activated K⫹ channels present in
the endothelium to cause vasodilation of mesenteric arteries
(107). Two additional studies have confirmed the involvement of TRPM4 channels in cerebral myogenic tone, in this
case in the cerebral intraparenchymal arteriolar network
(37, 188). The involvement of TRPM4 in pressure-induced
vasoconstriction suggests that this channel is mechanically
activated. Although one study reported that application of
negative pressure to the recording electrode activated single
cation channels in vascular smooth muscle cells with properties much like those in HEK cells overexpressing TRPM4
channels (234), a more recent study failed to detect inherent
mechanosensitivity of TRPM4 (118). Recent work points
to involvement of purinergic or ANG II type 1 receptor
(AT1R) GPCRs in arterial pressure-sensing. Along these
lines, a compelling case has been made that TRPM4 channels are activated downstream of a mechanosensitive losartan-sensitive receptor (likely AT1R) in cerebral artery
smooth muscle cells (118) (FIGURE 6). Mechanoactivation
of P2Y4 and P2Y6 purinergic receptors and downstream
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
activation of TRPM4 channels in cerebral intraparenchymal arteriolar myocytes have also been proposed (188).
Two studies have investigated the effect of TRPM4 activity
on cardiovascular control in vivo. Using a knockdown approach, Reading et al. (282) demonstrated that TRPM4
channels in cerebral artery myocytes directly contribute to
autoregulation of cerebral blood flow in intact animals,
presumably through control of myogenic tone. However,
another study (214) reported no difference in agonist- or
pressure-induced contractile responses of the aorta or in
hindlimb vascular preparations isolated from wild-type and
TRPM4⫺/⫺ mice. It is unclear if these discrepancies are due
to differences in vascular beds or experimental preparations, or possible compensatory expression of other cation
channels in the TRPM4⫺/⫺ animals. Furthermore, the
TRPM4⫺/⫺ mice used for this study were mildly hypertensive, likely due to increased release of catecholamines from
chromaffin cells. Thus it is possible that elevated catecholamine levels in TRPM4⫺/⫺ mice compensate for the loss of
TRPM4-induced smooth muscle contractility. Additional
studies are needed to determine if vascular function in specific areas of the circulation, such as in the brain, is altered
in TRPM4⫺/⫺ animals, preferably using conditional and
cell-specific deletion approaches.
TRPM4 activity also promotes excitability of detrusor
smooth muscle in the urinary bladder (258, 321, 322), suggesting that this channel could have a broad role in the
regulation of smooth muscle contractility.
D) TRPP CHANNELS.
The TRPP subfamily consists of TRPP1
(PKD2), TRPP2 (PKD2L1), and TRPP3 (PKD2L2). TRPP1
is a Ca2⫹-permeable, nonselective cation channel with a
large unitary conductance (135–175 pS) that may be localized to the plasma membrane or intracellular organelles.
PKD1 is sometimes referred to as TRPP1, but it is structurally distinct from the other TRPP proteins and does not
form a cation-permeable ion channel. PKD1 can interact
with TRPP1 and may be a mechanically sensitive regulatory
subunit. PKD1 and TRPP1 are both expressed in vascular
smooth muscle cells (121, 243, 354). Early reports suggested that PKD1 and TRPP1 physically interacted and
might be involved in regulating the development and organization of the cytoskeletal elements of vascular myocytes
(308). A report by Shaif-Naeini et al. (308) demonstrated
that overexpression of TRPP1 in COS-7 cells diminished
the stretch-induced currents present in native cells. Mechanosensitive currents were restored by coexpression of PKD1
and TRPP1. Selective deletion of PKD1 in smooth muscle
cells (Pkd1SMdel/del) decreased stretch-activated currents in
mesenteric artery smooth muscle cells and diminished myogenic constriction of isolated mesenteric arteries. Surprisingly, knockdown of TRPP1 expression in Pkd1SMdel/del
mice restored stretch-activated currents in isolated smooth
muscle cells and myogenic constriction of mesenteric arter-
ies. The authors of this study proposed that, in the absence
of PKD1, TRPP1 inhibits mechanosensitive ion channels
involved in the vascular smooth muscle depolarization associated with myogenic tone and that these responses are
restored by physical interaction of PKD1 with TRPP1.
Phenylephrine-induced aortic and mesenteric resistance artery contractions are enhanced in TRPP1⫹/⫺ mice due to
increased actin and myosin expression (72, 279), suggesting
that TRPP1 may exert inhibitory effects on vascular tone
through inhibition of contractile protein structure and function. An age-dependent vascular phenotype was observed in
PKD1⫺/⫹ mice, with old mice (40 wk), but not young mice,
exhibiting an increase in phenylephrine-mediated aortic
contractility (133). This finding also suggests vascular bedspecific roles for TRPP isoforms, although clarification of
this possibility will require additional detailed studies. It has
also been proposed that the physical interaction of PKD1
with TRPP1, and related membrane association of these
proteins, is an important element and causal factor in some
of the vascular pathologies observed in patients with polycystic kidney disease (280).
Jaggar et al. (243) reported a different take on TRPP1 channels, concluding that TRPP1 cation currents directly activate myogenic tone in the cerebral circulation. This study
showed that TRPP1 and PKD1 are present in cerebral artery
myocytes and localize to the plasma membrane, and further
indicated that TRPP1 mRNA is much more abundant than
PKD1 mRNA. Knockdown of TRPP1 reduced swellingactivated cation currents in isolated vascular smooth muscle cells and suppressed the development of myogenic tone
in isolated cerebral arteries. The reasons for the apparently
divergent functions of TRPP1 channels in cerebral and mesenteric arteries in particular, and the physiological roles of
TRPP proteins in vascular smooth muscle cells in general,
are not clear and will require further investigation.
E) TRPV1.
Expression of TRPV1 channels in aortic and skeletal muscle arteriolar myocytes from rats has been reported
(201, 158, 355). These studies showed that the TRPV1
activator capsaicin increases hindlimb vascular resistance in
vivo and causes substantial endothelium-independent vasoconstriction of skeletal muscle arterioles in vitro. Vasoconstriction of isolated canine mesenteric arteries following
direct activation of TRPV1 channels has also been reported
(273). More recent work has revealed the presence of
TRPV1 channels in murine arteriolar myocytes located in
thermoregulatory tissues and has demonstrated a role for
these channels as mediators of myocyte Ca2⫹ entry and
contraction in intact auricular arterioles (46).
F) TRPV2.
TRPV2 channels are present in murine aortic, mesenteric, and basilar artery smooth muscle cells (238). Hypotonic cell swelling of aortic myocytes was shown to activate nonselective cation channels and Ca2⫹ entry, effects
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659
SCOTT EARLEY AND JOSEPH E. BRAYDEN
that were suppressed by treatment with TRPV2 antisense
oligonucleotides. These findings led the authors of this
study to propose a role for TRPV2 channels in myogenic
vasoconstriction; however, functional studies investigating
the role of TRPV2 channels in intact arteries have not been
reported.
dition, as noted above, several lines of evidence argue
against the involvement of TRPC channels in SOCE in vascular smooth muscle and other cell types. (68, 274). While
the issue remains controversial, there is some evidence supporting the involvement of TRPC channel activity in SOCE
in smooth muscle cells, as discussed below.
G) TRPM8.
A) TRPC1. The contribution of TRPC1 to SOCE activity has
been investigated using siRNA-mediated knockdown in
A7r5 cells (40) and neutralizing antibody approaches (30,
402) in smooth muscle from several tissues, including pulmonary arteries (245–247) and the portal vein (14). SOCE
via TRPC1 channels is also thought to be involved in proliferation of pulmonary artery myocytes in culture systems
(216, 348). However, conflicting evidence is provided by
studies using TRPC1⫺/⫺ mice, which found no evidence for
the involvement of TRPC1 in SOCE in myocytes isolated
from cerebral arteries or the thoracic aorta (69). Work by
Saleh et al. (292) suggests that TRPC1 currents activated by
the SERCA pump inhibitor cyclopiazonic acid (interpreted
as SOCE) require PIP2 for activation.
TRPM8 channels are thought to be involved primarily in sensory nervous system signaling activated by cold
and menthol. A single study showed that TRPM8 mRNA
and protein are present in rat aortic, mesenteric, tail, and
femoral artery myocytes (151). In most reported experiments, menthol caused a dilatory response in the aorta, as
well as in mesenteric and tail arteries, that was at least
partly endothelium independent. However, in some cases
menthol caused small contractions of endothelium-dependent aortic rings. The exact role of TRPM8 channels in the
vasculature remains undefined.
H) TRPC1. TRPC1 channels are frequently linked to SOCE
(30, 145), although this function has been disputed (68, 69,
274), and a few studies have investigated ROCE through
TRPC1 channels in vascular smooth muscle cells. For example, ET-1 was shown to induce VDCC- and SOCE-independent Ca2⫹ entry in cerebral arteries (124) and in cultured aortic smooth muscle cells (344) that may be mediated by TRPC1 channels (344). A series of studies by Large,
Albert, and collegues investigated receptor-operated regulation of TRPC1 and heteromultimeric channels containing
TRPC1 subunits in vascular smooth muscle cells (291, 309,
310, 312). These studies suggested that TRPC1 channels
are activated downstream of PLC- and PKC-dependent signaling pathways. A model proposed in another report suggests an association between TRPC1, IP3Rs, and STIM1
that could account for Ca2⫹ influx and contraction of vascular smooth muscle independent of SOCE (34), although
supporting experimental evidence is meager. The biophysical properties of TRPC1 channels suggest that the fractional
Ca2⫹ component of TRPC1 currents under physiological
conditions is very small. This argues against involvement of
direct Ca2⫹ influx, but is it possible that TRPC1-mediated
cation influx could cause membrane depolarization and
Ca2⫹ influx through VDCCs in excitable cells. However,
vasoconstriction in response to elevated intravascular pressure (69), adrenoceptor activation, or K⫹-induced depolarization (304) is unaltered in TRPC1⫺/⫺ mice.
2. SOCE in vascular smooth muscle cells
A few studies have attempted to link SOCE through TRPC1
with smooth muscle cell contraction. In the pulmonary vasculature, SOCE has been shown to cause vasoconstriction
(174), and some evidence suggests that hypoxia-induced
pulmonary vasoconstriction could be mediated by SOCE
through TRPC1 channels (187, 205). One report suggested
that SOCE via TRPC1 leads to the activation of large-conductance, Ca2⫹-sensitive K⫹ (BK) channels and relaxation
of arterial smooth muscle (176). In this study, smooth muscle cell hyperpolarization and decreased global Ca2⫹ in response to activation of SOCE were greatly inhibited by a
TRPC1-specific antibody or by a selective inhibitor of BK
channels. Coimmunoprecipitation and immunohistochemical analyses indicated that TRPC1 and BK channels are
colocalized in arterial myocytes. The authors of this study
proposed that TRPC1 and BK channels form a vasodilatorsignaling complex in vascular smooth muscle cells, a conclusion supported by the subsequently reported interaction
between TRPC1 and BK channels in aortic smooth muscle
cells (166). Although it remains difficult to reconcile how
the small fractional Ca2⫹ currents generated by TRPC1
channels (⬃2 pS; Ref. 177) could produce localized domains with Ca2⫹ levels high enough to activate BK channels, such physical interactions may provide an enabling
mechanism.
Many studies have investigated the contribution of TRPCmediated SOCE in vascular smooth muscle cells to cellular
differentiation, proliferation, and excitation-contraction
coupling (183). In considering these reports, it is important
to bear in mind that SOCE is not active in all types of native,
contractile smooth muscle cells, and its presence may be an
indicator of phenotypic differentiation to a proliferative
state or adaptation to cell culture conditions (274). In ad-
B) TRPC4. Two studies in cultured cells, one using aortic and
one using mesenteric artery cells, suggested the involvement
of TRPC4 channels in SOCE in smooth muscle cells. Cyclic
stretch of these cells in culture reportedly caused a decrease
in TRPC4 expression that was correlated with a decrease in
SOCE (197). A second report from this group demonstrated
that downregulation of TRPC4 was associated with reduced SOCE in mesenteric artery smooth muscle cells from
660
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
normotensive WKY rats, but not spontaneously hypertensive rats (SHR) (196).
C) TRPC5.
Some evidence supporting a role for TRPC5 in
SOCE in vascular smooth muscle cells, likely in heteromultimeric combinations with TRPC1, TRPC6, and/or
TRPC7, has been presented. For example, an anti-TRPC5
antibody has been demonstrated to have a significant inhibitory effect on store-depletion-induced cation currents in
freshly isolated cerebral pial arterioles (403). Similarly,
Saleh et al. (294) showed that currents evoked by administration of the SERCA pump inhibitor cyclopiazonic acid
were attenuated by anti-TRPC5, anti-TRPC1, and antiTRPC7 antibodies in smooth muscle cells from coronary
and mesenteric arteries as well as the portal vein (294).
3. TRPV4 channels in vascular smooth muscle cells
and vasodilation
The presence of TRPV4 in cerebral artery smooth muscle
cells has been demonstrated by immunohistochemistry
(211) and RT-PCR (79). Earley et al. (79) reported the
surprising finding that Ca2⫹ influx through TRPV4 channels in cerebral arterial myocytes caused vasodilation rather
than vasoconstriction. This study showed that Ca2⫹ influx
through TRPV4 channels stimulated by 11,12-EET activated Ca2⫹-induced Ca2⫹ release (CICR) from SR stores in
the form of Ca2⫹ sparks. Ca2⫹ sparks evoked by 11,12-EET
activated BK channels, leading to myocyte hyperpolarization and decreased Ca2⫹ entry though VDCCs, causing vasodilation. Thus, although previous reports clearly demonstrated a key role for BK channels in the mechanism of
vasodilation induced by EETs (42, 143), an indirect activation pathway involving TRPV4 channels appears to be a
major mechanism of EET-induced activation of BK channels. A similar role for smooth muscle TRPV4 channels in
regulating mesenteric artery contractile activity in vitro and
in vivo has been described (81). In this study, ⬃50% of
11,12 EET-induced vasodilation was due to activation of
TRPV4 channels in the endothelium and 50% was due to
activation of the channel in vascular smooth muscle cells. A
subsequent study by Macado et al. (221) demonstrated that
ANG II acts through AT1R to increase TRPV4 activity,
recorded in the form of TRPV4 sparklets, in isolated cerebral artery smooth muscle cells. In intact cerebral arteries,
Ca2⫹ influx through TRPV4 channels opposes the vasoconstrictor effects of ANG II through the Ca2⫹ spark-dependent mechanism described above. Using selective pharmacology and an elegant optogentic approach, these authors demonstrated that the effects of AT1R activation
on TRPV4 sparklet activity are dependent on PKC. This
study further demonstrated that the dynamic interactions
between TRPV4 channels and PKC necessary for AT1Rdependent regulation of TRPV4 sparklets are coordinated
by AKAP150 (a kinase anchoring protein 150). Collectively,
these findings reveal the formation of an AKAP150-organized
subcellular signaling complex involving AT1Rs, PKC, and
TRPV4 channels that is capable of finely tuning local [Ca2⫹]
and CICR to influence vascular contractility (FIGURE 7).
B. TRP Channels in the Vascular
Endothelium
The vascular endothelium is composed of a single layer of
metabolically dynamic endothelial cells that line all of the
blood vessels in the body. This tissue is located between the
vascular lumen and underlying smooth muscle cells, allowing the endothelium to sense blood-borne substances and
shear stress resulting from fluid flow along the vessel wall.
The principal functions of the endothelium are to promote
smooth muscle cell relaxation and arterial dilation, control
vascular permeability, exert an antithrombotic effect, and
regulate angiogenesis. Endothelial dysfunction is a hallmark of common cardiovascular diseases such as hyperten-
AngII
11,12-EET
K+
Ca2+
AT1R
Gq
TRPV4
PLC
DAG
BK
PKC
+
AKAP
150
+
RyR
Ca2+
Smooth muscle cell
SR
FIGURE 7. Ca2⫹ influx through TRPV4
channels relaxes cerebral artery smooth
muscle cells. Stimulation of Ca2⫹ influx
through TRPV4 channels directly with 11,12EET or through activation of Gq-coupled
AT1Rs with ANG II, leading to generation of
DAG by PLC and AKAP150-dependent PKC
activity, causes an increase in the frequency
of Ca2⫹ sparks mediated by ryanodine receptors (RyRs) on the SR through a CICR mechanism. Increased generation of Ca2⫹ sparks
causes smooth muscle cell membrane hyperpolarization and relaxation by increasing K⫹
efflux through BK channels. [Modified from
Earley et al. (79), with permission from Wolters Kluwer Health, and Mercado et al.
(221).]
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
Table 3. TRP channels in the vascular endothelium
TRP
Function
TRPC1
TRPC3
TRPC4
Angiogenesis, permeability
Angiogenesis, endothelium-dependent dilation
Angiogenesis, endothelium-dependent dilation,
permeability
Angiogenesis
Angiogenesis, permeability
Endothelium-dependent dilation
Endothelium-dependent dilation
Angiogenesis, endothelium-dependent dilation,
permeability
Endothelium-dependent dilation
TRPC5
TRPC6
TRPV1
TRPV3
TRPV4
TRPA1
Tissue
Embryonic tissues (411), lung (39, 218)
Mesentery (198, 306), mammary artery (102)
Umbilical vein (19), aorta (97), lung (59, 352)
Umbilical vein (110)
Umbilical vein (110), epidermis (128)
Mesentery (57, 406), heart (36, 123)
Brain (78)
Aorta (381), mesentery (81, 295, 324, 417), skeletal muscle (169),
heart (220, 371), lung (15), brain (132, 211)
Brain (77, 281), Sullivan et al., unpublished data
Reference numbers are given in parentheses.
sion, atherosclerosis, and stroke, demonstrating the importance of this tissue for vascular health. Several TRP channels, acting though regulation of the global intracellular
[Ca2⫹] in endothelial cells and the generation of subcellular
Ca2⫹ signals, are important for endothelial cell function
(336). Approximately 20 different TRP channel family
members are reported to be expressed in cultured and native
endothelial cells (336), and the functional significance of
nine of these channels–TRPC1 (47, 103), TRPC3 (47, 103),
TRPC4 (97, 103), TRPC5 (103), TRPC6 (103), TRPV1,
TRPV3 (78), TRPV4 (392), and TRPA1 (77)– has been investigated (TABLE 3).
The architecture of the sites of interaction between smooth
muscle and endothelial cells within the vascular wall facilitates EDH. Smooth muscle cells and endothelial cells are
separated by a structure composed primarily of elastin and
collagen called the internal elastic lamina (IEL). Projections
of the endothelial cell plasma membrane protrude into fenestrations in the IEL, providing an interface between endothelial cell and smooth muscle cell plasma membranes.
Some, but not all, of these sites, collectively termed myoendothelial projections (MEPs), contain gap junctions between smooth muscle and endothelial cells. Those sites that
1. TRP channels and endothelium-dependent
vasodilation
The endothelium exerts a profound relaxing effect on underlying smooth muscle cells that, balanced by tonic constrictor influences, maintains optimal vascular tone (FIGURE 8).
Nitric oxide (NO) (100) and prostacyclin (PGI2) are wellcharacterized vasoactive substances produced by the endothelium that diffuse to and relax smooth muscle to cause
arterial dilation. Endothelium-dependent vasodilation persists when NO and PGI2 production are blocked, a response
initially attributed to an unidentified “endothelium-derived
hyperpolarizing factor” (EDHF) (48). The molecular basis
of EDHF is now thought to encompass multiple diffusible
substances, including K⫹ (86), EETs (42, 94), C-type natriuretic peptide, carbon monoxide, hydrogen peroxide
(H2O2), hydrogen sulfide (H2S), and others (87). In addition, vasodilatory responses originally attributed to EDHF
are now known to result from direct electrotonic spread of
membrane hyperpolarization from endothelial cells to
smooth muscle cells through myoendothelial gap junctions
(FIGURE 8). This factorless mechanism is referred to as endothelium-dependent hyperpolarization (EDH) to avoid
confusion with EDHF-type pathways involving diffusible
substances (106).
662
Ca2+
COX
EC
eNOS
NO
ΔEm
K+ EDHF PGI2
IEL
MEGJ
Smooth muscle cell
EDH
FIGURE 8. Mechanisms of endothelium-dependent vasodilation. In
endothelial cells (EC), Ca2⫹ influx stimulates the activity of eNOS to
generate NO, which diffuses through the IEL to cause relaxation of
underlying vascular smooth muscle cells (SMC). COX activity generates PGI2, which also relaxes SMC. A variety of other substances,
collectively known as EDHF, can cause smooth muscle cell hyperpolarization and relaxation. K⫹ efflux hyperpolarizes the endothelial cell
plasma membrane (⌬Em). Electrotonic spread of this influence
through myoendothelial gap junctions (MEGJs) promotes EDH, causing SMC relaxation.
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
contain gap junctions have been referred to as myoendothelial junctions (MEJs), whereas those that lack gap junctions
have been termed myoendothelial close contacts (MECCs).
Connexin proteins (MEJs only), intermediate-conductance
Ca2⫹-activated K⫹ (IK) channels (77, 298), IP3Rs (180),
TRPA1 (77), TRPV4 (325), and other signaling proteins are
present within MEPs. In mesenteric arteries, transient, localized Ca2⫹ signals are generated within MEPs by release
of Ca2⫹ from the ER through IP3Rs. These signals, which
occur both spontaneously and in response to activation of
GPCRs, are called Ca2⫹ pulsars and stimulate EDH by
activating SK (small-conductance) and IK Ca2⫹-activated
K⫹ channels (180) (FIGURE 9).
A) TRPV1.
TRPV1 channels are activated by capsaicin, a substance found in hot peppers, and by noxious heat (45). The
single-channel conductance of TRPV1 is 35 pS at ⫺60 mV
and 77 pS at ⫹60 mV. TRPV1 channels are selective for
Ca2⫹ versus Na⫹ (PCa:PNa ⬃9.6). Several studies have provided evidence of TRPV1 involvement in endothelium-dependent vasodilation. Poblete et al. (271) demonstrated
that activation of TRPV1 channels with anandamide increased the release of endothelium-derived NO but did not
cause dilation of rat mesenteric arteries. Work from Yang et
al. (406) demonstrated that capsaicin stimulated Ca2⫹ influx and endothelial nitric oxide synthase (eNOS) phosphorylation, and elevated NO production. Capsaicin also
caused endothelium-dependent relaxation of isolated mesenteric arteries, an effect that was absent in arteries from
TRPV1⫺/⫺ mice. In addition, this study showed that endothelial function was improved and systolic blood pressure
was slightly lower in SHRs receiving dietary administration
of capsaicin for 7 mo compared with animals fed a normal
diet. A subsequent study by Ching et al. (57) reported that
capsaicin-induced Ca2⫹ influx through TRPV1 channels
The Ca2⫹ influx pathways that stimulate endothelium-dependent vasodilation remained largely unknown until the
discovery of TRP channels (248). To date, TRPV1, TRPV3,
TRPV4, TRPA1, TRPC3, and TRPC4 channels have been
reported to be involved in endothelium-dependent vasodilation (TABLE 3) (77, 78, 307, 416, 417).
A
- 1.5
- 1.0
- 0.5
-0
- 2.0
- 1.5
- 1.0
- 0.5
-0
B
Endothelium
GPCR
PLC
r
ulsa
Ca P
2+
IP3
IP3R
2+
ulsar
Ca P
IEL
Smooth
muscle
Endothelial cell
IP3R
Endothelium
IP3R
ER
Hyperpolarization
K+
+
K
K+
K+
KCa3.1
KCa3.1
K+
K
Ca2+
eNOS
PLA3
IEL
VDCC
+
Gap
Junction
Channels
K+
Gap junctions
VSM cell
Ca2+
Kir2.1
FIGURE 9. Ca2⫹ pulsars in the endothelium. A:
time course of a three-dimensional Ca2⫹ pulsar originating from within a hole in the IEL (white circle)
shown in the leftmost image. Scale bar, 5 ␮m. B:
Ca2⫹ pulsars recorded from a pressurized artery
(80 mmHg) expressing the Ca2⫹-indicator protein
GCaMP2. An endothelial cell and its nucleus are outlined (dotted lines), with the initiation sites indicated
by red arrows. Scale bar, 10 ␮m. C: model of the
functional effects of endothelial Ca2⫹ pulsars. IP3Rdense ER stores follow portions of the endothelial cell
membrane that evaginate through holes in the IEL
and interface with underlying SM cell membranes.
Repetitive localized Ca2⫹ events (pulsars) originate
from these deep Ca2⫹ stores, which are regionally
delimited to the myoendothelial junction and the base
of the endothelial cell. These ongoing dynamic Ca2⫹
signals are driven by constitutive IP3 production and
are inherently dependent on the level of endothelial
stimulation. The left detail depicts a single endothelial
projection through the IEL. IK (KCa3.1) channels in
the plasma membranes of these endothelial projections are in very close proximity to Ca2⫹ pulsars,
eliciting persistent Ca2⫹-dependent hyperpolarization
of the membrane potential at the myoendothelial
junctions. The right detail illustrates the endothelial
influence on the smooth muscle membrane potential
at the myoendothelial interaction site where Ca2⫹
pulsars activate KCa3.1 channels and hyperpolarize
the endothelial membrane. This hyperpolarization
can be transmitted to the SM through gap junction
channels or by activation of SM Kir channels by K⫹
ions released by endothelial KCa3.1 channels. Membrane hyperpolarization promotes relaxation of SM
through a decrease in VDCC open probability. [From
Ledoux et al. (180).]
RyR
Smooth muscle
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
caused TRPV1 to physically interact with eNOS through a
pathway requiring phosphatidylinositol 4,5-bisphosphate
3-kinase, Akt, and calmodulin-dependent protein kinase II
(CamKII), resulting in increased NO production (57). Bratz
et al. (36) demonstrated that capsaicin dilated coronary
arteries from pigs and showed that this response was diminished by the TRPV1 antagonist capsazepine and inhibition
of NO production and was blunted in coronary arteries
from obese swine. Further work from this group showed
that capsaicin increased myocardial blood flow in mice in
vivo, a response that was absent in TRPV1⫺/⫺ animals and
blunted in the db/db model of type II diabetes (123). Together, these studies support a model in which Ca2⫹ influx
through TRPV1 channels increases the production of endothelium-derived NO to cause vasodilation. This mechanism
may be disrupted during metabolic syndrome and diabetes,
contributing to the endothelial dysfunction associated with
these conditions.
A study by Chen et al. (50) showed that capsaicin caused an
endothelium-dependent dilation of mesenteric arteries that
was absent in TRPV1⫺/⫺ mice. In contrast, capsaicin had
no TRPV1-dependent effects on the main renal arteries,
suggesting a heterogeneous distribution of TRPV1 channels
in the endothelium throughout the vasculature.
B) TRPV3. TRPV3 channels have a large unitary conductance
(⬃150 –200 pS) (58) and are highly permeable to Ca2⫹
(PCa:PNa ⬃12:1) (401). TRPV3 is activated by innocuous
heat (401) and dietary monoterpenes such as carvacrol (derived from oregano), eugenol (clove oil), and thymol (found
in thyme) (400). The channel is present in the skin and oral
and nasal epithelium and is involved in chemosensation
(400). Earley et al. (78) found that TRPV3 channels are also
present in the endothelium of rat cerebral arteries and
showed that carvacrol activated ruthenium red-sensitive
cation currents and increased intracellular Ca2⫹ levels in
native cerebral artery endothelial cells. Carvacrol administration evoked an endothelium-dependent dilation of isolated cerebral arteries that was not altered by NOS or cyclooxygenase (COX) inhibition; however, it was sensitive
to block of SK, IK, and inwardly rectifying K⫹ (KIR) channels, and was associated with smooth muscle cell hyperpolarization. These data suggest that Ca2⫹ influx through
TRPV3 activates EDH (78). The cardiovascular effects of
TRPV3 knockout have not been reported, and the functional significance and endogenous regulation of TRPV3 in
the endothelium remain unknown.
TRPV4 channels are more permeable to Ca2⫹ than
Na (PCa:PNa ⬃6:1) (332) and are activated by cell swelling
(193), warm temperatures, and chemical agonists (351,
381). The unitary conductance of the TRPV4 channel is
⬃30 – 60 pS at ⫺60 mV and ⬃88 –100 pS at ⫹60 mV. The
involvement of TRPV4 channels in endothelium-dependent
vasodilation has been extensively studied, in part because of
C) TRPV4.
⫹
664
the availability of selective pharmacological activators, including 4␣-phorbol 12,13-didecanoate (4␣-PDD) (381)
and GSK1016790A (351), and blockers such as RN-1732
(371) and HC-067047 (89).
I) TRPV4 and EET-induced dilation. Wantanabe and coworkers discovered that TRPV4 channels are present in
freshly isolated mouse aortic endothelial cells and could be
activated by the phorbol compound 4␣-PDD (381), heat
(25– 43°C) (383), and EETs (382). The EETs are a family of
four regioisomers, 5,6-EET, 8,9-EET, 11,12-EET, and
14,15-EET, generated from arachidonic acid by cytochrome P-450 (CYP) epoxygenase enzymes. These compounds have significant biological activity in many tissues,
and when produced by endothelial cells may act as EDHFs
in some vascular beds (42, 80, 88, 94). Earley et al. (81)
demonstrated that exogenous administration of 11,12-EET
dilated isolated mesenteric arteries from wild-type, but not
TRPV4⫺/⫺, mice. Approximately 50% of this response was
lost when endothelial function was disrupted, suggesting
that EETs can act on TRPV4 channels in both endothelial
cells and smooth muscle cells (81). This study also showed
that 11,12-EET-induced vasodilation was associated with
smooth muscle cell hyperpolarization and was not affected
by NOS or COX inhibition, but was blocked by inhibition
of BK, IK, or SK channels, supporting an EDHF or EDH
mechanism. A subsequent study by Vriens et al. (372) established that EETs generated by the CYP2C9 epoxygenase
isoform could activate TRPV4 in an autocrine manner in
cultured endothelial cells, providing support for the hypothesis that endogenously produced EETs activate Ca2⫹
influx through TRPV4 channels in an autocrine/paracrine
manner to elicit dilation.
II) TRPV4 and flow-induced dilation. Increases in blood
flow velocity or blood viscosity augment the level of laminar
shear stress at the vascular wall. The endothelium senses
this stimulus, initiating a vasodilator response known as
“flow-induced dilation” (66). The molecular nature of the
flow-sensing mechanism remains incompletely understood.
An early study showing that TRPV4 was activated by hypotonic conditions, possibly by stretch of the plasma membrane associated with cell swelling (193), stimulated interest in this channel as a flow sensor in the endothelium.
However, a subsequent study applying a cell-attached
patch-clamp approach reported that TRPV4 channels were
not directly activated by suction applied to the plasma
membrane (332), suggesting that cell swelling activates the
channel indirectly through a force-sensitive signaling cascade.
A number of studies support the possibility that TRPV4
channels are involved in flow-induced dilation. Kohler et al.
(169) reported that administration of the TRPV4 agonist
4␣-PDD or application of shear stress dilated rat gracilis
arteries, and showed that these responses were blunted by
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
the nonselective TRPV blocker ruthenium red. NOS inhibition attenuated flow-induced dilation but did not affect 4␣PDD-induced dilation, which was diminished by block of
SK and IK channels. These results suggest that shear stressinduced Ca2⫹ influx through TRPV4 channels causes dilation through increased NO production, but pharmacological activation of the channel initiates EDHF or EDH pathways (169). A further study by this group demonstrated
that carotid arteries from TRPV4⫺/⫺ mice lack shear stressinduced dilation (132). Zhang and colleagues (220) reported that TRPV4 is critically important for flow-mediated dilation of mouse mesenteric arteries and showed that
shear stress induces Ca2⫹ influx through TRPV4 channels
in human coronary artery endothelial cells. Bubloz and coworkers showed that flow-induced dilation of human coronary arteries was blunted by ruthenium red, the selective
TRPV4 blocker RN-1732 (371), and siRNA-mediated
downregulation of TRPV4 expression (41), providing
strong evidence for a TRPV4 influence in shear stress-induced responses in this vascular bed.
It remains unclear if TRPV4 channels are directly activated
by shear stress or respond to signaling pathways activated
by a flow-sensing apparatus. In support of the latter possibility, a few studies have provided evidence that TRPV4
channels are activated by metabolites produced in response
to laminar shear stress. Loot et al. (204) showed that inhibition of CYP epoxygenase attenuated flow-induced dilation of mouse carotid arteries, suggesting that EETs produced in response to shear stress are responsible for
TRPV4-mediated dilation. These findings are supported by
reports showing that cell-swelling-induced activation of
TRPV4 is blocked by selective inhibition of CYP2C9 (372)
and flow-induced dilation of mouse carotid arteries is impaired by inhibition of PLA2 (132), an enzyme that liberates
arachidonic acid from the plasma membrane. Together,
these studies support a mechanism in which TRPV4 is indirectly activated by shear through conversion of PLA2liberated arachidonic acid to EETs by CYP2C9. In this
scheme, the molecular nature of the endothelial cell flow
sensor responsible for activation of TRPV4 in response to
increased shear remains unresolved. The AT1R, which is
present in the endothelium and can be activated by a mechanical stimulus independently of ANG II, is a potential
candidate for this function. AT1Rs are functionally coupled
to TRPV4 channels in vascular smooth muscle cells (221),
but this linkage has not yet been demonstrated in endothelial cells.
III) TRPV4 and agonist-induced dilation. Several studies
have demonstrated involvement of TRPV4 channel activity
in endothelium-dependent relaxation of systemic arteries in
response to muscarinic and purinergic receptor agonists.
Saliez et al. (295) reported that EDHF/EDH and NO-dependent relaxation in response to carbachol are diminished
in mesenteric arteries from TRPV4⫺/⫺ mice compared with
arteries from wild-type animals, indicating that TRPV4
channels are involved in the response. Using pressure myography and in vivo approaches, Gutterman and colleagues
(417) confirmed that acetylcholine (ACh)-induced dilation
was blunted in mesenteric arteries from TRPV4⫺/⫺ animals.
This study also demonstrated that ACh-induced Ca2⫹ influx and NO production in endothelial cells isolated from
TRPV4⫺/⫺ mice were decreased compared with that in
wild-type controls (417). Senadheera et al. (306) showed
that block of TRPV4 with RN-1734 blunted ACh-induced
dilation of radial uterine arteries, producing a greater effect
in arteries from pregnant rats. Marrelli et al. (211) found
that UTP-induced endothelial cell Ca2⫹ influx and dilation
of cerebral arteries was blunted by ruthenium red and block
of PLA2, suggesting that PLA2 may serve to generate arachidonic acid, which activates TRPV4-mediated Ca2⫹ influx and leads to arterial dilation through an EDHF or EDH
mechanism. Chen et al. (50) showed that GSK1016790A
induced a dilation of isolated renal arteries and perfused
kidneys that was largely endothelium dependent and sensitive to NOS inhibitors.
TRPV4 channels are also present in the pulmonary artery
endothelium. The selective TRPV4 agonist GSK1016790A
relaxes pulmonary artery rings in an endothelium-dependent manner, an effect that is blocked by the selective
TRPV4 antagonist HC-067047 and is largely dependent on
SK and IK channel activity. These data indicate that pharmacological activation of TRPV4 causes EDH in pulmonary arteries. However, ACh-induced relaxation of pulmonary artery rings was not affected by block of TRPV4 channels with HC-067047 or RN1734 in this study, indicating
that TRPV4 channels are not involved in muscarinic agonist-induced responses in isolated pulmonary arteries. Similar results have been obtained in vivo (257), indicating that
TRPV4 channels are probably not involved in dilation of
pulmonary arteries in response to muscarinic agonists.
IV) TRPV4 channels and subcellular Ca2⫹ signaling in the
endothelium. An elegant study by Nelson and colleagues
(324) reported that TRPV4 sparklets could be recorded
from the intact endothelium of mesenteric arteries isolated
from mice expressing a genetically encoded Ca2⫹ indicator
protein exclusively in the endothelium. The amplitudes of
TRPV4 sparklets stimulated by 4␣-PDD, 11,12-EET, or
GSK1016790A displayed a quantal distribution and exhibited cooperative gating, with simultaneous activation of
two, three, or four channels occurring more frequently than
predicted by binomial distribution models (324). Events
with amplitudes greater than four quantal levels were not
observed, suggesting that TRPV4 is present in a four-channel metastructure in endothelial cells. This coupled gating
arrangement amplifies the initial Ca2⫹ signal created by
TRPV4 channel activation and, combined with the biophysical properties of the channel, which favor Ca2⫹ influx,
produces large-amplitude Ca2⫹ signals in the endothelium.
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
Although the amplitude of individual TRPV4 sparklets is
quite large, the total number of active sites per cell is surprisingly low. Both Sonkusare et al. (324) and Sullivan et al.
(337) showed that only a few (⬃3– 8 sites) TRPV4 sparklets
are active in individual endothelial cells during maximal,
agonist-induced dilation. TRPV4 sparklets stimulated IK
and SK currents in native endothelial cells, indicating that
TRPV4 sparklets activate an EDH-dependent vasodilator
pathway (324). Muscarinic receptor activation was shown
to increase TRPV4 sparklet activity, supporting the hypothesis that large-amplitude Ca2⫹ signals generated by coupled
TRPV4 channels activate proximal SK and IK channels to
cause vasodilation (FIGURE 10) (324).
A report from the Dora lab (22) suggested that Ca2⫹ influx
through TRPV4 channels could trigger CICR in the endothelium. This study showed that TRPV4 channels cluster in
MEPs in mesenteric arteries and are spatially coupled with
Ca2⫹ pulsars sites. Ca2⫹ pulsar activity was greater at very
low intraluminal pressure (5 mmHg) compared with physiological levels (80 mmHg). The effects of low pressure on
Ca2⫹ pulsar activity were attenuated by TRPV4 inhibition.
Block of TRPV4 channels also increased myogenic tone at
TRPV4
EC
IEL
SMC
Ca2+
K+
TRPV4
Ca2+
Vm
Kca
Gap
Junction
K+
Myoendothelial Junction
FIGURE 10. TRPV4 Ca2⫹ sparklets in endothelial cells initiate
vasodilation. TRPV4 Ca2⫹ influx from the extracellular space through
as few as 3– 4 TRPV4 channels activates Ca2⫹-activated K⫹ (KCa)
channels, resulting in K⫹ efflux from the cell. This hyperpolarizes the
endothelial cell (EC) membrane, which directly hyperpolarizes
smooth muscle cells (SMCs) via gap junctions located within myoendothelial junctions. IEL, internal elastic lamina; Vm, membrane potential. [From Sullivan and Earley (336).]
666
low levels of intraluminal pressure (20 and 40 mmHg),
suggesting that the channel mediates a Ca2⫹-pulsar-dependent vasodilator influence at these pressures. The authors
proposed that Ca2⫹ influx through TRPV4 channels stimulated by low intraluminal pressure is amplified by CICR
from IP3Rs to generate large-amplitude Ca2⫹ pulsars that
promote EDH and thereby suppress myogenic constriction.
The significance of these data is unclear, but it is conceivable that activation of endothelial cell TRPV4 channels at
low intraluminal pressure and subsequent EDH and/or increases in vascular permeability contribute to the precipitous drop in blood pressure observed during hypovolemic
shock. In contrast to the findings of Bagher et al. (22),
Senadheera et al. (306) did not detect enriched TRPV4 expression within IEL fenestrations of uterine arteries from
control or pregnant rats, suggesting that the site of TRPV4
expression in endothelial cells may differ between vascular
beds.
V) TRPV4 function in vivo. Many of the studies cited above
used ex vivo preparations to demonstrate that TRPV4
channels present in the endothelium contribute to vascular
regulation in response to shear stress, receptor-dependent
agonists, and autocrine/paracrine factors. These data are at
odds with a number of in vivo studies showing little involvement of TRPV4 channels in cardiovascular control. Two
reports showed that systemic activation of TRPV4 with
GSK1016790A caused a significant drop in mean arterial
pressure (MAP) that was independent of changes in cardiac
output (257, 389), indicating that activation of the channel
lowered total peripheral resistance in whole animals. These
findings suggest that loss of TRPV4 activity in vivo should
increase total peripheral resistance and MAP. However,
basal MAP did not differ between wild-type and TRPV4⫺/⫺
mice (342) or was slightly lower in TRPV4⫺/⫺ mice (254).
Furthermore, acute or chronic (8 days) administration of
the selective TRPV4 blocking compounds GSK2193874 and
GSK2263095 had no effect on MAP or heart rate, indicating
that inhibition of the channel has no significant effect on basal
cardiovascular regulation (350). GSK2193974 also had no
effect on decreases in MAP following intravenous injection
of ACh, suggesting that TRPV4 channels are not involved in
systemic muscarinic receptor-induced vasodilation in vivo
(257). The only reported cardiovascular phenotype of
TRPV4⫺/⫺ mice is a mildly enhanced sensitivity to hypertensive stimuli (81, 254). Together, these in vivo studies
suggest that TRPV4 channels in the endothelium are essentially inactive in healthy animals under basal conditions and
exert only a modest effect on vascular resistance and MAP.
The reason for the inconsistency between in vivo and ex
vivo data is not clear, but it is possible that the influence of
TRPV4 channels is confined to specific vascular beds, with
other segments rapidly compensating for the loss of channel
activity during pharmacological inhibition in whole animal
studies.
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
D) TRPA1.
TRPA1 channels have a large unitary conductance
(⬃98 pS) (241), are highly permeability to Ca2⫹ (PCa:PNa
⬃7.9) (157), and are activated by electrophilic compounds,
including the dietary molecules allicin (208), found in garlic, and allyl isothiocyanate (AITC) (24), derived from mustard oil. The channel acts as a receptor for these pungent
substances in sensory neurons and may also be involved in
responses to noxious cold (329). Recent studies have demonstrated that TRPA1 contributes to vascular regulation
(for review, see Ref. 75). Bautista et al. (27) reported that
allicin dilated mesenteric arteries by activating TRPA1
channels in perivascular nerves to cause the release of calcitonin gene-related peptide (CGRP). Earley and co-workers subsequently found that TRPA1 channels are present in
the cerebral artery endothelium of rodents and humans
(77), but were not detectable in mesenteric, coronary, dermal, or renal arteries (338). This study further reported that
AITC caused a robust endothelium-dependent dilation and
smooth muscle cell membrane hyperpolarization in pressurized cerebral arteries that was blocked by the selective
TRPA1 inhibitor HC-030031. These AITC-induced responses were insensitive to NOS and COX blockade but
were diminished by inhibition of SK, IK, or KIR channels
(77). This study also showed that TRPA1 channels are
abundant in MEPs in the cerebral endothelium and are
colocalized with IK channels in this tissue (77). Further
insight into TRPA1-mediated cerebral artery dilation was
provided by Qian et al. (281), who demonstrated that activation of TRPA1 channels provoked dilation by recruiting
large, dynamic Ca2⫹ signals in the cerebral artery endothelium. These signals, generated by the release of Ca2⫹ from
IP3Rs present on the endothelial cell ER, are distinguished
from sparklets and Ca2⫹ pulsars by amplitude, spatial
spread, and duration and appear to mediate AITC-induced
vasodilation.
It is unlikely that TRPA1 channel activity in the cerebral
endothelium is regulated by AITC or allicin under physiological conditions. TRPA1 channels are activated by reactive oxygen species (ROS), such as H2O2, and ROS-derived
metabolites, including 4-hydroxy-2-nonenal (4-HNE),
4-oxo-nonenal (4-ONE), and 4-hydroxylhexenal (4-HHE)
that are generated by peroxidation of ␻6 polyunsaturated
fatty acids (17, 363). Sullivan et al. (338) showed that
TRPA1 channels and the ROS-generating enzyme NOX2
(NADPH oxidase isoform 2) colocalize in the endothelium
of cerebral arteries. This study also showed that the amplitude of TRPA1 sparklets recorded from cerebral artery endothelial cells was approximately twice that of TRPV4
sparklets, consistent with the channel’s larger unitary conductance (⬃98 vs. 60 pS) and higher Ca2⫹ selectivity
(⬃7.9:1 vs. 6:1). Stimulation of NOX2 activity increased
the frequency of TRPA1 sparklets and dilated isolated cerebral arteries. These responses were dependent on the generation of extracellular H2O2 and hydroxyl radicals replicated by administration of exogenous 4-HNE. Together,
these data suggest that superoxide anions (O2⫺) generated
by NOX2 activity proximal to TRPA1 channels increase
TRPA1 sparklet activity to cause vasodilation. This mechanism appears to be present only in the cerebral circulation
and could provide compensatory increases in blood flow
during cerebrovascular diseases associated with oxidative
stress.
E) TRPC3.
TRPC3 channels have a unitary conductance of
⬃60 pS, are essentially equally permeable to Ca2⫹ and Na⫹,
and are more permeable to Na⫹ than K⫹ (156). The channel
is activated by GPCR pathways, is directly sensitive to DAG
(and stable analogs), and may be stimulated by elevated
intracellular [Ca2⫹]. Reports from several laboratories suggest involvement of TRPC3 in endothelium-dependent vasodilation. An early study of isolated rat mesenteric arteries
showed that injection of animals with TRPC3 antisense
oligonucleotides diminished flow- and bradykinin-induced
vasodilation (198). A later report by Gao et al. (102) provided evidence for expression of TRPC3 in the endothelial
and smooth muscle layers of the human internal mammary
artery (IMA), and showed that the TRPC3 blocker Pyr3
modestly diminished muscarinic agonist-induced relaxation of precontracted IMA rings. Kochukov et al. (166)
used knockout animals to demonstrate that TRPC1 and
TRPC3 subunits form heteromultimeric channels involved
in muscarinic agonist-induced Ca2⫹ influx and relaxation
of aortic ring segments. A subsequent study by Sandow et
al. (307) reported participation of TRPC3 channels in endothelium-dependent dilation of rat and mouse mesenteric
arteries. This latter study demonstrated that TRPC3 channels are present in the mesenteric arterial endothelium, with
⬃70% of the channels localizing to MEPs. The selective
TRPC3 blocker Pyr3 blunted acetylcholine-induced vasorelaxation and endothelial cell hyperpolarization in the presence of the NOS inhibitor L-NAME, the guanylyl cyclase
inhibitor ODQ, and the COX inhibitor indomethacin. The
IK channel blocker TRAM34 and the SK channel blocker
apamin also attenuated vasodilatory responses. Together,
these data suggest that TRPC3 channel activity stimulates
EDH in mesenteric arteries following muscarinic receptor
activation. A study by Kirby et al. (161) reported the presence of TRPC3 as well as SK and IK channels within IEL
fenestrations of rat popliteal and first-order skeletal muscle
arteries from the gastrocnemius muscle, but the function of
TRPC3 in this arterial bed was not assessed. Marrelli and
co-workers (165) demonstrated that TRPC3 channels contribute to EDH-mediated, SK and IK channel-dependent
dilation of cerebral arteries in response to ATP. Interestingly, this study provided evidence that receptor activation
leads to rapid recruitment of TRPC3 channels to the plasma
membrane (165).
Taken together, the findings cited above support a mechanism whereby activation of muscarinic GPCRs stimulates
TRPC3 activity, mostly likely through PLC-dependent gen-
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
eration of DAG. TRPC3 activity leads to increases in intracellular Ca2⫹ that activate SK and IK channels to cause
EDH. However, the Ca2⫹ fraction of TRPC3-mediated
mixed cation currents is predicted to be small at the endothelial cell resting membrane potential, arguing against a
direct Ca2⫹ influx mechanism for SK and IK channel activation. The explanation for this apparent discrepancy is not
clear, but it is possible that TRPC3 subunits form part of a
heteromultimeric, Pyr3-sensitive channel with greater Ca2⫹
permeability. Another possibility, reviewed by Eder et al.
(84), is that Na⫹ influx through TRPC3 indirectly activates
Ca2⫹ influx through the Na⫹/Ca2⫹ exchanger (NCX) acting in reverse mode. This mechanism requires colocalization of NCX and TRPC3 to produce subcellular domains
with locally elevated [Na⫹] capable of activating reversemode Ca2⫹ entry without causing membrane depolarization, which would diminish the electrical driving force for
Ca2⫹ entry. Support for TRPC3-mediated NCX reversemode Ca2⫹ entry is provided by a study from Rosker et al.
(289), who showed that elevated extracellular [Na⫹] caused
Ca2⫹ influx in HEK cells overexpressing TRPC3 channels.
Na⫹-induced Ca2⫹ influx was blocked by KB-R7943, a
compound that preferentially inhibits reverse-mode NCX
activity. Using a coimmunoprecipitation approach, these
authors (289) also demonstrated that endogenously expressed NCX1 binds to the COOH terminus (amino acids
742– 848) of heterologously expressed TRPC3. It has also
been reported that TRPC3 associates with NCX1 and KBR7943 blocks ANG II-induced Ca2⫹ influx in adult cardiomyocytes (85). However, KB-R7943 has off-target effect:
it is a potent blocker of TRPC3, TRPC5, and TRPC6 channels (171) and a strong Ca2⫹-independent activator of BK
channels in smooth muscle cells (191). Thus a convincing
demonstration of the proposed TRPC3-NCX Ca2⫹-entry
pathway and possible functional consequences awaits the
development of more selective pharmacological agents.
TRPC4 channels are equally permeable to Na⫹
and Ca2⫹ and are activated by GPCR pathways, although
the specific mechanisms are not fully understood.
Freichel et al. (97) reported involvement of TRPC4 channels in endothelium-dependent vasodilation. This study
demonstrated that SOCE could not be evoked in endothelial cells isolated from TRPC4⫺/⫺ mice, and AChinduced Ca2⫹ entry in these cells was diminished compared with that in wild-type controls. Aortic ring sections
from TRPC4⫺/⫺ mice displayed blunted endotheliumdependent relaxation in response to ACh, suggesting that
TRPC4-mediated Ca2⫹ influx contributes to endothelium-dependent dilation in conduit arteries (97). The authors proposed that SOCE through TRPC4 stimulates
eNOS activity and NO production to induce vasodilation
(98). This conclusion is tempered by subsequent reports
demonstrating that TRPC4 channels are not necessary
for SOCE in endothelial cells (2, 316).
F) TRPC4.
668
2. Permeability of the vascular endothelium
The vascular endothelium forms a semi-permeable barrier
that regulates passage of water, ions, and macromolecules
between the blood and the interstitium (219, 222). The
degree of permeability varies considerably among vascular
beds and segments and is influenced by physiological and
pathophysiological conditions. Substances are transferred
through the vascular wall by passive diffusion and active
transport mechanisms, and through clefts between neighboring endothelial cells by bulk flow. Transport across the
endothelium by bulk flow is governed by mechanical coupling between adjacent endothelial cells imparted by two
types of protein complexes, tight junctions and adherens
junctions, linked to the actin cytoskeleton. Ca2⫹-dependent
cytoskeletal reorganization of these interendothelial cell
junctions dynamically regulates permeability of the endothelium in response to GPCR agonists such as thrombin,
bradykinin, and histamine. TRPC1, TRPC4, TRPC6, and
TRPV4 channels have been reported to influence endothelial permeability, as discussed below.
A) TRPC1. TRPC1 channels have a unitary conductance of ⬃5
pS and are not selective for Ca2⫹ relative to Na⫹ in heterologous expression systems. It is unclear if homomeric
TRPC1 channels form in native cells, but TRPC1 subunits
have been shown to form heteromultimeric channels with
TRPC5 (334), TRPC4 (334), TRPC3 (333), TRPP2 (164,
419), and TRPV6 (301). TRPC4, TRPC1, and/or heteromultimeric channels containing TRPC1 subunits have
been implicated in the control of endothelial permeability
in the pulmonary vasculature through regulation of
SOCE. Inhibition of SERCA pump activity causes a large
increase in the permeability of the pulmonary endothelium (56) that is associated with a change in the shape of
pulmonary artery endothelial cells (232). Brough et al.
(39) showed that SOCE in pulmonary artery endothelial
cells was diminished following antisense-mediated
knockdown of TRPC1 expression, suggesting involvement of the channel in this response. Mehta el al. (218)
provided evidence that SOCE in cultured endothelial
cells was diminished by inhibition of RhoA activity and
also showed that RhoA physically interacts with IP3Rs
and TRPC1 channels (218). In cells overexpressing
TRPC1, stimulation with thrombin was found to result
in PKC-␣-mediated phosphorylation of TRPC1 channels
in association with SOCE and increased transendothelial
permeability (6). In human umbilical cord endothelial
cells in culture, the cytokine tumor necrosis factor-␣
(TNF-␣) was shown to increase TRPC1 expression accompanied by increased SOCE and stress fiber formation
(261).
B) TRPC4.
Evidence supporting a role for TRPC4 channels in
the regulation of pulmonary endothelial permeability was
first provided by Tiruppathi et al. (352), who demonstrated
that sustained increases in intracellular Ca2⫹ in response to
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
thrombin receptor activation were diminished in endothelial cells from the lungs of TRPC4⫺/⫺ mice compared with
controls. Diminished thrombin-induced Ca2⫹ influx was
associated with decreased actin-stress fiber formation and
endothelial cell retraction. In isolated lung preparations,
basal microvascular permeability did not differ between
control and TRPC4⫺/⫺ mice, but thrombin receptor activation-induced increases in lung permeability were significantly diminished in lungs from TRPC4⫺/⫺ mice. The authors concluded that Ca2⫹ store depletion following thrombin receptor stimulation causes a TRPC4-mediated influx
of Ca2⫹ that contributes to cytoskeletal rearrangement and
formation of intercellular gaps in the endothelium. Cioffi et
al. (59) demonstrated that the interaction between spectrinprotein 4.1 complexes and TRPC4 channels was required
for SOCE in pulmonary artery endothelial cell cultures. The
formation of intercellular endothelial cell adhesions promotes recruitment of TRPC4 channels to the plasma membrane. The authors of this study speculated that Ca2⫹ influx
through TRPC4 may contribute to the maturation of junctional complexes (120).
C) TRPC6. Several studies have implicated TRPC6 channels in
the regulation of endothelial permeability. Singh et al. (320)
reported that TRPC6 knockdown diminished thrombin-induced stress fiber formation and endothelial barrier function in pulmonary artery endothelial cells. Samapati et al.
(297) provided evidence that TRPC6 channels contribute to
the regulation of pulmonary vascular permeability. This
study demonstrated that TRPC6 channels are enriched in
pulmonary endothelial cell caveoli. The TRPC6 activator
hyperforin (182, 237) elevated intracellular Ca2⫹ levels in
primary lung endothelial cells and increased the wet weight
of isolated perfused rat lungs. Furthermore, platelet-activating factor (PAF)- and sphingomyelinase (ASM)-induced
increases in endothelial cell Ca2⫹, lung vascular filtration
coefficient, and wet weight gain were absent in TRPC6⫺/⫺
mice. The effects of PAF on endothelial cell Ca2⫹ levels and
hyperforin-induced inward cation currents were diminished
by administration of an NO donor or a membrane-permeant cGMP analog, and hyperforin-induced increases in
the wet weight of isolated perfused lungs were augmented
by NOS blockade. The authors concluded that NO (via
cGMP) inhibits TRPC6 channels in endothelial cells. Stimulation of the PAF/ASM signaling pathway recruits TRPC6
channels to caveoli and silences eNOS activity, allowing
TRPC6-mediated Ca2⫹ influx to increase vascular permeability.
D) ARE TRPC1, TRPC4, AND TRPC6 CHANNELS NECESSARY FOR ENDO-
Although many studies cited above have
put forward the concept that SOCE mediated by TRPC1
and TRPC4 channels can increase the permeability of the
vascular endothelium, this interpretation is called into question by work from the Trebak lab showing that endothelial
cell SOCE does not require TRPC1, TRPC4 or TRPC6
THELIAL CELL SOCE?
expression (2, 316), despite the fact that knockdown of
these TRPC channels blocks thrombin-induced increases in
endothelial permeability (316). This latter study also demonstrated that STIM1 independently controls endothelial
barrier function by a mechanism that does not require
TRPC1 or TRPC4 channels, Orai1, or Ca2⫹ entry (316).
These data indicate that TRPC channels contribute to the
regulation of endothelial permeability by a mechanism that
is independent of SOCE. A conflicting study by Cioffi et al.
(60) reported that heteromultimeric channels containing
TRPC4 and TRPC1 subunits are present in pulmonary artery endothelial cells and physically interact with Orai1 via
TRPC4; they further showed that Oria1 activity is necessary for the formation of interendothelial cell gaps. In a
similar vein, Sundivakkam et al. (341) showed that STIM1
interacts with TRPC1 and TRPC4 to form a store-operated
channel in cultured endothelial cells. Although the contradictory results regarding TRPC1 and TRPC4 channels and
SOCE currently remain unresolved, strong evidence supports the concept that endothelial cell SOCE can occur
without TRPC1, TRPC4, and TRPC6 expression (359).
The findings of the Trebak laboratory are supported by the
results of unbiased high-throughput siRNA screens, which
revealed the necessity for STIM1 and Orai1 in SOCE but
did not demonstrate the involvement of any TRP channels
(370). It has also been reported that Ca2⫹ release from
internal stores persists in Purkinje neurons from TRPC1/
C4/C6 triple-knockout mice, suggesting that these channels
are not required for the maintenance of intracellular stores
via SOCE (131). Furthermore, the biophysical properties of
TRPC channels suggest that fractional Ca2⫹ currents are
probably very small under physiological conditions. It
should be noted that many of the studies linking SOCE with
endothelial permeability have presented only data showing
changes in global intracellular Ca2⫹ and provide little or no
evidence for direct TRPC channel activation using biophysical or electrophysiological methods. For SOCE studies that
have used the patch-clamp technique, the apparent involvement of TRPC channels under the conditions used could
reflect PLC-mediated activation of TRPC channels downstream of store depletion (2). In addition, many of the studies supporting a role for TRPC channels in SOCE-mediated
Ca2⫹ influx were performed using endothelial cells in culture, and in some cases, overexpression systems. A study by
Bergdahl et al. (31) showed that culture conditions significantly increased TRPC1 and TRPC6 expression and SOCE
in smooth muscle cells. The effects of culture conditions on
endothelial cell TRPC4 expression have not been reported,
but phenotypic transformations that influence SOCE and
permeability regulation are possible. Additional work using
intact artery preparations is needed to fully resolve these
issues.
E) TRPV4.
TRPV4 is present in pulmonary endothelial cells in
both humans and rodents (15), and studies from several
laboratories have convincingly demonstrated an important
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
role for the channel in the regulation of pulmonary vascular
permeability. A study from by Townsley and colleagues
(15) showed that treatment of isolated lung preparations
from wild-type mice with 4␣-PDD, 5,6-EET, or 14,15-EET
increased microvascular permeability. That this increased
microvascular permeability was mediated by TRPV channels, specifically TRPV4, was confirmed by the fact that it
was attenuated by the nonselective TRPV blocker ruthenium red and was unaltered in response to 4␣-PDD in lungs
from TRPV4⫺/⫺ mice. However, SOCE-induced permeability responses did not differ between TRPV4⫺/⫺ and wildtype mice. The overall 4␣-PDD and EET-induced increases
in permeability were comparable to those induced by
SOCE, but these treatments influenced different vascular
segments. TRPV4 agonists primarily affected alveolar septal microvessels, whereas the effect of SOCE was greatest in
extra-alveolar vessels. Moreover, TRPV4 agonists caused
breaks in the barrier and blebbing of endothelial cells,
whereas SOCE caused the formation of gaps at cellular
junctions. This study clearly showed that TRPV4-mediated
Ca2⫹ influx and SOCE have differential effects on the pulmonary endothelium.
The concept of functional specificity of Ca2⫹-influx pathways in the pulmonary endothelium was further explored in
a study that compared the effects of endothelial cell Ca2⫹
entry through T-type Ca2⫹ channels with that of TRPV4.
This study showed that Ca2⫹ entry via T-type Ca2⫹ channels, but not TRPV4-mediated Ca2⫹ entry, induced surface
expression of the adhesion molecule P-selectin in pulmonary capillary endothelium. In contrast, TRPV4-dependent,
but not T-type Ca2⫹ channel-dependent, Ca2⫹ influx increased capillary permeability (397). Inhibition of myosin
light-chain kinase (MLCK) was shown to diminish cell surface levels of TRPV4 protein and decrease 4␣-PDD-induced
Ca2⫹ influx and cation currents in pulmonary vascular endothelial cells (264), suggesting that MLCK activity is involved in trafficking of TRPV4 channels to the plasma
membrane in the pulmonary endothelium. Using real-time
Ca2⫹ imaging in isolated perfused lungs, Yin et al. (410)
provided evidence that TRPV4 channels mediate increases
in endothelial cell [Ca2⫹] in response to elevations in left
atrial pressure. Increases in NO production induced by increases in left atrial pressure were absent in TRPV4⫺/⫺
mice, and wet-to-dry weight ratios of lungs subjected to
pressure elevation were smaller in TRPV4⫺/⫺ mice compared with those in wild-type animals. This study also
showed that inward cation currents stimulated by 4␣-PDD
in pulmonary microvascular endothelial cells were attenuated by pretreatment with a cGMP analog. The authors
proposed that pressure-induced activation of TRPV4 channels causes Ca2⫹ influx, which elevates endothelial permeability and increases NO production. NO, acting through
cGMP, acts as a negative-feedback mechanism to govern
TRPV4-mediated Ca2⫹ influx.
670
3. Angiogenesis
Angiogenesis, the process of new blood vessel development,
occurs in a tightly regulated manner during embryonic
growth and wound healing, and during pathological conditions such as diabetes, arthritis, and cancer (96). Angiogenesis is initiated by remodeling of the basement membrane
followed by proliferation and migration of endothelial cells,
resulting in the generation of new capillary tubes. The angiogenic process is stimulated by several growth factors,
including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and epidermal growth factor
(EGF). Blocking Ca2⫹ entry diminishes endothelial cell adhesion, motility, and tube formation in culture and angiogenesis in vivo (170), but the influx pathways responsible
are not well characterized. However, a few studies have
demonstrated the involvement of TRPC1, TRPC3, TRPC4,
TRPC5, TRPC6, and TRPV4 channels.
A) TRPC1.
Using an in vivo zebrafish model, Yu et al. (411)
demonstrated that knockdown of TRPC1 disrupted angiogenesis in developing larvae. This study showed that
TRPC1 is a downstream target of VEGF-a, and its expression is necessary for extracellular signal-regulated kinase
(ERK) activation. In contrast to these findings, knockdown
of TRPC1 expression had no significant effect on the in
vitro formation of human umbilical vein-derived endothelial tubes (19), and the vasculature was reported to develop
normally in TRPC1⫺/⫺ mice (304).
B) TRPC3, TRPC4, AND TRPC5.
Antigny et al. (19) reported that
siRNA-mediated silencing of TRPC3, TRPC4, or TRPC5
expression diminished spontaneous Ca2⫹ oscillations in human umbilical vein-derived endothelial cells and inhibited
endothelial tube formation in vitro, suggesting a possible
role for these TRPC channels in angiogenesis.
C) TRPC6. Endothelial cell TRPC6 channels are activated by
binding of the pro-angiogenic factor VEGF to VEGF2 receptors (54), suggesting that TRPC6-mediated cation currents could be involved in the angiogenic response. In agreement with this possibility, Hamdollah Zadeh et al. (128)
reported that VEGF-induced migration and tube sprouting
of microvascular endothelial cells was diminished in cultures expressing a dominant-negative form of TRPC6
(110). Further support is provided by a study by Kini et al.
(160), who showed that TRPC6 associates with PTEN
(phosphatase and tensin homologue) protein in human pulmonary artery endothelial cells and demonstrated that this
interaction is required for surface expression of TRPC6 and
thrombin-induced proliferation and tube formation. In
contrast, neither TRPC6 gain-of-function mutations in humans (391) nor TRPC6 gene deletion in mice (70) was
reported to have any overt effect on blood vessel structure.
D) TRPV4. Several studies have examined the concept that
activation of TRPV4 channels contributes to angiogenesis.
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
Knockdown of TRPV4 expression was shown to prevent
cyclic strain-induced Ca2⫹ influx and capillary endothelial
cell reorientation, suggesting involvement of the channel in
migration and sprouting associated with angiogenesis
(349). Interestingly, administration of the TRPV4 agonist
4␣-PDD enhanced collateral growth of cerebral blood vessels, suggesting involvement of the channel in cerebral angiogenesis (300). Total and surface TRPV4 expression and
channel activity were found to be elevated in breast tumorderived endothelial cells compared with normal dermal microvascular endothelial cells (92), and activation of TRPV4
channels with arachidonic acid promoted migration of tumor-derived, but not normal endothelial cells. Collectively,
these observations suggest that TRPV4 channels may be
involved in tumor angiogenesis.
C. TRP Channels in Perivascular Cells
TRP channels are present in perivascular neurons and astrocytes. Several studies have indicated that TRPV1,
TRPA1, and perhaps TRPV4 channels in perivascular neurons can cause vasodilation, and one study demonstrated
that TRPV4 channels present in astrocytic endfeet enveloping parenchymal cerebral arteries influence neurovascular
coupling.
1. TRPV1 in perivascular sensory nerves
Neuronally evoked vasodilation is a common feature of the
vasculature in many organ systems, including the brain,
cutaneous tissues, and the gastrointestinal tract (33). A
model describing a vasodilator role for TRPV1 channels
located on perivascular sensory nerves has emerged during
the past decade. Data from several research groups have
demonstrated that activation of TRPV1 channels causes
release of the potent vasodilator CGRP from sensory
nerves, leading to vasodilation. This interaction between
TRPV1 channels and CGRP activity is most frequently reported for the mesenteric resistance vasculature (335, 430,
376, 378), but also clearly applies to other vascular beds,
such as the dural (7) and cerebral (430) circulations. The
endogenous endocannabinoid anandamide has been
strongly implicated as a potential physiological activator of
the above TRPV1/CGRP vasodilator pathway, acting
through direct activation of TRPV1 channels on sensory
nerves rather than through activation of cannabinoid-specific receptors (7, 38). A similar vasodilatory mechanism in
the coronary circulation, initiated by ethanol, has been described (109). Activation of TRPV1 channels has also been
reported to inhibit sympathetic vasoconstrictor tone
through the release of CGRP (412). Similar roles for TRPA1
(27, 429) and TRPV4 (101) channels in mesenteric perivascular sensory nerves have been reported (FIGURE 11).
2. TRPV4 channels and neurovascular coupling
TRPV4 activity in astrocytes has been linked to the local
control of cerebral blood flow. Brain parenchymal astro-
cytes form close associations with neurons and with penetrating arterioles that link pial arteries on the surface of the
brain with the subsurface microcirculation. Astrocytes have
been proposed to serve as “middlemen” in the functional
hyperemic process of neurovascular coupling, which closely
matches blood perfusion with neuronal activity (318). Astrocytic endfeet envelop the parenchymal arterioles and can
mediate profound vasodilation in response to elevated synaptic activity. A number of vasoactive factors, including
EETs, NO, and K⫹, have been implicated in this response
(74). Work from the Nelson laboratory has shown that K⫹
efflux through BK channels present on astrocytic endfeet
acts through elevation of [K⫹] in the restricted space between the endfoot and the arteriolar wall to activate KIR
channels on smooth muscle cells, causing membrane hyperpolarization and vasodilation (91) (FIGURE 11). This response is dependent on increased astrocytic intracellular
[Ca2⫹] (330). Ca2⫹ entry through activated TRPV4 channels located in astrocytic endfeet (29) stimulates CICR from
intracellular stores and enhances arterial dilation (73).
TRPA1 (314, 315) and TRPC3 (331) channels are also
present in astrocytes and regulate intracellular Ca2⫹, but
their involvement in neurovascular regulation has not been
reported.
IV. INVOLVEMENT OF TRP CHANNELS IN
VASCULAR PATHOLOGIES
Given the clear physiological roles for TRP channels in
vascular smooth muscle, the endothelium, and perivascular
cells, it is not surprising that there are multiple examples
where the function or dysfunction of various TRP channels
contributes to cardiovascular disease (see TABLE 4). The
literature describing the involvement of TRP channels in
vascular-related diseases is discussed below.
A. Hypertension
Although the causes of essential hypertension are still not
established, alterations in central cardiovascular control
mechanisms as well as cardiovascular, hormonal, and fluid
and electrolyte adjustments typically associated with renal
disease are clearly involved in the development of this disease (44). Specific features of vascular dysfunction are prevalent in virtually all situations where sustained blood pressure elevations are present. The primary manifestation of
hypertension in the vasculature is an increase in peripheral
resistance resulting from enhanced vascular contractility,
differing degrees of vascular remodeling in the resistance
vasculature, or both. Although TRP channel dysfunction
may be involved in hypertension driven by neural (339),
humoral (214), or renal (395) mechanisms, the following
discussion will focus entirely on evidence that TRP channels
in the vasculature contribute to the pathophysiological processes associated with elevated blood pressure.
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
Ca2+
A
Artery
TRPV1
CGRP
TRPA1
Ca2+
B
Synapse
Astrocyte
Cerebral
arteriole
Endfoot
EETs
Ca2+
BK
TRPV4
K+
K+
mGluR
Ca2+
IP3
ER
FIGURE 11. TRP channels in perivascular
cells. A: Ca2⫹ influx through TRPV1 or TRPA1
channels in perivascular nerves can stimulate
the release of CGRP to cause arterial dilation.
B: TRPV4 channels contribute to neurovascular coupling. Stimulation of metabotropic glutamate receptors (mGluR) on parenchymal astrocytes leads to the generation of IP3, which
causes the release of Ca2⫹ from the endoplasmic reticulum (ER), stimulating K ⫹ efflux
through BK channels. The resulting increase in
[K⫹] in the space between the astrocytic endfoot and cerebral arteriole activates K⫹ efflux
through KIR channels present on vascular
smooth muscle cells to cause dilation. Production of EETs or prostaglandins (e.g., PGE2) may
also contribute. Ca2⫹ influx through TRPV4
channels contributes to the propagation of
Ca2⫹ waves throughout the endfoot.
KIR
K+
Ca2+
Ca2+
Ca2+
TRPV4
1. TRPC channels
There are multiple examples of altered TRPC channel expression and function, primarily TRPC3 and TRPC6, associated with hypertension.
A) TRPC3. Several studies have reported that TRPC3 expression is upregulated in arteries from hypertensive animals, a
phenomenon most clearly documented in the SHR model of
hypertension. Increased TRPC3 expression in vascular
smooth muscle cells isolated from SHRs is correlated with
enhanced ANG II-induced Ca2⫹ influx that is independent
of VDCCs (199). A similar increase in carotid artery
smooth muscle TRPC3 channel expression in SHRs accompanied by enhanced vascular contractility has also been
672
described (256). Interestingly, this latter study found that
TRPC1 expression was decreased. Others have reported
increased expression of TRPC3 as well as TRPC1 and
TRPC5 in mesenteric arterioles from SHRs compared with
normotensive controls, with an associated increase in vasomotion (51). Enhanced ET-1-induced contractions of SHR
mesenteric resistance arteries were apparently due in part to
increased TRPC3 expression, which was associated with
greater coupling of ET-1 receptor activity to Ca2⫹ entry
through direct physical interactions between IP3Rs and
TRPC3 channels (4). Although there are no specific reports
of TRPC3 upregulation in the vascular smooth muscle cells
of humans with hypertension, TRPC3 levels were found to
be elevated in monocytes from patients with essential hypertension in association with enhanced migration activity
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
Table 4. TRP channels involved in vascular pathologies
TRP
Pathology
Tissue
TRPC1
Hypertension, vascular remodeling/VSM proliferation
TRPC3
TRPC4
Hypertension, vascular remodeling/VSM proliferation
Diminished risk of MI, vascular remodeling/VSM
proliferation
Hypertension, I/R-induced pulmonary edema,
vascular remodeling/VSM proliferation
Hypertension, chronic hypoxia-induced proliferation,
cardiac and cerebral I/R injury, neurogenic
inflammation
Cerebral I/R injury
Hypertension, APP-associated endothelial
dysfunction, pulmonary edema
Oxidative stress response, neurogenic inflammation
Lung (113, 194, 200, 374), large veins (184, 347),
brain (31)
Mesentery (4, 51), brain (256), lung (413)
Endothelium (153), lung (421)
TRPC6
TRPV1
TRPV3
TRPV4
TRPA1
TRPM2
TRPM4
TRPM7
Oxidative stress response
Cerebral ischemia
Hypertension, neuronal death following cerebral
ischemia, vascular remodeling/VSM proliferation
Mesentery (277, 428), lung (194, 413)
Systemic (185, 379), lung (380), heart (375), brain
(239, 265)
Brain (235)
Brain (418), lung (127), mesentery (325)
Brain (Sullivan et al., unpublished data), sensory
nerves (18, 244, 346, 361)
Endothelium (135, 345)
CNS microvessels (112), brain (203)
Mesentery (357, 358), brain (339), epidermis (134)
VSM, vascular smooth muscle; MI, myocardial infarction; I/R, ischemia reperfusion; CNS, central nervous
system. Reference numbers are given in parentheses.
of these cells (424). Also, although TRPC3 expression levels
and hypertension were positively correlated in each of these
studies, cause and effect analyses, for instance, examination
of TRPC3 expression in prehypertensive or anti-hypertensive-treated SHRs, have not been reported. See Reference
377 for a comprehensive review of TRPC3 and hypertension.
B) TRPC6. Upregulation of TRPC6 in vascular smooth muscle
in association with hypertension has also been reported. In
a rat model of ouabain-induced hypertension, cellular levels
of TRPC6, TRPC1, the ␣2 subunit of the Na⫹ pump, and
the Na⫹-Ca2⫹ exchanger are increased in mesenteric artery
vascular smooth muscle cells (277). Increased and coordinated activity among these various Ca2⫹ transport systems
is correlated with increases in [Ca2⫹]i, which could increase
vascular contractility and contribute to elevated blood pressure. Similar changes in the expression of TRPC6 and the
Na⫹-Ca2⫹ exchanger in mesenteric myocytes, and altered
Ca2⫹ homeostasis occur in Milan hypertensive rats (195,
428). MAP is slightly elevated in TRPC6⫺/⫺ mice compared
with wild-type animals (70). This is unexpected, but may be
due to the (apparently compensatory) upregulation of
TRPC3 channels observed in TRPC6⫺/⫺ mice.
Pulmonary hypertension is characterized by pulmonary
arterial pressures greater than 25 mmHg and is due in
part to heightened contractility of pulmonary artery
smooth muscle cells as well as substantial cellular proliferation and remodeling of the pulmonary arterial wall.
Both processes contribute to sustained increases in pulmonary artery resistance. Several studies have indicated
that both TRPC6 and TRPC1 channels contribute to the
mechanisms underlying chronic hypoxia-induced pulmonary hypertension (194, 374). For example, chronic (3– 4
wk) hypoxia in rats was shown to increase TRPC6 and
TRPC1 channel expression in pulmonary artery smooth
muscle cells by two- to threefold (194). Chronic hypoxia
also increased ROCE and SOCE; these responses were
correlated with enhanced pulmonary artery contractile
activity (399) and were inhibited by molecular or genetic
suppression of TRPC6 and TRPC1 expression (194,
399). Others have demonstrated a central role for EETs,
in particular, 11,12-EET, in the increased expression and
activity of TRPC6 channels in this system, predominantly through enhanced trafficking and localization of
TRPC6 channels to the plasma membrane in response to
hypoxia (159, 272). A similar upregulation of TRPC6
and TRPC3 channels has been demonstrated in pulmonary artery myocytes from humans with idiopathic pulmonary hypertension (413, 414). Suppression of TRPC6
expression in pulmonary vascular smooth muscle cells
cultured from these patients was shown to substantially
reduce cellular proliferation. A role for TRPC6 in hypoxiainduced pulmonary hypertension is less apparent in mice,
where TRPC1 may make a significant contribution (209,
385). TRPC1 channel expression and Ca2⫹ entry are both
elevated in pulmonary arteries of rats with monocrotalineinduced pulmonary hypertension (200). Taken together,
the preceding observations suggest that the common theme
of upregulated TRPC6 and/or TRPC1 channel function and
accompanying heightened vascular reactivity is associated
with nearly all forms of hypertension.
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
2. TRPV channels
A) TRPV1.
TRPV1 channel function appears to be altered in
response to hypertension and has a significant impact on the
regulation of MAP. The TRPV1 channel agonist capsaicin
was shown to decrease MAP in normal Wistar rats (185)
and salt-sensitive Dahl rats (379) fed a high-salt diet;
TRPV1 channel expression was also increased in these latter animals. Interestingly, MAP was further elevated in hypertensive rats on a high-salt diet following treatment with
the TRPV1 channel antagonist capsazepine. These observations have led to the conclusion that TRPV1 receptors,
likely those present predominantly on perivascular sensory
nerves, are activated in animals with salt-sensitive hypertension. The associated release of CGRP from sensory nerve
terminals provides a compensatory vasodilator response
that opposes the elevation of blood pressure in this model of
hypertension. There is no significant difference in systolic or
diastolic blood pressures in TRPV1⫺/⫺ compared with
wild-type mice (212). This supports the hypothesis that altered TRPV1 activity occurs primarily in response to elevated blood pressure, but probably is not involved in the
etiology of hypertension.
B) TRPV4.
A few studies have indicated the involvement of
TRPV4 channels in pulmonary and systemic hypertension.
In rat pulmonary arteries, chronic hypoxia-induced pulmonary hypertension was shown to upregulate TRPV4 channels (407). This increase in TRPV4 expression produced a
feed-forward effect on pulmonary hypertension whereby
Ca2⫹ entry through the expanded population of TRPV4
channels in pulmonary artery myocytes increased global
[Ca2⫹]i, which enhanced pulmonary vascular contractility
and pulmonary hypertension.
port in numerous tissue types, including vascular smooth
muscle. Recent observations point to a substantial role for
TRPM channels, particularly TRPM7, and altered TRPM
channel function in various forms of hypertension. TRPM7
expression and activity, as well as [Mg2⫹]i, are reduced in
mesenteric artery smooth muscle from SHR compared with
WKY rats (358). Decreased intracellular Mg2⫹ is associated
with heightened contractility, which contributes to hypertension. Disrupted Mg2⫹ regulation involving altered
TRPM7 activity is also correlated with changes in cellular
inflammation, proliferation, and remodeling, each of which
is associated with vascular disease in hypertension (357).
B. Vascular Remodeling, Intimal
Hyperplasia, and Vascular Occlusive
Disease
Vascular remodeling occurs in the context of a number of
cardiovascular diseases, including hypertension, coronary
artery disease, atherosclerosis, vascular injury, and inflammatory diseases. Increased intracellular [Ca2⫹] and activation of the ␦ isoform of Ca2⫹/CamKII seems to be a common mechanism associated with this type of cellular proliferative response. Signaling mechanisms downstream of
CamKII activation are highly diverse, but likely involve altered expression of inhibitors and activators of cell-cycle
progression, and altered gene transcription regulated by
nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) (319). TRP channels can facilitate increases in intracellular Ca2⫹ associated
with activation of CamKII that are linked to vascular disease and remodeling.
1. TRPC1
Elegant work from Nelson and colleagues (325) has shown
that the coupling efficiency between endothelial cell TRPV4
channels and activation of EDH and dilation of mesenteric
resistance arteries is disrupted in a mouse model of ANG
II-induced chronic hypertension. Much of this effect was
attributed to a profound decrease in the PKC-anchoring
protein AKAP150 in the vicinity of myoendothelial gap
junctions in arteries from normotensive mice, and the resulting disruption in the AKAP150-dependent clustering of
TRPV4 channels that is responsible for the high degree of
cooperative gating of localized TRPV4 channels. The authors of this groundbreaking study proposed that these hypertension-induced changes in TRPV4 function could contribute to the mechanisms by which high blood pressure
alters local blood flow regulation.
3. TRPM7
Abundant evidence supports the hypothesis that altered
regulation of Mg2⫹ transport contributes to the development and maintenance of hypertension (reviewed in Ref.
326). TRPM6 and TRPM7 play key roles in Mg2⫹ trans-
674
Several studies provide evidence for the involvement of
TRPC1 channels in disease-associated smooth muscle cell
proliferation. In cultured human pulmonary artery myocytes, stimulation with serum-derived growth factors was
shown to increase TRPC1 expression and Ca2⫹ influx in
association with enhanced cellular proliferation (113). In
cultured human saphenous vein myocytes, TRPC1 channel
activity has been linked to smooth muscle cellular proliferation, an effect that is partly dependent on interactions with
STIM1 (184). Other studies have shown that TRPC1 channel expression is upregulated in rodent models of vascular
injury and myocyte proliferation in association with enhanced Ca2⫹ entry (31, 173). In the latter of these two
studies, administration of an antibody targeting an extracellular loop of TRPC1 substantially reduced smooth muscle proliferation and Ca2⫹ entry in cultured human saphenous veins. Similarly, chronic stimulation of cultured human coronary artery smooth muscle cells with ANG II was
shown to increase TRPC1 expression and SOCE in association with cellular hypertrophy, effects that were suppressed by siRNA-mediated knockdown of TRPC1 (347).
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
2. Other TRPCs
A few studies have implicated other members of the TRPC
subfamily in smooth muscle cell proliferation. One report
demonstrated that the proliferative response of human pulmonary artery smooth muscle to extracellular ATP, a mitogenic stimulant, occurred via CREB-mediated upregulation
of TRPC4 and enhanced SOCE (421). Pulmonary artery
smooth muscle from a subset of pulmonary hypertensive
patients with idiopathic pulmonary arterial hypertension
displayed enhanced expression of both TRPC3 and TRPC6.
Proliferation of pulmonary artery myocytes cultured from
these patients was greatly attenuated by suppression of
TRPC6 expression (413). In addition, one group demonstrated that TRPC3 contributes to the cellular proliferation
that occurs in the setting of stent-induced remodeling of
large arteries (168).
3. TRPV1
One group reported that the proliferative response of cultured human pulmonary myocytes to chronic hypoxia was
correlated with increased expression of TRPV1 channels
and elevated SOCE (380). A role for TRPV1 in the remodeling of venous tissue exposed to altered pressure and flow
has also been reported (52).
SHR model of hypertension. However, direct causal links
between TRPM7 expression, intracellular [Mg2⫹], and differential effects on vascular contractility or smooth muscle
proliferation are presently difficult to define given the celltype-dependent, model-related, and tissue-specific differences in these parameters that have been reported (189,
259).
An exciting recent report demonstrated a novel mechanism
for the regulation of gene expression, differentiation, and
development involving TRPM7 channels (172). In this
study, Krapivinsky et al. (172) showed that the COOHterminal serine/threonine kinase domain of TRPM7 is proteolytically cleaved from the cation-conducting domain and
translates to the nucleus in many different types of cells. The
kinase domain participates in chromatin remodeling by
phosphorylating histone proteins at specific sites important
for cell differentiation and development. This study also
showed that TRPM7 channels control cytosolic [Zn2⫹],
which in turn regulates binding of the cleaved kinase fragments to transcription factors containing zinc-finger domains. These data suggest that the kinase and ion-conducting domains of TRPM7 can together control epigenetic
modifications and gene expression. However, it is not currently known if this system is functional in smooth muscle
cells or participates in cellular proliferation.
C. Oxidative Stress and
Ischemia/Reperfusion Injury
4. TRPM6 and TRPM7
Numerous studies have demonstrated that Mg2⫹ have a
substantial impact on vascular smooth muscle and endothelial cell function (for review, see Ref. 326). In particular,
changes in cytosolic Mg2⫹ levels may be involved in regulating vascular proliferation. TRPM6 and TRPM7 are critically important for Mg2⫹ transport in vascular smooth
muscle and are involved in vascular diseases characterized
by cellular proliferation and inflammation. Exposure to
ANG II or aldosterone for 24 h was shown to substantially
increase TRPM7 mRNA and protein levels in primary cultured aortic and mesenteric arterial smooth cells, an increase that was correlated with an increase in intracellular
[Mg2⫹] (134). The effects of ANG II on TRPM7 expression,
Mg2⫹ influx, and accompanying cellular proliferation were
inhibited by siRNA-mediated TRPM7 downregulation
(134). The authors of this work concluded that a TRPM7dependent influx of Mg2⫹ is directly linked to the vascular
proliferative response to activation of ANG II receptors.
Interestingly, basal levels of TRPM7 and intracellular
[Mg2⫹] were found to be reduced in cultured mesenteric
vascular smooth muscle cells from SHRs compared with
those from WKY rats (358). Furthermore, ANG II was
shown to increase TRPM7 expression and intracellular
[Mg2⫹] in WKY, but not SHR, cells. These observations
suggest that changes in TRPM7 expression and Mg2⫹ homeostasis are better correlated with enhanced contractility
than with vascular smooth muscle cell proliferation in the
ROS generated by biological systems include H2O2, O2⫺,
and hydroxyl radicals (OH·). ROS act as signaling molecules, but when produced in excess can cause oxidative
damage to membrane phospholipids, proteins, and DNA
and diminish the bioavailability of endothelium-derived
NO in the vasculature. Oxidative stress, defined as an imbalance between the generation of ROS by a biological
system and the ability of that system to remove toxic metabolites and repair damage, is associated with numerous
pathological conditions, including common cardiovascular
diseases such as hypertension (65), atherosclerosis, and
stroke. Reperfusion of tissue following ischemia (IR injury)
is well recognized as a source of oxidative stress in the brain
following stoke and in the heart following myocardial infarction. Multiple TRP channels have been implicated in
responses to ROS and oxidative stress in various organ
systems.
1. TRPC4
A study by Jung et al. (153) identified a single-nucleotide
polymorphism (SNP) in the coding region of TRPC4
(I957V) that is associated with a diminished risk of myocardial infarction (MI) (153). Ca2⫹ imaging and patchclamp analysis indicated that the mutant channel is more
sensitive to muscarinic receptor stimulation compared with
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
the wild type. The authors speculate that elevated Ca2⫹
influx through the I957V variant lowered the incidence of
MI by enhancing endothelium-dependent relaxation in coronary arteries.
2. TRPC6
Weissmann et al. (386) demonstrated that pulmonary
edema caused by I/R injury is diminished in lungs from
TRPC6⫺/⫺ mice compared with wild-type controls and
TRPC1⫺/⫺ and TRPC4⫺/⫺ mice. This study further demonstrated that TRPC6 activation and I/R injury occur downstream of NOX2-generated ROS and PLC-␥-generated
DAG.
3. TRPV1
A study by Wang et al. (375) demonstrated that recovery of
cardiac function following I/R injury was diminished in
TRPV1⫺/⫺ mice or mice treated with the TRPV1 inhibitor
capsazepine compared with controls. Impairment of functional recovery was worse in animals treated with capsazepine compared with controls. These data suggest that
TRPV1 channel activity attenuates I/R-induced damage in
the heart. An apparent protective effect of TRPV1 channel
activation on I/R injury has also been demonstrated in the
kidney (269, 362, 364, 365).
ruthenium red, suggesting that activation of TRPV3 channels mediates a portion of this response. The mechanism of
action is unknown, but TRPV3 channels are present in the
cerebral artery endothelium, and activation of the channel
with carvacrol causes endothelium-dependent vasodilation
(78), consistent with the possibility that incensole protects
against ischemic damage by promoting cerebral blood flow.
5. TRPV4
Zhang et al. (418) showed that the selective TRPV4 activator GSK1016790A and the muscarinic receptor agonist
ACh caused endothelium-dependent dilation of mouse cerebral arteries, an effect that was blocked by the selective
TRPV4 antagonist HC-067047 and by inhibition of SK and
IK channels. Interestingly, block of TRPV4 had little effect
on ACh-induced dilation of arteries isolated from mice expressing mutant forms of the human amyloid precursor
protein (APP), constitutively active tumor growth factor
(TGF)-␤1, or both transgenes. This effect was reversed by
superoxide dismutase or catalase in arteries from APP mice,
but not those from other groups. The authors concluded
that endothelial TRPV4 function is impaired by the oxidative stress that is specifically associated with APP overexpression.
6. TRPA1
Several studies have examined the effects of TRPV1 activity
on I/R injury in the brain. Capsaicin administered 5 min
after restoration of flow was shown to diminish I/R injuryinduced neuronal damage in a global cerebral ischemia
model, suggesting that activation of TRPV1 in this context
is neuroprotective (265). Xu et al. (404) extended these
findings, showing that chronic dietary capsaicin diminished
hypertrophy of the cerebral vasculature and delayed onset
of stroke in stoke-prone SHRs, likely due to enhanced production of NO. In addition, a study by Muzzi et al. (239)
showed that systemic administration of the TRPV1 agonist
rinvanil following I/R injury caused hypothermia in mice in
association with diminished neuronal damage. In contrast,
Gauden et al. (108) reported that blocking TRPV1 with
capsazepine reduced I/R injury-induced increases in endothelial permeability in the brain, suggesting that TRPV1
activity can have negative consequences following cerebral
I/R injury (108).
TRPA1 is present in the endothelium of cerebral arteries
(77) and in the perivascular nerves of mesenteric arteries
(27). A number of studies have demonstrated that TRPA1
channels are able to detect cellular oxygen status and oxidative stress. Work by Takahashi et al. (346) demonstrated
that TRPA1 channels are activated by both hyperoxic and
hypoxic conditions, suggesting that the channel is intimately involved in oxygen sensing. TRPA1 channels are
also activated by substances such as 4-HNE, 4-ONE, and
4-HHE that are generated by ROS-induced peroxidation of
␻6 polyunsaturated fatty acids (17, 363). A study by Sullivan et al. (338) demonstrated that endogenously generated
oxidized lipids cause endothelium-dependent dilation of cerebral arteries, suggesting that TRPA1 channels may have a
protective role in the cerebral vasculature during oxidative
stress.
4. TRPV3
The unitary conductance of TRPM2 is ⬃52– 60 pS at negative holding potentials and ⬃76 pS at positive potentials.
Ionic selectivity is temperature dependent, with PCa:PNa ratios of ⬃0.7:1 at 20°C and ⬃6:1 at 35°C (267, 299, 353).
TRPM2, which is broadly expressed and is present in vascular smooth muscle and endothelial cells (135), is activated
by ADP-ribose (ADPR) (267, 299) and H2O2 (129, 384).
Several studies have indicated that TRPM2 is critically involved in ROS-mediated signaling and responses to oxida-
A study by Moussaieff and colleagues demonstrated that
incensole, a TRPV3-activating compound derived from
trees of Boswellia species (235), diminished brain infarct
volume and neurological deficits induced by I/R injury following carotid artery occlusion (236). The neuroprotective
effects of incensole were diminished in TRPV3⫺/⫺ mice and
in mice pretreated with the nonselective TRPV inhibitor
676
7. TRPM2
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
tive stress in a number of organ systems, including the vasculature (345). In cultured endothelial cells, high concentrations of H2O2 activated a cation current that was
diminished by siRNA-mediated knockdown of TRPM2
channels (135). H2O2-induced Ca2⫹ influx and apoptosis
of cultured endothelial cells is caused by PKC-␣-dependent
activation of TRPM2 (136, 340). The in vivo significance of
these findings has not been demonstrated.
8. TRPM4
Trauma-induced expression of TRPM4 channels in the endothelium has been linked to neuronal damage. Gerzanich
et al. (112) demonstrated that TRPM4 expression is increased in tissue surrounding the site of spinal cord injury,
particularly in microvessels and capillaries. Downregulation of TRPM4 expression reduced secondary hemorrhage
at the injury site and improved neurobehavioral performance. A study by Loh et al. (203) demonstrated that I/R
injury induced by middle cerebral artery occlusion
(MCAO) promoted the expression of TRPM4 in the cerebral artery endothelium. Knockdown of TRPM4 expression enhanced angiogenesis and reduced infarct volume after MCAO, effects that were accompanied by improved
motor function. These studies identify TRPM4 blockade as
a possible prophylactic strategy against injury- and strokeinduced neuronal damage.
9. TRPM7
A study by Aarts et al. (1) demonstrated that exposure of
cultured neurons to oxygen-glucose depravation (OGD),
an in vitro model of I/R injury, stimulated a cation current
that was diminished by siRNA-mediated knockdown of
TRPM7 expression. TRPM7 knockdown also decreased
OGD-induced Ca2⫹ influx and reduced cell death. A follow-up study by Sun et al. (339) used targeted injection of
adeno-associated virus to selectively diminish TRPM7 expression in CA1 hippocampal neurons in vivo. The resulting decrease in TRPM7 expression was associated with diminished delayed neuronal death following global cerebral
ischemia and improved neurological outcomes. These data
indicate an important role for TRPM7 in neuronal damage
following I/R injury and suggest that block of the channel
after stroke may be neuroprotective.
D. Pulmonary Edema
Pulmonary edema can result from congestive heart failure,
ventilator-induced mechanical injury, prolonged exposure
to high altitude, and other causes. Several studies have demonstrated that TRPV4 channels are involved in the development of pulmonary edema. A study by Hamanaka et al.
(127) showed that ventilation-induced lung injury was diminished in lungs from TRPV4⫺/⫺ mice compared with
controls, suggesting that TRPV4 channel activity contrib-
utes to mechanically induced vascular injury in the lung.
The authors proposed that stretch-induced activation of
TRPV4 contributes to this process. This report also demonstrated that infusion of macrophages isolated from wildtype mice into lungs isolated from TRPV4⫺/⫺ mice restored
the permeability response to high ventilation pressure, suggesting that mechanical activation of TRPV4 channels present in macrophages is responsible for high pressure injury
(126). Jian et al. (149) showed that elevated pulmonary
endothelial permeability resulting from high vascular pressures was diminished in lungs from TRPV4⫺/⫺ mice compared with controls. This response was blocked by inhibition of CYP activity, suggesting that biosynthesis of EETs
during exposure to high pressures induces activation of
TRPV4 channels in the pulmonary microvascular endothelium. Ventilator-induced lung edema was blocked by inhalation of nanoparticles that release the nonselective TRPV
blocker ruthenium red (155), further implicating TRPV4
channels in this response. An intriguing translational study
by Thorneloe et al. (350) demonstrated that novel orally
active TRPV4 blockers can prevent and resolve pulmonary
edema associated with heart failure in rats, suggesting that
block of TRPV4 channels may have important therapeutic
effects. TRPV4 blockade has also been shown to prevent
lung injury in mice exposed to acid and chlorine gas (23).
E. Neurogenic Inflammation
Neurogenic inflammation is characterized by vasodilation
and edema that results in redness, warmth, hypersensitivity,
and swelling. This condition is initiated by substances released from small-diameter sensory neurons, such as substance P, CGRP, glutamate, and prostaglandins, that act on
endothelial cells, mast cells, immune cells, and vascular
smooth muscle cells (284). Neurogenic inflammation is associated with many diseases, including migraine, arthritis,
asthma, inflammatory bowel disease, and chronic obstructive pulmonary disease. Several studies have implicated
TRPV1 and TRPA1 channels in neurogenic inflammation
(for review, see Refs. 20, 90).
1. TRPV1
Many studies have demonstrated that TRPV1 channels are
involved in neuropathic pain and neurogenic inflammation
(for review, see Ref. 111). Among these studies is a report
by Ji et al. (148) demonstrating that TRPV1 protein levels
were elevated by inflammatory stimuli in dorsal root ganglion (DRG) neurons, resulting in hypersensitivity to heat.
Genetic models have been used to show that mustard oil
(25) and capsaicin (356) induce significantly less inflammation in TRPV1⫺/⫺ mice than in wild-type mice. The signaling pathways responsible for TRPV1 channel activation by
inflammatory mediators have been extensively characterized and include sensitization mediated by PLC-, PKA-, and
PKC-dependent signaling cascades (270). There is consid-
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SCOTT EARLEY AND JOSEPH E. BRAYDEN
erable interest in TRPV1 blockade and desensitization as a
potential treatment for chronic pain and neurogenic inflammation (233, 343).
2. TRPA1
TRPA1 channels are often coexpressed with TRPV1 in primary sensory neurons and may play an important role in
the vascular component of neurogenic inflammation. This
possibility was first suggested by Trevisani et al. (361), who
showed that the TRPA1 agonist 4-HNE caused edema
when injected into the hind paws of rats. Additional evidence for an inflammatory role for TRPA1 was provided by
a study demonstrating that, in guinea pigs, tracheal plasma
extravasation resulting from cigarette smoke inhalation
was attenuated by inhibition of TRPA1 with HC-030031
(18). Furthermore, increases in capillary permeability were
absent in TRPA1⫺/⫺ mice administered an aqueous extract
of cigarette smoke (18). N-acetyl-p-benzo-quinoneimine
(NAPQI), a metabolite produced following administration
of large doses of acetaminophen, was shown to activate
TRPA1 channels and cause an extravasation of tracheal
plasma in rats and mice that was sensitive to HC-030031
and TRPA1 knockout (244). NAPQI application also induced an increase in plasma protein extravasation in the
skin conjunctiva of rats and mice that was attenuated by
TRPA1 blockade or gene knockout (244). These findings
suggest that inhibitors of TRPA1 may be useful for treating
increased capillary permeability and edema associated with
certain inflammatory conditions.
V. SUMMARY AND CONCLUSIONS
TRP channels are critically involved in normal and pathophysiological responses in the vasculature. Strong evidence
demonstrates that TRP channels contribute to mechanicaland agonist-induced vascular smooth muscle contractility
and proliferation. TRP channels present in endothelial cells
contribute to endothelium-dependent vasodilation, regulation of vascular permeability, and angiogenesis. TRP channels in other cell types, such as perivascular neurons and
parenchymal astrocytes, are also important in the regulation of vascular function, primarily through the control of
vascular tone. The primary regulatory paradigm for most of
these functions is the control of global [Ca2⫹]i or the propagation of subcellular Ca2⫹ signaling events that modulate
cellular activity. These effects can result directly from TRP
channel-mediated Ca2⫹ influx; can be secondary to cation
influx-induced membrane depolarization or activation of
GPCRs; or can be caused by CICR from internal sources.
Compelling evidence supports substantial roles for TRP
channels in vascular disease states such as hypertension,
neointimal injury, oxidative stress response, neurogenic inflammation, and pulmonary edema. These disease mechanisms involve increased expression or activation of TRP
channels leading to elevated contractility, vascular hyper-
678
trophy, hyperplasia, or other forms of remodeling within
the vascular wall. In light of these findings, selective pharmacological blockers of TRP channels may present exciting
opportunities for the development of new therapies to prevent and treat vascular-related diseases.
Considerable work is needed to more fully understand the
structural, functional, and mechanistic aspects of vascular
TRP channel biology. In particular, important features of
the organization and interactions between closely related,
colocalized TRP channels and other signaling modalities in
vascular cells remain largely unknown. Several recent studies have highlighted the importance of such signaling networks in native smooth muscle and endothelial cells, and
further advances in this area will greatly enrich our understanding of vascular function. Proteomic analyses and super-resolution imaging approaches may be useful in providing a more complete understanding of the interactions between TRP channels and cellular elements, such as the
cytoskeleton and membrane phospholipids. Addressing
controversies surrounding the relative fractional Ca2⫹ composition of TRP currents in vivo will require additional
efforts to record TRP currents from native vascular cells
under physiological ionic gradients. Such experiments may
help to resolve controversies surrounding the role of TRP
channels in SOCE. Successful completion of these studies
will likely require the development of a more extensive array of selective pharmacological tools for modifying TRP
channel activity, such as those that are currently available
for probing TRPV4 channel function. In addition, little is
currently known about how TRP channel gene expression is
regulated under normal conditions, during development, or
during disease. Finally, advanced genetic approaches, including the development of cell-specific, conditional knockouts targeted to the vasculature, genetically encoded indicators, and the application of optogenetic techniques to
modulate channel activity in specifically targeted locations,
are needed to address deeper questions regarding the physiological significance of TRP channels. These new approaches will allow a more comprehensive appreciation of
the molecular and cellular complexity of TRP channels in
the vasculature, providing substantial insight into fundamental mechanisms of vascular physiology and pathophysiology.
ACKNOWLEDGMENTS
We thank Drs. Thomas A. Longden and Fabrice Dabertrand, Michelle N. Sullivan, and Yao Li for critical comments on the manuscript and Dr. David Hill-Eubanks for
editorial assistance.
Address for reprint requests and other correspondence: J. E.
Brayden, Dept. of Pharmacology, Univ. of Vermont College
of Medicine, 89 Beaumont Ave., Burlington, VT 05405
(e-mail: Joe.Brayden@uvm.edu).
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TRANSIENT RECEPTOR POTENTIAL CHANNELS IN THE VASCULATURE
GRANTS
This work was supported by National Heart, Lung, and
Blood Institute Grants R01HL091905 (to S. Earley) and
P01HL095488 (to J. E. Brayden).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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