Am J Physiol Renal Physiol 296: F1464–F1476, 2009.
First published February 25, 2009; doi:10.1152/ajprenal.90542.2008.
Attenuated, flow-induced ATP release contributes to absence
of flow-sensitive, purinergic Ca2⫹
signaling in human ADPKD
i
cyst epithelial cells
Chang Xu,1,2 Boris E. Shmukler,1,2 Katherine Nishimura,1,2 Elzbieta Kaczmarek,3 Sandro Rossetti,4
Peter C. Harris,4 Angela Wandinger-Ness,5 Robert L. Bacallao,6 and Seth L. Alper1,2
1
Molecular and Vascular Medicine and Renal Divisions, Beth Israel Deaconess Medical Center and Departments
of 2Medicine and 3Surgery, Harvard Medical School, Boston, Massachusetts; 4Departments of Medicine and Biochemistry,
Mayo Medical School, Rochester, Minnesota; 5Department of Pathology, University of New Mexico School of Medicine,
Albuquerque, New Mexico; and 6Department of Medicine, University of Indiana School of Medicine, Indianapolis, Indiana
Xu C, Shmukler BE, Nishimura K, Kaczmarek E, Rossetti S,
Harris PC, Wandinger-Ness A, Bacallao RL, Alper SL. Attenuated,
flow-induced ATP release contributes to absence of flow-sensitive, purisignaling in human ADPKD cyst epithelial cells. Am J
nergic Ca2⫹
i
Physiol Renal Physiol 296: F1464 –F1476, 2009. First published February 25, 2009; doi:10.1152/ajprenal.90542.2008.—Flow-induced cytososignaling in renal tubular epithelial cells is mediated in
lic Ca2⫹ Ca2⫹
i
part through P2 receptor (P2R) activation by locally released ATP.
The ability of P2R to regulate salt and water reabsorption has
suggested a possible contribution of ATP release and paracrine P2R
activation to cystogenesis and/or enlargement in autosomal dominant
polycystic kidney disease (ADPKD). We and others have demonstrated in human ADPKD cyst cells the absence of flow-induced Ca2⫹
i
signaling exhibited by normal renal epithelial cells. We now extend
these findings to primary and telomerase-immortalized normal and
ADPKD epithelial cells of different genotype and of both proximal
and distal origins. Flow-induced elevation of Ca2⫹
concentration
i
([Ca2⫹]i) was absent from ADPKD cyst cells, but in normal cells was
mediated by flow-sensitive ATP release and paracrine P2R activation,
modulated by ecto-nucleotidase activity, and abrogated by P2R inhibition or extracellular ATP hydrolysis. In contrast to the elevated ATP
release from ADPKD cells in static isotonic conditions or in hypotonic conditions, flow-induced ATP release from cyst cells was lower
than from normal cells. Extracellular ATP rapidly reduced thapsigargin-elevated [Ca2⫹]i in both ADPKD cyst and normal cells, but cyst
cells lacked the subsequent, slow, oxidized ATP-sensitive [Ca2⫹]i
recovery present in normal cells. Telomerase-immortalized cyst cells
also exhibited altered CD39 and P2X7 mRNA levels. Thus the loss of
flow-induced, P2R-mediated Ca2⫹
signaling in human ADPKD cyst
i
epithelial cells was accompanied by reduced flow-sensitive ATP
release, altered purinergic regulation of store-operated Ca2⫹ entry,
and altered expression of gene products controlling extracellular
nucleotide signaling.
autosomal dominant polycystic kidney disease; telomerase; monocilium; shear stress; luciferase; fura 2
AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is
characterized by progressive enlargement of fluid-filled cysts
originating from only 1–5% of nephrons. The disease affects
between 1:400 and 1:1,000 people, and leads to end-stage renal
disease in half of those affected. Between 70 and 85% of cases
are caused by mutations in the PKD1 gene encoding the 4,302
Address for reprint requests and other correspondence: S. L. Alper, Molecular and Vascular Medicine and Renal Divs., Beth Israel Deaconess Medical
Center, 330 Brookline Ave., E/RW763, Boston, MA 02215 (e-mail: salper
@bidmc.harvard.edu).
F1464
amino acid (aa) protein polycystin-1 (PC1). PC1 includes a
⬃3,000 aa NH2-terminal extracellular domain, ⬃ 11 transmembrane spans, and a ⬃200 aa COOH-terminal cytoplasmic
tail. Most or all other cases of ADPKD are caused by mutations
in the 968 aa polycystin-2 (PC2) (52), a cation channel interacting with and regulated by PC1, and forming heteromers
with the TRPV4 and TRPC1 cation channels (1, 24, 53).
ATP release is a property shared by most cell types subjected to mechanical deformation (10, 41, 55). Epithelial
monolayers of renal, respiratory, and gastrointestinal origin
release ATP from both apical and basolateral surfaces (14, 20,
22, 28). The released nucleotides achieve concentrations sufficient to activate purinergic receptors, in turn regulating secretion and reabsorption of ions and water (26, 27, 35, 55).
These effects of ATP on solute and water transport have
encouraged the hypothesis that ATP release from cyst epithelial cells regulates cyst enlargement and possibly also cystogenesis in ADPKD (48, 60).
ADPKD cyst epithelial cells grown on permeable supports
in primary culture released ATP at higher rates at rest and
under hypotonic stress than did cells isolated from normal
kidneys (60). ATP release can be detected in both apical and
basolateral compartments. Cyst epithelial cells express P2X
and P2Y receptors, which, upon activation, elevated cytosolic
Ca2⫹ concentration ([Ca2⫹]i) and stimulated Cl⫺ secretion by
confluent cyst cell monolayers. Cysts microdissected from
ADPKD kidneys also released ATP, and cyst fluid contained as
much as 10 ␮M ATP, suggesting that ATP release from cyst
epithelial cells might regulate cyst enlargement in ADPKD
(48, 60). However, purinergic signaling may produce opposing
effects on models of dominant and recessive cystic kidney
disease (18, 54), and the role of purinergic signaling in early
cystogenesis remains unclear.
signaling in epithelial monolayers
Flow stimulates Ca2⫹
i
(42) by mechanisms proposed to include bending of the primary cilium (40, 43). Flow also regulates [Ca2⫹]i in intact
renal tubules (14, 22, 27), contributing to modulation of ion
and water transport in response to changes in luminal solute
load and/or concentration (47, 55). The flow-induced Ca2⫹
i
signal evident in primary renal cortical epithelial cell monolayers isolated from mouse (33) and human kidney (34, 63)
was absent from Pkd1⫺/⫺ mouse embryonic collecting duct
epithelial cells or cells isolated from human ADPKD cysts
expressing lectin markers of nominal distal or proximal origin
(33, 34, 63). Isolated, perfused collecting ducts from the
0363-6127/09 $8.00 Copyright © 2009 the American Physiological Society
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Submitted 11 September 2008; accepted in final form 24 February 2009
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
METHODS
Cell culture. Discarded portions of human kidney cortex were
harvested according to CCI/IRB protocols reviewed and approved at
the Indiana University School of Medicine and Beth Israel Deaconess
Medical Center. NK cortical tubular epithelial cells from individual
NK57M03 and ADPKD cyst cells from an anonymized ADPKD
patient undergoing nephrectomy were grown in primary culture as
described (63). Aliquots of NK cells from the same individual
(NK57M03) and aliquots of ADPKD cyst cells from a different,
anonymized ADPKD patient kidney were similarly grown in primary
culture. These pools of primary cells were referred to as NK and PKD
cells (Supplemental Table 1; all supplementary material for this article
are accessible on the journal web site).
AJP-Renal Physiol • VOL
Primary NK and PKD cells were subjected to fluorescence-activated
cell sorting (FACS). Cells were separated according to intensity of the
bound fluorophore-conjugated lectins Lotus tetragonolobus agglutinin
(LTA; a marker of proximal nephron origin) and Dolichos biflorus
agglutinin (DBA; a marker of distal nephron origin; Vector Laboratories,
Burlingame, CA) (6, 49, 63). Marker-enriched, sorted cell pools were
transformed with human telomerase (hTERT) cDNA as previously described (5, 65). The EcoRI fragment from pGRN145 encoding hTERT
cDNA was subcloned into retroviral vector pBABEneo. Recombinant
viruses were generated by electroporation-transfection of the ecotropic
packaging line PE501 followed by selection with 400 ␮g/ml G418.
Supernatants containing amphotropic virus released from confluent cultures were filtered through 0.45-␮m filters. This virus preparation was
used to infect NK and PKD cells, which were clonally selected in G418
and expanded.
Cloned cells were maintained in Clonetics REGM Renal Epithelial
Cell Medium (Lonza, Portsmouth, NH) in the presence of penicillin/
streptomycin and G418. After seven passages, G418 was removed
from the growth medium. Cells for experiments were transferred to
glass coverslips coated with Vitrogen collagen (Conhesion Technologies, Palo Alto, CA), fed every other day with REGM, and grown to
confluence 5– 6 days after plating, at which time they were used for
experiments. Primary cells were used through passage 6 only. The
hTERT-transformed cells were passaged weekly and maintained a
stable growth rate and phenotype through passage 34 and beyond
(Bacallao R, unpublished observations). These hTERT-immortalized
cell lines were referred to as LTA⫹-NKTERT, DBA⫹-NKTERT,
LTA⫹-PKDTERT, and DBA⫹-PKDTERT (Supplemental Table 1).
Genomic DNA analysis. Genomic DNA was extracted from LTA⫹PKDTERT cells by the salting-out method. All coding exons and splice
junctions of the PKD1 and PKD2 genes were amplified by PCR from
genomic DNA (46, 63) and directly sequenced on both strands. The
duplicated region of the PKD1 gene was amplified with rTth DNA
polymerase (PerkinElmer Applied Biosystems, Foster City, CA) in
the supplied DMSO-containing buffer. Primers were either anchored
in the single-copy region of the gene or mismatched with the homologous gene sequences to generate five, locus-specific Long Range
PCR fragments of 3–9 kb in length. The DNA sequencing allowed
identification of heterozygous variation in gene sequence. Any mutation found was confirmed with a second, independent amplification.
RNA preparation and RT-PCR. Total RNA from NK and PKD cyst
cells was isolated with an RNeasy kit (Qiagen, Valencia, CA). Human
kidney total RNA was purchased from Ambion (Woodlands, TX).
Reverse transcription was performed with a Retroscript first-strand
cDNA synthesis kit (Ambion) using 1 ␮g of total RNA. Of the
reaction volume, 5% was used for PCR with HotStart DNA polymerase (Qiagen) in a total reaction volume of 50 ␮l in the supplier’s
recommended buffer. cDNA fragments were amplified using specific
primer pairs and annealing conditions for P2Y and P2X receptor
products (Supplemental Table 2) and for CD39 family ecto-nucleoside triphosphate diphosphohydrolase products (NTPDases; Supplemental Table 3). Enzyme activation and initial template denaturation
were at 95°C for 15 min, followed by 35–38 cycles of 45 s at 94°C,
2 min at 52– 60°C, and 2 min at 72°C, with a 7-min final extension
step at 72°C. PCR products were separated on 1% agarose gels,
visualized with ethidium bromide, purified with a Qiagen Gel Extraction kit, and validated by DNA sequence analysis. PCR product
abundance was documented (GelDoc, Bio-Rad) and semiquantitated
with ImageJ (National Institutes of Health). PCR cycle number was
adjusted to ensure detection of low-abundance transcripts and to
maintain higher abundance transcripts within the log-linear range of
amplification.
Confocal immunofluorescence microscopy. Cell monolayers grown
on glass coverslips were fixed for 30 min at room temperature with
140 mM NaCl and 10 mM sodium phosphate, pH 7.4 (PBS) containing 3% (wt/vol) paraformaldehyde. Fixed cells were extensively
rinsed with PBS, quenched with 50 mM lysine HCl, pH 8.0, exposed
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orpk/orpk mouse model of recessive polycystic kidney disease exhibited attenuated flow-induced [Ca2⫹]i signaling
apparent by postnatal day 14 (29). Reduced or absent
flow-induced [Ca2⫹]i signaling also characterized primary
embryonic orpk/orpk collecting duct epithelial monolayers,
albeit accompanied by increased resting rates of apical Ca2⫹
entry (21, 50). In contrast, primary human ARPKD cyst
epithelial cell monolayers exhibited enhanced flow-induced
[Ca2⫹]i signaling (44).
Since flow stimulates both ATP release and elevation of
[Ca2⫹]i, the relationship between these two events is of interest. Flow-stimulated elevation of [Ca2⫹]i in isolated, perfused
mouse medullary thick ascending limb (MTAL) was attenuated
by P2R blockade and by extracellular ATP hydrolysis (22), and
was reduced in MTAL from P2y2⫺/⫺ mice (22). P2R inhibition and extracellular ATP hydrolysis reduced spontaneous
[Ca2⫹]i oscillations in resting Madin-Darby canine kidney
(MDCK) monolayers and in perfused mouse MTAL. Spontaneous [Ca2⫹]i oscillation amplitude was also reduced in amplitude in a P2R-deficient cell line and in the perfused P2y2⫺/⫺
MTAL (14). P2R inhibition and ATP hydrolysis nearly abolished flow-induced elevation of [Ca2⫹ ]i in established human
and rat cholangiocyte cell lines (62).
We therefore tested the hypotheses that the normal flowinduced elevation of [Ca2⫹]i that is deficient in primary human
ADPKD cyst epithelial cells is mediated by paracrine purinergic signaling, and that defective purinergic signaling might
explain in part the loss of flow-sensitive Ca2⫹
elevation in
i
ADPKD cells.
We found that flow-induced Ca2⫹
signaling of primary
i
human epithelial cells is preserved in telomerase-immortalized
normal kidney epithelial (NK cell lines), and its absence in
ADPKD cyst cells is preserved in telomerase-immortalized cell
lines of defined germline PKD1 genotype. ADPKD cyst cells
exhibited enhanced resting and hypotonicity-induced ATP release. In contrast, flow-induced ATP release from ADPKD cyst
cells was markedly reduced compared with that of normal renal
epithelial cells, despite near-normal nucleotide-induced peak
[Ca2⫹]i elevations in cyst cells. Purinergic control of [Ca2⫹]i in
thapsigargin-pretreated cells was also altered in ADPKD cyst
cells, and thapsigargin-sensitive stores were reduced. Variations in P2R and ecto-nucleotidase gene expression by cyst cell
lines were also detected. We conclude that loss of flowsensitive Ca2⫹
signaling in human ADPKD cyst epithelial cells
i
is due in part to reduced flow-sensitive ATP release and is
accompanied by altered purinergic responsiveness in the setting of nominal store depletion.
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F1466
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
AJP-Renal Physiol • VOL
during the 15-min preincubation period before the initiation of flow).
At both 0.75 dyne/cm2 (flow of 320 ␮l/min) and 10 dyne/cm2 (flow of
5,000 ␮l/min), the collection period was 1 min.
Tonicity-regulated ATP release was measured after 30-min exposure of 5 ⫻ 105 confluent cells in 35-cm culture dishes to isotonic
Hanks’ solution or to a hypotonic 4:6 vol/vol mixture of Hanks’
solution and distilled water, with a relative osmolarity of 40%. ATP
concentration was measured by luciferin-luciferase assay (Sigma).
Duplicate 100-␮l aliquots of perfusate or incubation medium were
added to glass tubes containing 100 ␮l ATP assay mix dilution buffer
(25-fold dilution), and luminescence intensity was measured (Lumat
LB 9507, Berthold, Oak Ridge, TN). Flow-induced ATP release from
5 ⫻ 104 cells was expressed as picomoles per minute. Tonicityregulated ATP release from 5 ⫻ 105 cells during a 30-min period was
expressed as picomoles. Identity of the ATP-associated luciferase
signal was confirmed by its complete abolition after apyrase treatment.
ATP concentrations of samples were extrapolated from a calibration curve constructed with ATP standards in isotonic Hanks’ solution. Although chloride can inhibit luciferase (8, 19), several subsequent studies of ATP release by renal and respiratory epithelial cells
exposed to low [Cl⫺] hypotonic solutions have shown luciferase light
emission across this range of [Cl⫺] to not differ significantly (21, 45,
51, 60) or to differ by ⬍10% (35).
Statistics. Data are presented as means ⫾ SE for n measurements.
Means of the data were compared by Student’s two-tailed unpaired
t-test, with P ⬍ 0.05 defined as the threshold for significance.
RESULTS
Shear stress elevates [Ca2⫹]i in both primary and telomerase-immortalized NK epithelial cells. We showed previously
that confluent primary human NK cells expressing predominantly the proximal nephron marker LTA exhibit flowsensitive Ca2⫹
signaling, whereas predominantly LTA⫹
i
primary ADPKD cyst cells harboring the germline mutation
⌬L2433 lack this response (63). Previous reports have
suggested that shear sensitivity of human and mouse NK
cells expressing the distal marker DBA (33, 34) is higher
than that of human primary LTA⫹-NK cells (63). We
therefore generated and studied hTERT-immortalized clonal
isolates of NK and ADPKD cyst cells of nominal proximal
origin (LTA⫹) and distal origin (DBA⫹). The latter are
shown in Supplemental Fig. 1.
LTA⫹-PKDTERT cells harbor the heterozygous C-to-T transition at nucleotide 12010 of PKD1 cDNA (counting A of the ATG
imitator codon as ⫹1), converting amino acid residue Q4004 of
polycystin-1 (PC1) to a nonsense codon (Q4004X; Supplemental
Fig. 2). The previously reported PKD1 Q4004X mutation (39)
encodes a polypeptide lacking the COOH-terminal 300 aa of PC1,
a region encompassing terminal transmembrane spans and the
COOH-terminal cytoplasmic tail. No mutation was found in the
coding region of the PKD2 gene of LTA⫹-PKDTERT cells.
LTA⫹-NKTERT cells subjected to superfusion at low (Fig. 1A,
0.75 dyne/cm2) and high (Fig. 1C, 10 dyne/cm2) levels of shear
stress transiently elevated [Ca2⫹]i, whereas LTA⫹-PKDTERT
cyst cells lines failed to elevate [Ca2⫹]i in response to superfusion
at either low (Fig. 1B) or high (Fig. 1D) shear stress. Similar
differences between LTA⫹-NKTERT and LTA⫹-PKDTERT cells
were observed at shear stress levels of 0.32 and 2.3 dyne/cm2
(not shown). [Ca2⫹]i elevation in NK cells remained at a
submaximal plateau value in the presence of higher shear stress
(Fig. 1C). Thus hTERT immortalization and cell cloning did
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to 1% SDS in PBS for 15 min for permeabilization and epitope
unmasking, and blocked for 15 min in PBS with 1% bovine serum
albumin and 0.05% saponin. For lectin staining, cells were incubated
for 2 h with FITC-conjugated LTA or with FITC-conjugated DBA,
then washed. For PC1 and PC2 staining, after 4°C overnight incubation with 1:100 dilutions of rabbit polyclonal antibody NM005 against
human PC1 COOH-terminal aa 4070 – 4302 or rabbit polyclonal
antibody against GST fusion protein with human PC2 C-terminal aa
687–968, coverslips were incubated with Cy3-conjugated donkey
anti-rabbit Ig secondary antibody (1:500, Jackson ImmunoResearch,
West Grove, PA) for 2 h at room temperature as described (63). Some
coverslips immunostained and fixed as above for PC1 or PC2 were
subsequently stained with mouse monoclonal antibody to the ciliary
marker N-acetylated ␣-tubulin (1:500, Sigma, St. Louis, MO). P2X7
immunostaining was performed by overnight incubation (1:100) with
polyclonal antibody to rat P2X7 intracellular COOH-terminal tail aa
576 –595 KIRKEFPKTQGQYSGFKYPY (Alomone, Jerusalem, Israel). Anti-CD39 immunostaining with mAb clone BU-61 (Bayport,
MN) was performed by overnight incubation of fixed cells, followed
by PBS wash and visualization with Cy3-conjugated donkey antimouse Ig secondary antibody (1:500). In some experiments as indicated, antibodies were pre- and coincubated with excess specific
peptide antigen. Immunostained cells on coverslips were imaged with
a Zeiss LSM 510 Meta confocal laser scanning microscope.
Measurement of [Ca2⫹]i. NK and PKD cells cultured to confluence
on collagen-coated 35-mm glass coverslips were loaded with 5 ␮M
fura 2-AM (Molecular Probes, Eugene, OR) in HEPES-buffered
HBSS (pH 7.4) at 37°C for 30 min. Extracellular fura 2-AM was
removed by washing twice with HEPES-HBSS. Coverslips were then
mounted into the base of a parallel-plate flow chamber 0.5 cm in width
and 0.0254 cm in depth (GlycoTech, Gaithersburg, MD) and perfused
with a Harvard Syringe pump at 37°C using a calibrated, in-line heater
(WPI, Sarasota, FL), with monitoring of inflow and outflow temperatures. Perfusion medium (Hanks’ solution) composition was (in mM)
127 NaCl, 5.4 KCl, 1.27 CaCl2, 1 MgCl2, 5.6 glucose, and 11.6
HEPES, final pH 7.4. [Ca2⫹]i was measured by fluorescence ratio
imaging with a Metafluor digital imaging system (Universal Imaging,
West Chester, PA) equipped with an Olympus IMT-2 inverted microscope, and a CoolSNAP CCD camera (Photometrics, Tucson, AZ).
Fura 2 emission images were recorded at 510 nm during alternating
excitation at 340 and 380 nm. Fura 2 fluorescence ratio values
determined by in situ calibration in immortalized epithelial cells did
not differ from values determined by in vitro calibration for [Ca2⫹]i,
and so were calibrated in vitro with the same experimental settings for
the imaging system, using a Ca2⫹ Calibration Kit No. 2 (Molecular
Probes) with concentrations between 36 nM and 4 ␮M (32). The
minimal fluorescence ratio (Rmin) was determined at “zero Ca2⫹”
(⬍10 nM) and the maximal fluorescence ratio (Rmax) at 4 ␮M total
Ca2⫹. The equilibrium constant (Kb) was determined by fitting experimental fluorescence ratio R values at various free [Ca2⫹] with the
equation [Ca2⫹]free ⫽ Kb (Sf2/Sb2)[(R ⫺ Rmin)/(Rmax ⫺ R)], where
the factor Sf2/Sb2 corrects for fura 2 ion selectivity at 380 nm. For
each coverslip, one visual field was selected as a region of interest,
recorded before and during imposition of a uniform rate of fluid flow.
Shear stress ␶␻ was calculated as ␶␻ ⫽ 6␮Q/a2b, where ␮ ⫽ apparent
viscosity of superfusate (1.00 for H2O at 20°C, 0.70 at 37°C), Q ⫽
volumetric flow rate (ml/s), and a and b ⫽ flow chamber depth and
width, respectively.
Measurement of ATP release. To measure flow-induced ATP
release, confluent monolayers of 5 ⫻ 104 NK or PKD cells mounted
in the Glycotech chamber were subjected to defined laminar flow rates
at 37°C as described above. Perfusate volumes of at least 200 ␮l were
collected at a single time for each flow rate. At 0.1 dyne/cm2 (flow of
50 ␮l/min), the collection period was 6 min (including 2 min to fill the
100 ␮l of initially empty postchamber dead space at the start of flow,
followed by 4 min to collect 200 ␮l containing ATP released under
flow, in addition to that accumulated in the 25-␮l chamber volume
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
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not change the flow-sensitivity phenotypes of either LTA⫹-NK
or LTA⫹-PKD cells.
DBA⫹-NKTERT cells also elevated [Ca2⫹]i in response to
low levels of shear stress (Fig. 2A), with a peak response at
lower shear stress (2.3 dyne/cm2) than that exhibited by primary (63) and by LTA⫹-NKTERT cells at 10 dyne/cm2 (Fig. 1).
In contrast, [Ca2⫹]i in DBA⫹-PKDTERT cells was unresponsive
to either low (Fig. 2B) or high levels (Fig. 2D) of shear stress.
As was true for primary NK and ⌬L2433 PKD cells (63),
resting [Ca2⫹]i did not differ between LTA⫹-NKTERT and
Q4004X LTA⫹-PKDTERT cyst cells (not shown). Thus the loss
of flow-induced [Ca2⫹]i elevation characterizes primary and
immortalized ADPKD cyst cells of distinct nephron segment
origin, and bearing distinct heterozygous germline mutations.
AJP-Renal Physiol • VOL
LTA⫹-PKDTERT cells form primary cilia lacking PC1.
Among their multiple sites of subcellular localization (25),
PC1 and PC2 are expressed along with many other cystic
kidney disease gene products in the primary cilium (33, 34,
63). Figure 3 shows PC1 (Fig. 3A, left) and PC2 (Fig. 3A, right)
in the cilium of LTA⫹-NKTERT cells. However, PC1 was
undetectable (Fig. 3B, left) and PC2 nearly so (Fig. 3B, right)
in LTA⫹-PKDTERT cell cilia 5.0 ⫾ 0.2 ␮m in length (n ⫽ 11),
indistinguishable from the 4.8 ⫾ 0.3-␮m (n ⫽ 19) length of
LTA⫹-NKTERT cilia. Ciliary PC2 localization in the x-y plane
is shown in Supplemental Fig. 3. DBA⫹-NKTERT cell cilia of
8.7⫹0.4 ␮M in length (n ⫽ 21), and the slightly shorter
DBA⫹-PKDTERT cilia of 7.4 ⫾ 0.3 ␮M in length (n ⫽ 25; P ⬍
0.05 compared with NK) each were significantly longer than
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Fig. 1. Human telomerase (hTERT)-immortalized, clonal Lotus tetragonolobus agglutinin (LTA⫹) autosomal dominant polycystic kidney disease (ADPKD) cyst
cells (LTA⫹-PKDTERT) lack the flow-sensitive elevation in cytosolic Ca2⫹ concentration ([Ca2⫹]i) exhibited by LTA⫹-NKTERT cells, where NK refers to normal
kidney. Imposition of laminar flow at t ⫽ 0 increases [Ca2⫹]i in LTA⫹ NKTERT cells at low (0.75 dyne/cm2; A) and high levels of shear stress (10 dyne/cm2;
C). In contrast, laminar flow fails to increase [Ca2⫹]i in LTA⫹-PKDTERT cells in response to shear stress at low (B) or high levels (D). Values are means ⫾ SE
for (n) coverslips, with fura 2 ratio recorded from a single ⫻20 visual field encompassing 40 – 60 confluent cells on each coverslip.
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PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
LTA⫹-TERT cells (P ⬍ 0.01). Thus clonal PKDTERT cells
heterozygous for the PC1 germline mutation Q4004X resemble
mixed primary PKD cyst cells with the PC1 germline mutation
⌬L2433 in their absence of detectable ciliary PC1, but they are
more severely impaired in ciliary PC2 localization than were
⌬L2433 primary cyst cells, 30% of which expressed apparently
normal ciliary levels of PC2 (63). Extraciliary localization of
PC1 and PC2 in NKTERT and ADPKDTERT cells did not differ
(Supplemental Fig. 4).
Shear stress-induced net ATP release is selectively reduced
in PKD cyst cells compared with NK cells. NKTERT cells
released ATP in response to various uncontrolled types of
mechanical stimulation (not shown), including complete reAJP-Renal Physiol • VOL
placement of medium, tilting of the monolayer, and touching of
the cell monolayer surface with a glass pipette, as reported with
other cell types (16, 59). Although slow superfusion at 0.1
dyne/cm2 produced little net ATP release into the medium, the
net ATP release at this low flow rate from primary ⌬L2433
cells (Fig. 4A) and from Q4004X PKDTERT cells (Fig. 4, B and
C) exceeded that from NK (Fig. 4A) or NKTERT cells (Fig. 4,
B and C). This elevated net ATP release was especially marked
in DBA⫹-PKDTERT cells (Fig. 4C), consistent with previous
reports of elevated resting net ATP release by primary ADPKD
cyst cells (48). Low (0.75 dyne/cm2) and high flow (10 dyne/
cm2) elicited higher magnitudes of net ATP release from
primary NK and PKD cells (Fig. 4A) and from LTA⫹ (Fig.
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Fig. 2. hTERT-immortalized, clonal Dolichos biflorus agglutinin (DBA⫹) ADPKD cyst cells (DBA⫹-PKDTERT) lack the flow-sensitive elevation in [Ca2⫹]i
exhibited by DBA⫹-NKTERT cells. Imposition of laminar flow at t ⫽ 0 increases [Ca2⫹]i in DBA⫹-NKTERT cells at low levels of shear stress (0.75 or 2.3
dyne/cm2; A). Higher shear stress (10 dyne/cm2; C) slightly reduces the magnitude of peak [Ca2⫹]i, but prolongs peak duration. In contrast, laminar flow fails
to increase [Ca2⫹]i in DBA⫹-PKDTERT cyst cells in response to shear stress at low (B) or high levels (D). Values are means ⫾ SE for (n) coverslips, with 40 – 60
confluent cells/⫻20 visual field on each coverslip.
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
F1469
4B)-or DBA⫹ (Fig. 4C)-NKTERT and PKDTERT cells. Primary
⌬L2433 ADPKD cyst cells (Fig. 4A) and Q4004X LTA⫹-PKD
cells (Fig. 4B) released substantially less net ATP in response
to 10 dyne/cm2 shear stress than did the corresponding NK
cells (P ⬍ 0.05). Q4004X DBA⫹-PKD cells (Fig. 4C) released
less net ATP in response to 2.3 dyne/cm2 shear stress than did
NK cells. In contrast, primary ⌬L2433 PKD cyst cells at rest in
isotonic and hypotonic conditions released significantly more
net ATP than did primary NK cells (Fig. 4D; P ⬍ 0.05).
Apyrase treatment of the collected perfusates abolished the
luciferase signal, confirming its source as released ATP (n ⫽ 3,
data not shown). The applied shear stresses and times did not
detach, loosen, or morphologically alter the cells under study,
and cessation of flow returned [Ca2⫹]i to preflow levels in all
cases. The results demonstrate a selective defect in shear
stress-induced net ATP release from both primary ⌬L2433
primary PKD cells and Q4004X PKDTERT cells.
Shear stress-induced ATP release mediates flow-induced
Ca2⫹
in NK cells. We hypothesized that flow-induced ATP
i
release might mediate shear stress-induced [Ca2⫹]i increase in
NK cells. Thus primary NK cells were pretreated with P2
receptor blockers or with the ATP hydrolase apyrase for 10
min before the onset of flow at 10 dyne/cm2 shear stress in the
continued presence of the drugs. As shown in Fig. 5A, flowinduced elevation in [Ca2⫹]i was abolished by apyrase (5
U/ml) and by the semiselective P2Y receptor antagonist
suramin (300 ␮M). Figure 5B shows that the semiselective
P2X receptor antagonist PPADS (100 ␮M) also abolished the
flow-induced [Ca2⫹]i increase. Suramin also greatly reduced
flow-induced [Ca2⫹]i elevation in LTA⫹-NKTERT cells (Fig.
5C) and abolished it in DBA⫹-NKTERT cells (Fig. 5D). Conversely, inhibition of ATP hydrolysis with the 5⬘-ecto-nucleotidase inhibitor ARL67156 (300 ␮M) prolonged the plateauphase duration of [Ca2⫹]i elevation in response to both high
and low shear stress (Fig. 6, A and B). This potentiation
suggests that released ATP is actively degraded by cellular
nucleotidase activities and that the values of net ATP released
AJP-Renal Physiol • VOL
in response to hypotonic swelling (Fig. 4D) and in response to
shear stress (Fig. 4, A–C) are underestimates. The data indicate
that released ATP is a crucial intermediate in the transduction
of a flow signal leading to elevation of [Ca2⫹]i in NK cells.
Defective flow-sensitive autocrine/paracrine ATP release by
PKD cyst cells likely contributes to their inability to elevate
[Ca2⫹]i in response to flow.
PKDTERT cyst cells exhibit intact ATP-induced [Ca2⫹]i elevation but modestly reduced [Ca2⫹]i elevation in response to
other nucleotide P2 receptor ligands. Since PKDTERT cells
lack flow-responsive Ca2⫹
signaling, and exhibit decreased
i
flow-responsive net ATP release, we tested the possibility that
ATP responsiveness was also deficient. Exogenous extracellular ATP triggered similar [Ca2⫹]i responses in primary NK and
⌬L2433 PKD cells (63). Exposure to extracellular ATP elevated
[Ca2⫹]i in both LTA⫹-NKTERT and Q4004X LTA⫹-PKDTERT
cells, and differed only in the more prolonged and stable plateau
phase of the PKDTERT cells (Fig. 7; P ⫽ 0.03 at 90 s, 0.01 at 100 s
compared with NKTERT). Nominally Ca2⫹-free medium did not
change peak ATP-induced [Ca2⫹]i in PKDTERT cells but reduced the peak by ⬃30% in NKTERT cells. Ca2⫹-free medium
also accelerated the [Ca2⫹]i decline from peak ATP-stimulated
values in both NKTERT and PKDTERT cells (Fig. 7). Similar
[Ca2⫹]i differences between cell lines and conditions were
evident at 20°C (not shown). The nucleotide-induced Ca2⫹
i
increase in primary NK cells was elicited by nucleotides in a
concentration-dependent manner with the rank order of potency UTP ⬎ ATP ⬎ ADP (Supplemental Fig. 5A) and in
⌬L2433 PKD cells UTP ⫽ ATP ⬎ ADP (Supplemental Fig.
5B). Similar peak [Ca2⫹]i elevations and rank orders of
potency in response to 10 ␮M UTP, ADP, and ATP were
measured in LTA⫹ (Suppl. Fig. 5C) and DBA⫹ (Suppl Fig.
5D) clones of NKTERT and Q4004X PKDTERT cells. These
findings are consistent with the previously reported expression of multiple functional P2X and P2Y receptor subtypes
in human primary cultures of normal and ADPKD renal
epithelia (48).
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Fig. 3. Ciliary localization of polycystins PC1
and PC2 is reduced or absent in PKDTERT
cells. Confocal x-z plane reconstructions of
LTA⫹-PKDTERT cells show undetectable ciliary PC1 (B, left) and reduced levels of ciliary
PC2 (B, right), as judged by colocalization
with N-acetylated ␣-tubulin in contrast to
the presence of PC1 (A, left) and PC2 (A,
right) in cilia of LTA⫹-NKTERT cells.
Scale bar ⫽ 10 ␮m.
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PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
P2 receptor mRNA expression in NKTERT and PKDTERT cyst
cells. The apparent role of purinergic signaling in renal epithelial cell flow sensitivity prompted RT-PCR survey screening of
P2 receptor mRNA expression in NKTERT and PKDTERT cells.
Among P2Y receptors, amplification of P2Y1, P2Y2, P2Y4,
P2Y6, P2Y11, P2Y13, and P2Y14 mRNAs yielded single
bands (Fig. 8, A and B). P2Y12 mRNA was undetectable in
NKTERT and PKDTERT cells (after 38 cycles), although present
in the human kidney (Fig. 8B). Among P2X receptors, P2X1
and P2X2 mRNAs were undetected in NKTERT and PKDTERT
cells, despite their presence in the human kidney (Fig. 8C).
Consistent with the absence of P2X1 mRNA, neither LTA⫹NKTERT nor LTA⫹-PKDTERT cells elevated [Ca2⫹]i in response to the P2X1 agonist ␤-methyl-ATP (100 ␮M; n ⫽ 3– 4,
not shown). mRNAs encoding P2X4, P2X5, P2X6, and P2X7
mRNAs were detected in NKTERT and PKDTERT cells of both
lectin types (Fig. 8, C–E).
Human kidney mRNA consistently yielded two P2X7 amplification products of similar intensity (Fig. 8E). The larger
product included intron 10 and encoded a P2X7 polypeptide
with a severely truncated COOH-terminal cytoplasmic tail
(data not shown), encoding a polypeptide retaining partial
AJP-Renal Physiol • VOL
cation channel function but unable to mediate the slower
nonspecific pore formation (3) and endowed with dominant
negative properties (2, 9). RT-PCR amplification product
bands of each P2X7 transcript were of lower intensity in
LTA⫹- and DBA⫹-PKDTERT cells than in NKTERT cells of
corresponding lectin status (Fig. 8, E and F). Two previously
reported transcript variants of P2X6 (7) were also detected, but
neither RT-PCR amplification product band varied consistently
between NKTERT and PKDTERT cells (n ⫽ 3, not shown).
P2X7 protein expression in NKTERT and PKDTERT cells.
Both DBA⫹-NKTERT (Fig. 9A) and DBA⫹-PKDTERT cells
(Fig. 9B) revealed a punctate distribution of P2X7 immunostaining at the cell periphery, with greater signal intensity in
intracellular compartments. Preadsorption of the P2X7 antibody with its peptide antigen completely abolished these signals (Fig. 9C). P2X7 staining intensity was lower in DBA⫹PKDTERT cells than in DBA⫹-NKTERT cells (Fig. 9, A and B).
Similar differences were noted between LTA⫹-NK and LTA⫹PKD cells (not shown). Thus PKDTERT cells express reduced
levels of P2X7 mRNA and polypeptide. The [Ca2⫹]i response
to ATP was examined in thapsigargin-treated DBA⫹-NKTERT
and DBA⫹-PKDTERT cells (Fig. 9E) to isolate a P2X receptor
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Fig. 4. Shear stress-induced net ATP release from ADPKD cyst cells is greater than from NK cells. Primary NK and PKD cells (5 ⫻ 104; A), LTA⫹-NKTERT
and LTA⫹-PKDTERT cells (B), or DBA⫹-NKTERT and DBA⫹-PKDTERT cells (C) were subjected to shear stress at the indicated levels. PKD cells of each type
exhibited more net ATP release than did corresponding NK cells at the lowest tested flow. In contrast, PKD cells of each type exhibited far less net ATP release
than did corresponding NK cells in response to the highest tested flow. D: hypotonic stress increased net ATP release from primary NK and primary PKD cells
(5 ⫻ 105 cells). In both isotonic and hypotonic static (no-flow) conditions, net ATP release from primary PKD cells exceeded that from primary NK cells. As
noted in the description of the luciferase assay in METHODS, the values for ATP release into hypotonic (low [Cl⫺]) medium may overestimate ATP release
compared with isotonic values by as much as 10% or more. Black bars, NK cells; gray bars, ADPKD cells. *P ⬍ 0.05 for PKD or PKDTERT cells compared
with NK or NKTERT cells in the same condition. #P ⬍ 0.05 for cells at intermediate or high shear stress compared with the same cell type at 0.1 dyne/cm2.
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
F1471
component of Ca2⫹ signaling in these cells (64). The thapsigargin-induced elevation of [Ca2⫹]i in NK cells (E) was
attenuated in PKDTERT cells (F), as observed previously in
primary PKD cells (63). NKTERT cell pretreatment with the
P2X7 antagonist oxidized ATP (O-ATP; 300 ␮M, ‚) enhanced
the rate of [Ca2⫹]i rise elicited by thapsigargin, but attenuated
the magnitude of the plateau phase. Subsequent exposure to 1
mM ATP, a concentration sufficient for near-maximal P2X7
activation, produced a biphasic effect on [Ca2⫹]i. In the first
phase, ATP acutely reduced the thapsigargin-elevated [Ca2⫹]i
Fig. 6. Ecto-nucleotidase inhibition prolongs the flow-induced elevation of [Ca2⫹]i. NK cell [Ca2⫹]i increase in response to 10 (A) or 0.75 dyne/cm2 (B) shear
stress was monitored in absence (E) or continued presence of the 5⬘-ectonucleotidase inhibitor ARL67156 (300 ␮M, F).
AJP-Renal Physiol • VOL
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Fig. 5. ATP released in response to flow mediates flow-induced Ca2⫹
signaling by NK cells. A: primary NK cell [Ca2⫹]i increase in response to 10 dyne/cm2
i
shear stress was monitored in absence or continued presence of the nucleotidase apyrase (5 U/ml) or the P2Y receptor antagonist suramin (300 ␮M). B: primary
NK cell [Ca2⫹]i increase in response to 10 dyne/cm2 shear stress was monitored in absence or continued presence of the P2X receptor antagonist PPADS (100
␮M). C: LTA⫹-NKTERT cell [Ca2⫹]i increase in response to 10 dyne/cm2 shear stress was monitored in the absence or continued presence of suramin. D:
DBA⫹-NKTERT cell [Ca2⫹]i increase in response to 2.3 dyne/cm2 shear stress was monitored in the absence or continued presence of suramin.
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PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
pression of CD39 gene family members encoding nucleoside
triphosphate diphosphohydrolases expressed in the kidney (11,
23, 56). Comparative RT-PCR screening (Supplemental Fig. 6)
revealed greatly reduced band intensities of CD39 RT-PCR
amplification products in both LTA⫹- and DBA⫹-PKDTERT
cells compared with lectin-matched NKTERT cells. In contrast,
band intensities of RT-PCR amplification products encoding
CD39L2, CD39L3, and CD39L4 did not differ consistently
between NK and ADPKD cells of either lectin type. Immunoreactive CD39 was less abundant in PKDTERT than in NKTERT
cells of LTA⫹ (Supplemental Fig. 7) and DBA⫹ lectin types
(not shown).
DISCUSSION
to near resting levels. In NKTERT cells, this rapid fall in [Ca2⫹]i
was followed within ⬃30 s by a slower secondary recovery
phase, during which [Ca2⫹]i regained its plateau value (Fig.
9E). The recovery phase was strongly inhibited to similar
extents by both P2X7 antagonists O-ATP and (not shown)
1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4phenylpiperazine (KN-62; 10 ␮M; n ⫽ 3). This secondary
recovery phase was severely attenuated in PKDTERT cells (Fig.
9D), a phenotype consistent with reduced P2X7 expression in
these cyst cells.
CD39 gene family mRNA expression in NKTERT and PKDTERT
cells. Since pharmacological inhibition of ecto-nucleotidase
activity markedly prolonged the plateau phase of flow-induced
[Ca2⫹]i elevation in NKTERT cells (Fig. 6), we assessed ex-
Fig. 8. RT-PCR analysis of P2 receptor mRNA expression in NK and ADPKD cells. mRNA expression of P2Y (A and B) and P2X receptor subtype mRNAs
(C–E) in human kidney (K) and in confluent 5-day cultures of LTA⫹-NK (NL) and LTA⫹-PKD (PL) was assessed by RT-PCR. D and E: P2X6 and P2X7 mRNA
levels were also assessed in DBA⫹-NK (ND) and DBA⫹-PKD cells (PD). F: summary of similar experiments, measuring intensity of both amplification products.
Values are means ⫾ SE; n ⫽ 3– 4. **P ⬍ 0.01.
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Fig. 7. NKTERT and PKDTERT cells exhibit similar [Ca2⫹]i elevation and
extracellular Ca2⫹ dependence of that elevation in response to exogenous ATP.
LTA⫹-NKTERT (filled symbols) and LTA⫹-PKDTERT cells (open symbols)
were treated at t ⫽ 0 with 10 ␮M ATP in the presence (circles) or nominal
absence of extracellular Ca2⫹ (triangles) as indicated.
This report presents several novel findings. hTERT-immortalized ADPKD cyst cells heterozygous for the germline mutation
Q4004X share the loss of normal flow-sensitive [Ca2⫹]i signaling
that characterizes primary ADPKD cyst cells heterozygous for the
distinct germline mutation ⌬L2433 (63) and SV40-transformed
cells hetero- or hemizygous for the germline mutation Q2556X
(34). hTERT-immortalized clonal NK cell lines (NKTERT) selected for expression of proximal or distal markers exhibited
slightly different flow sensitivities, but hTERT-immortalized
clonal ADPKD cyst cell lines (PKDTERT) expressing either
marker were similarly unresponsive to flow. These immortalized cyst cells possessed cilia of nearly normal length, but
lacked ciliary localization of PC1 and PC2. Despite elevated
net ATP release in resting and hypotonic conditions, primary
and immortalized ADPKD cyst cells of both lectin types
exhibited markedly reduced flow-sensitive ATP release. Released ATP acts through P2Y and P2X receptors to mediate
flow-sensitive Ca2⫹
signaling, and ecto-nucleotidase inhibition
i
prolonged the plateau phase of the flow-induced Ca2⫹
signal.
i
However, Ca2⫹
signaling amplitude in response to exogi
enously added ATP was only moderately reduced in PKDTERT
cells. These changes in cyst cells were accompanied by re-
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
F1473
duced mRNA and protein levels of P2X7 and CD39 and by a
reduction in a pharmacological index of P2X7 activity. These
observations are the first to demonstrate paracrine purinergic
mediation of flow-sensitive Ca2⫹ signaling in human NK cells
and to show deficient flow-sensitive net ATP release from
ADPKD cells.
Role of genotype and nephron segment in flow-sensitive
Ca2⫹
signaling. The present work adds to the range of defined
i
PKD1 genotypes associated with loss of flow-sensitive Ca2⫹
i
signaling to include heterozygosity for the mutation Q4004X in
addition to the previously reported heterozygosity for the
mutation ⌬L2433 (63) and heterozygosity and likely hemizygosity for the germline mutation Q2556X (34). These germline
mutations together encompass a range of total PC1 polypeptide
expression that is normal or elevated (this work and Ref. 63),
or reduced, or absent (34). They also now extend across the
gamut of primary cells, SV40-transformed cell lines, and
hTERT-immortalized cell lines. The defective flow phenotype
is present in cells of both proximal and distal origin, as judged
by lectin expression. Thus loss of flow-sensitive [Ca2⫹]i signaling has proven to be a robust phenotype of ADPKD cyst
AJP-Renal Physiol • VOL
cells in culture, thus far regardless of germline genotype or of
nephron segment of apparent origin.
The emerging picture is more complicated in recessive
cystic disease. Embryonic orpk/orpk collecting duct cells with
stunted cilia exhibited a moderate reduction of the flowinduced [Ca2⫹]i elevation observed in Tg737/polaris-rescued
cells. This reduction was noted in the context of increased
“basal” Ca2⫹ permeability and elevated apical PC2 expression
(50). Similar reduction in flow-triggered [Ca2⫹]i elevation was
evident in isolated, perfused orpk/orpk collecting ducts from
2-wk-old, but not 1-wk-old mice (29). Small interference RNA
knockdown (⬃90%) of the ARPKD gene fibrocystin produced
reductions of comparable magnitude in flow-induced [Ca2⫹]i
elevation in mIMCD3 and murine embryonic collecting duct
cells (58). In contrast, both clonal and pooled SV40-immortalized human ARPKD cyst cells responded to flow with [Ca2⫹]i
elevations twofold higher than those of immortalized collecting
duct cells from age-matched normal kidneys (44).
The reported requirements of flow-sensitive Ca2⫹
signaling
i
for extracellular Ca2⫹ entry and for Ca2⫹ release from intracellular stores have also varied. In human ADPKD cyst cells
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Fig. 9. Reduced P2X7 protein and a reduced index of P2X7 function in ADPKD cyst cells. P2X7 immunostaining in DBA⫹-NK cells (A), DBA⫹-PKD cells
(B), and DBA⫹-NK cells in the added presence of peptide antigen (C). Scale bar ⫽ 10 ␮m. D: biphasic effect of 1 mM extracellular ATP on [Ca2⫹]i in 500
nM thapsigargin-treated NKTERT cells (E) and PKDTERT cells (F) and in thapsigargin-treated NKTERT cells pretreated with and in the continued presence of the
P2X7 antagonist oxidized ATP (O-ATP; 300 ␮M, ‚).
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PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
AJP-Renal Physiol • VOL
Normal and ADPKD kidneys express a wide range of
purinergic receptor and ecto-nucleotidase mRNAs and immunoreactive polypeptides (27, 48, 55). The absence in
LTA⫹-NK and PKD cyst cells of some P2R subtype mRNAs
expressed in the kidney (Fig. 8) may reflect either axial
heterogeneity of P2R expression or alterations in gene expression
accompanying immortalization. Although ATP-responsive
[Ca2⫹]i elevation was normal or modestly reduced in NK and
ADPKD cyst cells, cyst cell responses to UTP and ADP were
reduced to greater extents (Supplemental Fig. 5). PKDTERT cells
displayed lower mRNA levels and immunostaining intensity
for P2X7 and CD39 than detected in NKTERT cells of either
lectin type (Figs. 8 and 9; Supplemental Figs. 6 and 7).
ADPKD cyst cells also exhibited a delayed and attenuated
[Ca2⫹]i recovery following the profound reduction in [Ca2⫹]i
induced by 1 mM ATP, a recovery sensitive to the P2X7
antagonists O-ATP and KN-62 (Fig. 9). Thus reductions in
flow-induced net ATP release and flow-induced, extracellular
ATP-dependent Ca2⫹
signaling in PKDTERT cells were assoi
ciated with apparent decreases in P2X7 expression and in Ca2⫹
i
signaling sensitive to P2X7 inhibitors.
The ATP-induced rapid fall in thapsigargin-elevated
[Ca2⫹]i observed in NKTERT cells has been observed in
other cell types. In thapsigargin-pretreated CFPAC-1 cells,
10 ␮M ATP rapidly reduced [Ca2⫹]i by 40% without decreasing plasmalemmal Ca2⫹ entry (as measured by Mn2⫹
quench of Ca2⫹
signal) and without increasing Ca2⫹
extrui
i
⫹
sion by either Na /Ca2⫹ exchange or vanadate-sensitive Ca2⫹ATPase (61). Similar inhibitory effects of ATP were noted in
HT-29 cells. In rat brown adipocytes, 10 ␮M ATP also rapidly
depressed thapsigargin-elevated [Ca2⫹]i by 92% (37) by a
mechanism insensitive to phorbol ester but sensitive to high
concentrations of suramin and of PPADS (36). The inhibitory
effect of ATP on thapsigargin-elevated [Ca2⫹]i correlated with
increased peripheral actin polymerization and was blocked by
pharmacological disruption of the actin cytoskeleton (38). The
mechanism of this inhibitory effect of ATP on [Ca2⫹]i in
NKTERT and PKDTERT cells, as well as in brown adipocytes,
CFPAC-1, and HT-29 cells, remains unclear. The secondary
elevation of depressed [Ca2⫹]i might be mediated by slower P2
receptor activation (or recovery from thapsigargin-associated
inactivation), with pharmacological properties suggestive of
P2X7.
Reduced expression of both P2X7 and CD39 in PKDTERT
cells suggests important roles for these proteins in normal
flow-sensitive Ca2⫹
signaling, but would be predicted to exert
i
opposing effects. Generation of adenosine by CD39 and other
ecto-nucleotidases may play an additional, potentially important role in terminating or otherwise modulating the flow
signal. However, suramin is a poor P2X7 antagonist (13), and
preliminary experiments suggest that neither P2X7 antagonist
O-ATP nor KN-62 can reproduce the inhibitory effects of
suramin and of PPADS on flow-induced Ca2⫹
signaling in
i
LTA⫹ or DBA⫹ NKTERT cells (Xu C and Alper SL, unpublished observations). In addition, primary ADPKD cyst cells
bearing the heterozygous germline mutation ⌬L2433 exhibited
increased levels of mRNA encoding P2X7 (n ⫽ 4) and CD39
(n ⫽ 2; not shown), in contrast to the decreased levels in
immortalized Q4004X ADPKD cyst cells (Fig. 8 and Supplemental Fig. 6). Thus levels of these mRNAs may vary as a
function of either genotype or immortalization state. Taken
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and in murine Pkd1⫺/⫺ embryonic kidney cells, Ca2⫹ entry
was required, and stores were ryanodine-sensitive (33, 34, 63).
MDCK cells (42) and intact or split-open rabbit collecting duct
(30) required Ca2⫹ entry, but the stores were inositol triphosphate (IP3)-sensitive. orpk/orpk tubules also required Ca2⫹
entry (29), but flow-induced elevation of [Ca2⫹]i in cell monolayers was bath Ca2⫹-independent (21). The pharmacosensitivity of flow-induced Ca2⫹
elevation reported in the orpk/orpk
i
tubule experiments did not define the nature of the Ca2⫹ stores
(29, 50). Flow-induced [Ca2⫹]i increase in the isolated, perfused mouse MTAL did not require luminal Ca2⫹ but did
require basolateral extracellular Ca2⫹ to sustain the post-peak
plateau response (22). These differences likely reflect species,
developmental stage, and differentiation state, with differing
contributions of shear stress, mechanical stretch, and hydrostatic pressure according to experimental system and geometry.
Role of ATP release in flow-sensitive Ca2⫹
signaling. The
i
selective reduction in flow-sensitive net ATP release observed
in ADPKD cyst cells (Fig. 4) suggests that it contributes to the
loss of flow-sensitive Ca2⫹
signaling in ADPKD cells. The role
i
of paracrine ATP signaling in flow-induced Ca2⫹ signaling has
been investigated in several cell types. Flow-induced [Ca2⫹]i
increase in perfused mouse MTAL was inhibited by apyrase
and suramin, mediated by P2y2 receptors on both apical and
basolateral membranes, and substantially reduced in P2y2⫺/⫺
tubules (22). P2y2 activation was also found to underlie temperature-dependent, spontaneous [Ca2⫹]i oscillations in MDCK
cells at rest (14). In orpk/orpk mouse collecting duct cells
rescued with the Tg737/polaris transgene, flow-induced elevation of [Ca2⫹]i was blocked by apyrase and suramin. In
contrast to the selective defect in flow-induced net ATP release
of ADPKD cyst cells, the unrescued orpk/orpk cells with
stubby cilia exhibited attenuated net ATP release in response to
three different stimuli: hypotonic shock, harsh pipetting, and
ionomycin (21).
Flow elevates [Ca2⫹]i in isolated rat bile duct segments and
in cholangiocytes by a cilium-dependent mechanism (31).
Flow also triggers cholangiocytes to release ATP, which mediates flow-induced Ca2⫹
elevation by paracrine P2 receptor
i
stimulation (62). P2R activation in cholangiocytes activates
anion channels in parallel with increased bicarbonate secretion
bile flow. However, flow-induced ATP release and subsequent
purinergic elevation of [Ca2⫹]i in some human cholangiocyte
cell lines was not cilium dependent (62). Thus the properties of
flow-stimulated net ATP release appear to differ among cell
types, may reflect predominant utilization of distinct ATP
release pathways, and may be differentially affected by mutations in different cystic disease genes.
Purinergic receptors and nucleotidases in flow sensing.
Intraluminal ATP concentrations in superficial proximal tubules of anesthetized rats have been estimated at 100 –300 nM,
several-fold higher than in Bowman’s space, and several-fold
higher than in superficial distal convoluted tubule (57). These
concentrations likely underestimate true juxtamembrane concentrations, in view of the high activities of both secreted and
apical membrane ecto-nucleotidases. Thus released luminal
ATP is believed to reach concentrations sufficient for activation of apical purinergic receptors (27, 55). A subset of
ADPKD cyst fluids contained ATP at the higher concentrations
of 0.5–10 ␮M (60).
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING
ACKNOWLEDGMENTS
We thank Drs. W. Junger and D. H. Friedman (Harvard) and O. Ibraghimova-Beskrovnaya (Genzyme) for antibodies and D. H. Friedman, W. Junger,
and S. A. Ness (University of New Mexico) for helpful discussions.
GRANTS
C. Xu was supported by postdoctoral fellowship F32 DK69049 from the
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
and by a postdoctoral fellowship from the Polycystic Kidney Disease Foundation. This work was additionally supported by NIDDK Grants R01DK57662 to S. L. Alper, R01 DK58816 to P. C. Harris, R01 DK50141 to A.
Wandinger-Ness, by a Polycystic Kidney Disease Foundation research grant to
R. Bacallao, and by Shared Instrument Grant S10-RR017927 to Beth Israel
Deaconess Medical Center.
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38.
PARACRINE PURINERGIC MEDIATION OF FLOW-INDUCED Ca SIGNALING