Page 1Articles
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in PresS. Am J Physiol Lung Cell Mol Physiol (June 29, 2007). doi:10.1152/ajplung.00218.2007
INTERRELATIONS/CROSSTALK BETWEEN TRANSCELLULAR TRANSPORT
FUNCTION AND PARACELLULAR TIGHT JUNCTIONAL PROPERTIES IN LUNG
EPITHELIAL AND ENDOTHELIAL BARRIERS
Willy Van Driessche1, James L. Kreindler2, Asrar B. Malik3, Susan Margulies4, Simon A.
Lewis5, and Kwang-Jin Kim6
1
Laboratory of Physiology, Campus Gasthuisberg O&N, K.U. Leuven, B-3000 Leuven, Belgium
(willy.vandriessche@med.kuleuven.ac.be)
2
Division of Pulmonary Medicine, Allergy, and Immunology, Department of Pediatrics,
Children’s Hospital of Pittsburgh / University of Pittsburgh, Pittsburgh, PA 15213
(james.kreindler@chp.edu)
3
Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA
(abmalik@uic.edu)
4
Department of Bioengineering, School of Engineering and Applied Science, University of
Pennsylvania, Philadelphia, PA 19104, USA (margulie@seas.upenn.edu)
5
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX
77555, USA (slewis@utmb.edu)
6
Will Rogers Institute Pulmonary Research Center, Departments of Medicine, Physiology and
Biophysics, Pharmacology and Pharmaceutical Sciences, and Biomedical Engineering,
Keck School of Medicine, School of Pharmacy, and Viterbi School of Engineering,
University of Southern California, Los Angeles, CA 90033, USA (kjkim@usc.edu)
Copyright © 2007 by the American Physiological Society.
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Abstract
In this synopsis of a symposium at EB 2007, we start with an overview of noise and
impedance analyses that have been applied to various epithelial barriers. Noise analysis yields
specific information about ion channels and their regulation in epithelial and endothelial barriers.
Impedance analysis can yield information about apical and basolateral membrane conductances
and paracellular conductance of both epithelial and endothelial barriers. Using a
morphologically-based model, impedance analysis has been used to assess changes in apical and
basolateral membrane surface areas and dimensions of the lateral intercellular space. Impedance
analysis of an in vitro airway epithelial barrier under normal, nucleotide-stimulated and cigarette
smoke exposed conditions yielded information on how activation and inhibition of secretion
occur in airway epithelial cells. Similarly, impedance analysis of primary rat alveolar epithelial
cell monolayer model under control and EGTA exposure conditions indicate that EGTA causes
decreases in resistances of tight junctional routes as well as apical and basolateral cell
membranes without causing much changes in cell capacitances. In a stretch-caused injury model
of alveolar epithelium, transcellular ion transport function and paracellular permeability of solute
transport appear to be differentially regulated. Finally, inhibition of caveolae-mediated
transcytosis in lung endothelium led to disruption of paracellular routes, increasing the physical
dimension and permeability of tight junctional region. These data together demonstrate the
crosstalk between transcellular and paracellular transport (function and routes) of lung epithelial
and endothelial barriers. Mechanistic (e.g., signaling cascades) information on such crosstalk
remain to be determined.
Introduction
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Epithelia and endothelia perform three basic functions. They separate two fluid
compartments, they selectively restrict the movement of some substances between these
compartments, and last they permit the movement of selected substances between the
compartments by either active transport or modification of the passive permeability. Various
techniques have been applied over the years to investigate how lung epithelia and endothelia
perform these basic functions. These techniques include but are not limited to fluorescence-based
techniques for unraveling Ca signaling mechanisms, Ussing chamber techniques for
characterization of active and passive ion transport properties, and patch clamp studies for
deducing kinetics of ion channels in both isolated and in situ cells of the lungs. In the past, the
transcellular and paracellular route were treated as two distinct and independent parallel entities.
It is becoming clear that this is not the case and to understand the physiology of solute and
solvent movement across the lung epithelia and endothelia, one must consider these two
pathways as two dynamic and interacting parallel pathways. Studies of transcellular/paracellular
crosstalk in lung epithelial and endothelial barriers are relatively new and interesting and
important observations are starting to emerge.
Transepithelial and endothelial noise and impedance measurements: strengths and
shortcomings
Noise analysis has been successfully applied to study ion channel activity in a variety of
tight epithelia. Two categories of fluctuation in current were considered: spontaneous noise
(SN) which is caused by the spontaneous open-close kinetics of ion channels, and blockerinduced noise (BIN) which is caused by random interaction of a blocker with the channel. SN
spectra are particularly useful to characterize ion conductive pathways that are not detectable
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with macroscopic current recordings. The benefits of studies of BIN spectra are: i) unbiased
measurement of the kinetics of the interaction of the blocker with the channel and ii)
determination of the single channel current and channel density. Requirements for a successful
application of BIN recording are that the induced noise gives rise to relaxation noise in the audio
frequency range (0.1 Hz to 1000 Hz) and that the magnitude of the inhibition of macroscopic
current component can be recorded. A separately but equally powerful technique of impedance
analysis of various epithelial and endothelial barriers has been utilized over the years. Impedance
of a biological barrier can be measured by imposing voltage (or current) pulses across the barrier
and recording the current (or voltage) responses in the time domain and then convert the voltage
and current data from the time domain to the frequency domain to yield the ratio of voltage and
current data in the frequency domain i.e. the impedance. Sinusoids whose frequencies vary from
very slow (e.g., 0.01 Hz) to very high (e.g., 20 kHz) can be used in lieu of voltage (or current)
pulses to estimate impedance, albeit the overall time to make one measurement may take more
than 100 sec during which some transport properties (e.g., fast acting or fast changing events)
may undergo changes. In part to circumvent such shortcomings and ensure relatively fast and
accurate measurements of impedance, multisinuoids (e.g., composite voltage superimposed with
sinusoids ranging from 1 Hz to 20 kHz) can be used as the input voltage (or current) and output
current (or voltage) response can then be measured with a relatively short time span (e.g., 20
msec). Another approach deals with white noise generated digitally as the input voltage (or
current) and output responses of current (or voltage) obtained. Transepithelial impedance
spectra, represented in a Cole-Cole diagram, have the shape of a single semicircle or the
superimposed two semicircles. They are affected by several components of the epithelial
structure, the lateral interspace (LIS) being the most important one. Narrowing the LIS will
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increase the access resistance to the lateral membrane and will give rise to an upwards shift of
the center of the semicircle (5). In addition, dielectric relaxation will also affect the impedance,
however this occurs in a frequency range that is higher than commonly used and will not be
considered further. Impedance analysis is particularly useful in studies of the basolateral barrier
where it provides a tool for monitoring the ratio of the apical versus basolateral resistance during
permeabilization of the apical barrier (15). Moreover, impedance analysis becomes an essential
tool in studies of transepithelial and transendothelial resistance (RT). When leaky epithelia, such
as small intestine, as well as endothelial monolayers, are studied in Ussing chambers, the
resistance of the bathing medium is generally larger than RT (12). Changes in RT will therefore
be masked by both the solution resistance (Rsol) in series with epithelial or endothelial barrier per
se and resistance of the substratum on which epithelial or endothelial cells grow or subepithelial
or subendothelial compartment comprised of connective tissues and other components (Rsub). As
illustrated in Figure 1, RT can be determined as the difference between the impedance at low and
high frequencies. This method has been successfully applied in studies of biopsies of human
colon to estimate RT (28).
Transport and barrier functional changes in airway epithelia revealed by transepithelial
impedance analysis
Polarized human bronchial epithelial (HBE) cells, established and refined by Gray et al
(13) following the procedures of Adler et al (1), have a mucociliary phenotype and recapitulate
many of the functions of the native epithelium including mucus secretion and vectorial ion
transport (8, 13). Individual membrane conductances and capacitances of HBE cells can be
estimated noninvasively utilizing the system of transepithelial impedance analysis engineered by
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Dr. Willie van Driessche in which current is passed across the epithelium at 100 frequencies, and
the lumped model of a simple epithelium is employed to fit the resulting data (25). When data
are expressed as Nyquist plots (alias Cole-Cole plots), two membranes can be distinguished after
the apical membrane conductance has been increased by activation of the cystic fibrosis
transmembrane conductance regulator (CFTR) (Figure 2). Therefore, transepithelial impedance
analysis can be used to estimate real-time conductance and capacitance changes induced by both
stimuli and inhibitors of ion transport in HBE cells. For example, when either ATP or UTP is
applied to the HBE cells the observed increase in capacitance appears to reflect degranulation
events at the apical cell membrane (7) (Figure 3). Conversely, in forskolin-stimulated HBE cells,
application of cigarette smoke results in cell swelling that can be observed by an increase in
apical cell membrane capacitance and a decrease in basolateral cell membrane capacitance
coupled with an acute increase in apical and basolateral cell membrane resistances (18). These
data demonstrate that transepithelial impedance analysis of HBE cells can be used to study both
activation and inhibition of secretion. Furthermore, real-time capacitance measurements may be
used to estimate morphological changes of the cell membrane.
Impedance analysis of primary rat alveolar epithelial cell monolayers
Using an apical permeabilization (i.e., invasive) technique, paracellular ion conductance
(Gj)/total ion conductance (Gdc) of primary rat alveolar epithelial cell monolayers (RAECM) has
been reported to be ~0.25 (16). RAECM impedance was recently estimated as a ratio between
multisinusoid input voltages and resultant current responses in the frequency domain (17, 29).
From the observed impedance data, fit to a lumped equivalent circuit model for epithelial
barriers, tight junctional resistance (Rj, k ) of ~14.8 and individual resistances (Ra and Rb, k )
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of ~0.88 and ~0.66 and capacitances (Ca and Cb, µF) of ~1.13 and ~1.38 for both apical (a) and
basolateral (b) cell membranes of RAECM were estimated, respectively. The overall monolayer
resistance (Rdc, k ) is ~1.38 and Gj/Gdc ~0.1. When the effects of a calcium chelator, EGTA
(2mM in both bathing fluids for 0.5 hr), were investigated, results indicate that EGTA exposure
led to decreases in Rdc, Ra, Rb and Rj without appreciable changes in Ca and Cb, while Gj/Gdc
increased to ~0.68. These data indicate that RAECM transcellular (Gc) and paracellular (Gj) ion
conductances are both increased by calcium chelation (with the increase in Gj greater than that in
Gc). EGTA activation of nonspecific cation channels may have been the cause for decreases in
both Ra and Rb. Non-invasive impedance analysis is expected to provide more reliable and
quantitative information on ion transport parameters for paracellular (i.e., tight junctional) and
transcellular pathways of alveolar epithelium under various experimental conditions including
inflammation and injury/repair.
Transcellular ion transport function vs paracellular transport properties in stretchinduced alveolar epithelial injury
Ventilator-induced lung injury is characterized by edema with and without increases in
alveolar epithelial permeability. Thus, it is reasonable to hypothesize that transcellular ion
transport properties (Na+-K+-ATPase activity) and epithelial paracellular permeability may both
be affected by increasing stretch magnitude. To test this hypothesis, isolated rat alveolar
epithelial cells were cultured on flexible substratum and stretched cyclically or statically in a
custom made stretching device described previously (27). Cells were cultured for either 2 or 5
days at 37°C under 5% CO2 in MEM with 10% FBS (3, 27). Cyclic stretch was applied to a peak
strain of 12%, 25% or 37% change in surface area ( SA; ~70%, ~90% or ~100% total lung
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capacity, respectively (26)) for 1 hr at 0.25 Hz. Additional unstretched cells from the same
isolation were used as controls. Alveolar epithelial cells maintained in culture for 2 days
significantly increased their ouabain-dependent Na pump activity (6) in a “dose-dependent”
manner (Figure 4) in response to increasing cyclic stretch magnitude (11). Moreover, these
stretched cells increase their Na pump activity by trafficking Na pump
1-subunit
protein from
the intracellular stores to the basolateral cell membrane (4, 11, 14). After 5 days in culture, cells
demonstrate a significant increase in permeability to uncharged, small, nonelectrolyte tracers
only at 37% SA (Figure 4), indicative of a decrease in monolayer barrier function near total
lung capacity compared to both unstretched cells and cells stretched at lower magnitudes (2).
However, tonic stretch (at 25% SA) for 1 hr caused no significant change in Na pump activity
or cell monolayer permeability, compared with unstretched controls (2, 11) (Figure 5).
Measurements of Na pump activity and tight junctional permeability to small nonelectrolyte
tracers demonstrate that Na transport increases with cyclic stretch amplitude, but permeability is
compromised only at high levels of cyclic stretch. Specifically, these findings support the theory
that when challenged by continuous cyclic stretch, even at low magnitudes, the alveolar
epithelium responds by trafficking Na pumps to the basolateral cell membrane to aid in edema
clearance. This response is enhanced with strain amplitude in a dose-dependent manner similar
to surfactant release (22). In contrast, injurious stretch effects (e.g., breakdown of tight
junctions) resulting in the increased epithelial permeability with mechanical destruction of the
cell perhaps, only begin to appear near 100% total lung capacity (TLC). These in vitro findings
are consistent with transport data from whole lung studies which demonstrate total loss of barrier
function at 100% TLC, as determined by high pulmonary permeability to macromolecule tracers
(9, 10). These findings, in conclusion, make important contributions to our understanding of the
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etiology of barrier dysfunction induced by mechanical ventilation at high tidal volumes.
Crosstalk between transcellular and paracellular endothelial permeability pathways
Since endothelial barrier function is a tightly regulated process requiring the signaling
machinery for both transcellular and paracellular permeability pathways, the suggestion has been
made that there may be crosstalk between these two routes of solute transport in the endothelial
barrier (20). There are two possible mechanisms by which endothelial permeability is regulated
by caveolae-mediated transcytosis, specifically affecting the paracellular or junctional
endothelial barrier. The first mechanism derives from recent studies in the caveolin-1 null
mouse model (20) in which an increase in the permeability of the junctional barrier to the
macromolecular of albumin is observed in the absence of caveolin-1. Studies of endothelial cells
depleted of caveolin-1 using siRNA approach have also shown a marked reduction in the number
of transporting caveolae-derived vesicles, resulting in the opening of the junctional pathway (20,
21) (Figure 5). This crosstalk between transcytosis pathway and junctional pathways was
dependent on the de-inhibited eNOS activity (as a result of caveolin-1 knockdown) and increase
in the eNOS-derived NO production (21), which mediated the opening of the junctional pathway.
However, the mechanisms of NO-mediated increase in paracellular permeability are complex
and remain controversial (23). Nevertheless, the possibility exists that NO is important in
mediating the increase in permeability in endothelial cells of caveolin-1 knockout mice or with
defective caveolin-1 function. This effect of NO may be the result of phosphorylation of VEcadherin/catenin junctional complex proteins and NO-mediated disassembly of the adherens
junctions. Another possible mechanism of integrating caveolae-mediated transport and
endothelial tight junctional barrier permeability involves the recently described role of the
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scaffold proteins, intersectins (20, 24). These proteins are present in abundance in endothelial
cells where they regulate the activity of dynamin and mediate Cdc42-mediated actin
polymerization. Studies showed that siRNA-induced knockdown of intersectin-2l resulted in
decreased caveolae-mediated internalization, but at the same time increased dimension of
junctional permeability pathway (Klein et al, unpublished observation). This was evident by the
decrease in the transendothelial electrical resistance measured in monolayers. The mechanism of
potentially important intersectin-mediated crosstalk is not clear. One possibility is that dynamin,
which binds to intersectins and controls the localization of dynamin to the neck region of
caveolae, is de-inhibited in the absence of intersectin. Since the activated GTP-bound dynamin
induces activation of eNOS and the production of NO (19), this may be a key mechanism of
increased NO production induced by dynamin that promotes disassembly of adherens junctions
and increased junctional permeability. Thus, caveolin-1, dynamin and intersectins may play a
crucial role in the mechanism of transcellular/paracellular crosstalk such that decreased
transcellular permeability of albumin results in increased paracellular permeability. This is
necessary to maintain appropriate tissue oncotic pressure and fluid balance. It is unclear,
however, whether this crosstalk mechanism constitutively regulates tissue fluid balance and is
activated in perturbed endothelial cells (e.g., decreased ATP supply causing a reduction in
caveolae-mediated transcytosis). Also the signaling mechanisms in mediating crosstalk between
these pathways are unknown. Their understanding is likely to be important in delineating how
transcytosis inversely regulates the paracellular junctional pathway and thereby exquisitely
controls transendothelial permeability and lung fluid balance.
SUMMARY AND FUTURE DIRECTIONS
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This symposium was designed to investigate crosstalk between the transcellular and
paracellular pathways in lung epithelia and endothelia. Before addressing this topic we had a
primer in the use of noise to determine membrane ion channel regulation and impedance analysis
using morphologically based models to determine the permeability and surface area of the apical
and basolateral membranes, the permeability of the paracellular pathway and dimensions of
lateral intercellular space. Impedance analysis was then used to address crosstalk in both
bronchial and alveolar epithelia. Cell biological, physiological and biochemical techniques were
also used to investigate transcellular and paracellular crosstalk in endothelia and alveolar
epithelia. In the future, combining modern cell biological techniques (e.g., knock-down / knockout / knock-in of specific gene(s) responsible for transport of water, ions, and solutes including
macromolecules), noise and impedance analyses using morphologically based models should
allow a dissection of the mechanisms underlying such crosstalk(s) in lung epithelial and
endothelial barriers under diverse experimental conditions (e.g., inflammation, injury/repair) and
in specific diseases (e.g., cystic fibrosis).
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Acknowledgments
This work was supported in part by the Hastings Foundation and research grants from the
National Institutes of Health (K08 HL081080 to JLK; HL07829, HL07239, HL45638, HL46350,
HL60678, HL64573, and HL77806 to ABM; HL57204 to SM; and HL38658 and HL64365 to
KJK). JLK acknowledges the contribution of Henry Danahay, Ph.D. to the work presented
herein on airway epithelial transport studies and also thanks Robert Bridges, Ph.D. for his
continued support and encouragement for the study of transepithelial ion transport physiology.
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Figure legends
Figure 1. Illustration of the impedance that is represented in a Cole-Cole plot. Due to the
distribution of the capacitance in the lateral interspace, the center of the semi-circle is depressed.
Data are calculated for RT = 20
, Rsol +Rsub= 40
, and transepithelial capacitance CT = 4 µF.
The upwards shift of the center of the semi-circle is simulated by a Cole-Cole dispersion with
= 0.95. At high frequencies, impedance equals Rsol + Rsub. At low frequencies, the impedance
becomes equal to RT + Rsol + Rsub. Thus, RT = impedance at lowest frequency utilized impedance at highest frequency utilized. Adapted from Ghanem et al., Am J Physiol
Gastrointest Liver Physiol 288:G972-G977, 2005, used with permission.
Figure 2. Representative Nyquist plot of a HBE cells after amiloride inhibition and
forskolin stimulation. The left locus represents the apical membrane, a conclusion that is
supported by the capacitance estimates and by manipulations of basolateral K+ conductances that
alter the right locus.
Figure 3. Capacitance estimates reveal real-time morphological changes in HBE cells. ATP
or UTP stimulation of HBE cells results in net insertion of mucin granules into the apical
membrane which are detected as an increase in apical membrane capacitance. Adapted from
Danahay et al., Am J Physiol Lung Cell Mol Physiol 290:L558-L569, 2006, used with permission.
Figure 4. Response of Na pump activity and paracellular permeability to stretch. Primary
culture rat alveolar epithelial cells grown on flexible substratum were utilized. See text for
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details. Bars represent mean ± standard error; * represents statistical significance, P < 0.05.
Because values are normalized by unstretched controls, the line at y=1 marks no change.
Figure 5. Caveolin-1 suppression induces dilation of interendothelial junctions (IEJs) and
reduction in caveolar number. Electron micrographs show lung capillaries of control mice (A),
mice at 96 h after siRNA injection (B), and mice at 192 h after siRNA injection (C). Caveolin-1
knockdown mice showed decreased number of caveolae and open IEJs. Caveolae reformed in
mice after the restoration of caveolin-1 expression. C, inset: IEJ recover to establish a normally
restrictive barrier. Thin arrows in A point to caveolar profiles in control cells. Thick arrows
indicate IEJs in A and B. Scale bars: A, 250 nm; B, 175 nm; and C, 120 nm. Adapted from
Miyawaki-Shimizu, et al., Am J Physiol Lung Cell Mol Physiol 290:L405-13, 2006, used with
permission.
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126x76mm (300 x 300 DPI)
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82x119mm (300 x 300 DPI)
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160x120mm (72 x 72 DPI)