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EB2007 SYMPOSIUM REPORT
1Laboratory of Physiology, Campus Gasthuisberg O&N, K.U. Leuven, Leuven, Belgium; 2Division of Pulmonary Medicine, Allergy, and Immunology, Department of Pediatrics, Children's Hospital of Pittsburgh/University of Pittsburgh, Pittsburgh, Pennsylvania; 3Department of Pharmacology, University of Illinois, Chicago, Illinois; 4Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania; 5Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas; and 6Will 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, California
ABSTRACT
In this synopsis of a symposium at EB2007, 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 change 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 cross talk between transcellular and paracellular transport (function and routes) of lung epithelial and endothelial barriers. Mechanistic (e.g., signaling cascades) information on such cross talk remain to be determined.
TRANSEPITHELIAL AND TRANSENDOTHELIAL 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 blocker-induced noise (BIN), which is caused by random interaction of a blocker with the channel. SN spectra are particularly useful in characterizing ion conductive pathways that are not detectable with macroscopic current recordings. The benefits of studies of BIN spectra are 1) unbiased measurement of the kinetics of the interaction of the blocker with the channel, and 2) 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 frequency range of 0.1–1,000 Hz and that the magnitude of the inhibition of macroscopic current component can be recorded. A separate but equally powerful technique of impedance analysis of various epithelial and endothelial barriers has been used 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, for which 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 s, 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, multi-sinusoids (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 ms). 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 increase the access resistance to the lateral membrane and will give rise to an upward 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-to-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 Fig. 1, R T 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).
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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 using the system of transepithelial impedance analysis engineered by Dr. Willy 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 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) (Fig. 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 (Ref. 7; Fig. 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.
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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 multi-sinusoid 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) of 14.8 k
and individual resistances (Ra and Rb) of 0.88 and 0.66 k and capacitances (Ca and Cb) of 1.13 and 1.38 µF for both apical (a) and basolateral (b) cell membranes of RAECM were estimated, respectively. The overall monolayer resistance (Rdc) is 1.38 k and Gj/Gdc is 0.1. When the effects of a calcium chelator, EGTA (2 mM in both bathing fluids for 0.5 h), were investigated, results indicate that EGTA exposure led to decreases in Rdc, Ra, Rb, and Rj without appreciable changes in Ca and Cb, whereas 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. Noninvasive 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 STRETCH-INDUCED 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 capacity (TLC), respectively; Ref. 26] for 1 h 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 (Fig. 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 (Fig. 4), indicative of a decrease in monolayer barrier function near TLC compared with both unstretched cells and cells stretched at lower magnitudes (2). However, tonic stretch (at 25% SA) for 1 h caused no significant change in Na pump activity or cell monolayer permeability compared with unstretched controls (2, 11) (Fig. 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% TLC. These in vitro findings are consistent with transport data from whole lung studies that 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 etiology of barrier dysfunction induced by mechanical ventilation at high tidal volumes.
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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 cross talk 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 macromolecule albumin is observed in the absence of caveolin-1. Studies of endothelial cells depleted of caveolin-1 using small interfering RNA (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; Fig. 5). This cross talk between transcytosis pathway and junctional pathways was dependent on the deinhibited endothelial nitric oxide synthase (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 vascular endothelial-cadherin/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 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 (I. Klein, S. Pedescu, A. B. Malik, unpublished observation). This was evident by the decrease in the transendothelial electrical resistance measured in monolayers. The mechanism of potentially important intersectin-mediated cross talk 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 deinhibited 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 cross talk 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 cross talk 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 cross talk 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
This symposium was designed to investigate cross talk between the transcellular and paracellular pathways in lung epithelia and endothelia. Before addressing this topic, we had a primer in the use of noise analysis 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 LIS. Impedance analysis was then used to address cross talk in both bronchial and alveolar epithelia. Cell biological, physiological, and biochemical techniques were also used to investigate transcellular and paracellular cross talk in endothelia and alveolar epithelia. In the future, combining modern cell biological techniques [e.g., knockdown/knockout/knock-in of specific gene(s) responsible for transport of water, ions, and solutes including macromolecules] and noise and impedance analyses should allow a dissection of the mechanisms underlying such cross talk(s) in lung epithelial and endothelial barriers under diverse experimental conditions (e.g., inflammation, injury/repair) and in specific diseases (e.g., cystic fibrosis).
GRANTS
This work was supported in part by the Hastings Foundation and by National Heart, Lung, and Blood Institute Grants K08-HL-081080 (to J. L. Kreindler), HL-07829, HL-07239, HL-45638, HL-46350, HL-60678, HL-64573, and HL-77806 (to A. B. Malik), HL-57204 (to S. Margulies), and HL-38658 and HL-64365 (to K.-J. Kim).
ACKNOWLEDGMENTS
J. L. Kreindler acknowledges the contribution of Dr. Henry Danahay to the work presented herein on airway epithelial transport studies and also thanks Dr. Robert Bridges for continued support and encouragement for the study of transepithelial ion transport physiology.
FOOTNOTES
Address for reprint requests and other correspondence: K.-J. Kim, Will Rogers Institute Pulmonary Research Center, Depts. of Medicine, Physiology and Biophysics, Pharmacology and Pharmaceutical Sciences, and Biomedical Engineering, Keck School of Medicine, School of Pharmacy, and Viterbi School of Engineering, Univ. of Southern California, Los Angeles, CA 90033 (e-mail: kjkim{at}usc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
F508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell Physiol 279: C461–C479, 2000.
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