Interrelations/Cross Talk Between Transcellular Transport Function and Paracellular Tight Junctional Properties in Lung Epithelial and Endothelial Barriers

Am J Physiol Lung Cell Mol Physiol 293: L520–L524, 2007. First published June 29, 2007; doi:10.1152/ajplung.00218.2007.

EB2007 SYMPOSIUM REPORT

Interrelations/cross talk between transcellular transport function and paracellular tight junctional properties in lung epithelial and endothelial barriers

Willy Van Driessche,1 James L. Kreindler,2 Asrar B. Malik,3 Susan Margulies,4 Simon A. Lewis,5 and Kwang-Jin Kim6 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

Van Driessche W, Kreindler JL, Malik AB, Margulies S, Lewis movement of some substances between these compartments, SA, Kim K-J. Interrelations/cross talk between transcellular trans- and, last, they permit the movement of selected substances port function and paracellular tight junctional properties in lung between the compartments by either active transport or modi- epithelial and endothelial barriers. Am J Physiol Lung Cell Mol fication of the passive permeability. Various techniques have Physiol 293: L520–L524, 2007. First published June 29, 2007; been applied over the years to investigate how lung epithelia doi:10.1152/ajplung.00218.2007.—In this synopsis of a symposium at EB2007, we start with an overview of noise and impedance analyses and endothelia perform these basic functions. These techniques that have been applied to various epithelial barriers. Noise analysis include but are not limited to fluorescence-based techniques for yields specific information about ion channels and their regulation in unraveling Ca-signaling mechanisms, Ussing chamber tech- epithelial and endothelial barriers. Impedance analysis can yield niques for characterization of active and passive ion transport information about apical and basolateral membrane conductances and properties, and patch-clamp studies for deducing kinetics of paracellular conductance of both epithelial and endothelial barriers. ion channels in both isolated and in situ cells of the lungs. In Using a morphologically based model, impedance analysis has been the past, the transcellular and paracellular routes were treated used to assess changes in apical and basolateral membrane surface as two distinct and independent parallel entities. It is becoming areas and dimensions of the lateral intercellular space. Impedance clear that this is not the case, and to understand the physiology analysis of an in vitro airway epithelial barrier under normal, nucle- of solute and solvent movement across the lung epithelia and otide-stimulated, and cigarette smoke-exposed conditions yielded information on how activation and inhibition of secretion occur in endothelia, one must consider these two pathways as two airway epithelial cells. Similarly, impedance analysis of primary rat dynamic and interacting parallel pathways. Studies of transcel- alveolar epithelial cell monolayer model under control and EGTA lular-paracellular cross talk in lung epithelial and endothelial exposure conditions indicate that EGTA causes decreases in resis- barriers are relatively new, and interesting and important ob- tances of tight junctional routes as well as apical and basolateral cell servations are starting to emerge. membranes without causing much change in cell capacitances. In a stretch-caused injury model of alveolar epithelium, transcellular ion TRANSEPITHELIAL AND TRANSENDOTHELIAL NOISE AND transport function and paracellular permeability of solute transport IMPEDANCE MEASUREMENTS: STRENGTHS appear to be differentially regulated. Finally, inhibition of caveolae- AND SHORTCOMINGS mediated transcytosis in lung endothelium led to disruption of paracellular routes, increasing the physical dimension and permeability of Noise analysis has been successfully applied to study ion tight junctional region. These data together demonstrate the cross talk channel activity in a variety of tight epithelia. Two categories between transcellular and paracellular transport (function and routes) of fluctuation in current were considered: spontaneous noise of lung epithelial and endothelial barriers. Mechanistic (e.g., signaling (SN), which is caused by the spontaneous open-close kinetics cascades) information on such cross talk remain to be determined. 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 EPITHELIA AND ENDOTHELIA PERFORM three basic functions. They pathways that are not detectable with macroscopic current separate two fluid compartments, they selectively restrict the recordings. The benefits of studies of BIN spectra are 1) unbiased measurement of the kinetics of the interaction of Address for reprint requests and other correspondence: K.-J. Kim, Will the blocker with the channel, and 2) determination of the single 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 The costs of publication of this article were defrayed in part by the payment School of Engineering, Univ. of Southern California, Los Angeles, CA 90033 of page charges. The article must therefore be hereby marked “advertisement” (e-mail: [email protected]). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(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 Fig. 1. Illustration of the impedance that is represented in a Cole-Cole plot. other components (Rsub). As illustrated in Fig. 1, RT can be Because the distribution of the capacitance in the lateral interspace, the center determined as the difference between the impedance at low and of the semicircle is depressed. Data are calculated for transepithelial resistance high frequencies. This method has been successfully applied in (RT) ϭ 20 ⍀, solution resistance (Rsol) ϩ subepithelial/subendothelial resis- studies of biopsies of human colon to estimate RT (28). tance (Rsub) ϭ 40 ⍀, and transepithelial capacitance (CT) ϭ 4 ␮F. The upward shift of the center of the semicircle is simulated by a Cole-Cole dispersion with TRANSPORT AND BARRIER FUNCTIONAL CHANGES IN ␣ϭ0.95. At high frequencies, impedance equals Rsol ϩ Rsub.Atlow frequencies, the impedance becomes RT ϩ Rsol ϩ Rsub. Thus RT equals AIRWAY EPITHELIA REVEALED BY TRANSEPITHELIAL impedance at lowest frequency used minus impedance at highest frequency IMPEDANCE ANALYSIS used. X, imaginary part or reactance. Reprinted with permission from Ghanem et al., Ref. 12. Polarized human bronchial epithelial (HBE) cells, estab- lished and refined by Gray et al. (13) following the procedures of Adler et al. (1), have a mucociliary phenotype and recapit- channel current and channel density. Requirements for a suc- ulate many of the functions of the native epithelium including cessful application of BIN recording are that the induced noise mucus secretion and vectorial ion transport (8, 13). Individual gives rise to relaxation noise in the frequency range of 0.1– membrane conductances and capacitances of HBE cells can be 1,000 Hz and that the magnitude of the inhibition of macro- estimated noninvasively using the system of transepithelial scopic current component can be recorded. A separate but impedance analysis engineered by Dr. Willy van Driessche, in equally powerful technique of impedance analysis of various which current is passed across the epithelium at 100 frequen- epithelial and endothelial barriers has been used over the years. cies, and the lumped model of a simple epithelium is employed Impedance of a biological barrier can be measured by imposing to fit the resulting data (25). When data are expressed as voltage (or current) pulses across the barrier and recording the Cole-Cole plots, two membranes can be distinguished after the current (or voltage) responses in the time domain and then apical membrane conductance has been increased by activation convert the voltage and current data from the time domain to of the cystic fibrosis transmembrane conductance regulator the frequency domain to yield the ratio of voltage and current (CFTR) (Fig. 2). Therefore, transepithelial impedance analysis data in the frequency domain, i.e., the impedance. Sinusoids, can be used to estimate real-time conductance and capacitance for which frequencies vary from very slow (e.g., 0.01 Hz) to changes induced by both stimuli and inhibitors of ion transport very high (e.g., 20 kHz), can be used in lieu of voltage (or in HBE cells. For example, when either ATP or UTP is applied current) pulses to estimate impedance, albeit the overall time to to the HBE cells, the observed increase in capacitance appears make one measurement may take more than 100 s, during to reflect degranulation events at the apical cell membrane which some transport properties (e.g., fast acting or fast chang- (Ref. 7; Fig. 3). Conversely, in forskolin-stimulated HBE cells, ing events) may undergo changes. In part to circumvent such application of cigarette smoke results in cell swelling that can shortcomings and ensure relatively fast and accurate measure- be observed by an increase in apical cell membrane capaci- ments of impedance, multi-sinusoids (e.g., composite voltage tance and a decrease in basolateral cell membrane capacitance superimposed with sinusoids ranging from 1 Hz to 20 kHz) can coupled with an acute increase in apical and basolateral cell be used as the input voltage (or current), and output current (or membrane resistances (18). These data demonstrate that trans- voltage) response can then be measured with a relatively short epithelial impedance analysis of HBE cells can be used to time span (e.g., 20 ms). Another approach deals with white study both activation and inhibition of secretion. Furthermore, noise generated digitally as the input voltage (or current) and output responses of current (or voltage) obtained. Transepithe- lial 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 Fig. 2. Representative Cole-Cole plot of human bronchial epithelial (HBE) provides a tool for monitoring the ratio of the apical-to- cells after amiloride inhibition and forskolin stimulation. The left locus basolateral resistance during permeabilization of the apical represents the apical membrane, a conclusion that is supported by the capac- barrier (15). Moreover, impedance analysis becomes an essen- itance estimates and by manipulations of basolateral Kϩ conductances that ⍀⅐ 2 tial tool in studies of transepithelial and transendothelial resis- alter the right locus. Units of resistance and impedance are cm . Ra and Rb, individual resistances; C and C , individual capacitances. Norm, measurement tance (R ). When leaky epithelia such as small intestine as well a b T of the quality of the mathematical fit; Rs, solution resistance; Rp, paracellular as endothelial monolayers are studied in Ussing chambers, the pathway resistance; ZR, impedance on the real axis; ZI, impedance on the resistance of the bathing medium is generally larger than RT imaginary axis.

AJP-Lung Cell Mol Physiol • VOL 293 • SEPTEMBER 2007 • www.ajplung.org L522 EB2007 SYMPOSIUM REPORT

Rdc, Ra, Rb, and Rj without appreciable changes in Ca and Cb, whereas Gj/Gdc increased to ϳ0.68. 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 (Naϩ-Kϩ-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; ϳ70%, ϳ90%, or ϳ100% 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” Fig. 3. Capacitance estimates reveal real-time morphological changes in HBE manner (Fig. 4) in response to increasing cyclic stretch mag- cells. ATP (A–D) or UTP (E–H) stimulation of HBE cells results in net nitude (11). Moreover, these stretched cells increase their Na insertion of mucin granules into the apical membrane, which are detected as an pump activity by trafficking Na pump ␣1-subunit protein from increase in apical membrane capacitance. Isc, short-circuit current. Reprinted the intracellular stores to the basolateral cell membrane (4, 11, with permission from Danahay et al., Ref. 7. 14). After 5 days in culture, cells demonstrate a significant increase in permeability to uncharged, small, nonelectrolyte ⌬ real-time capacitance measurements may be used to estimate tracers only at 37% SA (Fig. 4), indicative of a decrease in morphological changes of the cell membrane. monolayer barrier function near TLC compared with both unstretched cells and cells stretched at lower magnitudes (2). IMPEDANCE ANALYSIS OF PRIMARY RAT ALVEOLAR However, tonic stretch (at 25% ⌬SA) for 1 h caused no significant EPITHELIAL CELL MONOLAYERS change in Na pump activity or cell monolayer permeability compared with unstretched controls (2, 11) (Fig. 5). Measure- 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 ϳ0.25 (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)ofϳ14.8 k⍀ and individual resistances (Ra and Rb)ofϳ0.88 and ϳ0.66 k⍀ and capacitances (Ca and Cb)ofϳ1.13 and ϳ1.38 ␮F for both apical (a) and basolateral (b) cell membranes of RAECM were estimated, Fig. 4. Response of Na pump activity and paracellular permeability to stretch. respectively. The overall monolayer resistance (Rdc)isϳ1.38 ⍀ ϳ Primary culture rat alveolar epithelial cells grown on flexible substratum were k and Gj/Gdc is 0.1. When the effects of a calcium chelator, used. See text for details. Bars represent means Ϯ SE; *P Ͻ 0.05. Because EGTA (2 mM in both bathing fluids for 0.5 h), were investi- values are normalized by unstretched controls, the line at y ϭ 1 marks no gated, results indicate that EGTA exposure led to decreases in change. ⌬SA, change in surface area.

AJP-Lung Cell Mol Physiol • VOL 293 • SEPTEMBER 2007 • www.ajplung.org EB2007 SYMPOSIUM REPORT L523 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). Neverthe- less, 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 Fig. 5. Caveolin-1 suppression induces dilation of interendothelial junctions effect of NO may be the result of phosphorylation of vascular (IEJs) and reduction in caveolar number. Electron micrographs show lung endothelial-cadherin/catenin junctional complex proteins and capillaries of control mice (A), mice at 96 h after small interfering RNA NO-mediated disassembly of the adherens junctions. Another (siRNA) injection (B), and mice at 192 h after siRNA injection (C). Caveolin-1 knockdown mice showed decreased number of caveolae and open IEJs. possible mechanism of integrating caveolae-mediated transport Caveolae reformed in mice after the restoration of caveolin-1 expression. C, and endothelial tight junctional barrier permeability involves inset: IEJ recover to establish a normally restrictive barrier. Thin arrows in A the recently described role of the scaffold proteins, intersectins point to caveolar profiles in control cells. Thick arrows indicate IEJs in A and (20, 24). These proteins are present in abundance in endothelial B. Scale bars: A, 250 nm; B, 175 nm; and C, 120 nm. Reprinted with permission from Miyawaki-Shimizu et al., Ref. 21. 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 ments of Na pump activity and tight junctional permeability to small nonelectrolyte tracers demonstrate that Na transport in- increased dimension of junctional permeability pathway (I. creases with cyclic stretch amplitude, but permeability is compro- Klein, S. Pedescu, A. B. Malik, unpublished observation). This mised only at high levels of cyclic stretch. Specifically, these was evident by the decrease in the transendothelial electrical findings support the theory that when challenged by continuous resistance measured in monolayers. The mechanism of poten- cyclic stretch, even at low magnitudes, the alveolar epithelium tially important intersectin-mediated cross talk is not clear. responds by trafficking Na pumps to the basolateral cell mem- One possibility is that dynamin, which binds to intersectins and brane to aid in edema clearance. This response is enhanced with controls the localization of dynamin to the neck region of strain amplitude in a dose-dependent manner similar to surfactant caveolae, is deinhibited in the absence of intersectin. Since the release (22). In contrast, injurious stretch effects (e.g., breakdown activated GTP-bound dynamin induces activation of eNOS and of tight junctions), resulting in the increased epithelial permeabil- the production of NO (19), this may be a key mechanism of ity with mechanical destruction of the cell perhaps, only begin to increased NO production induced by dynamin that promotes appear near 100% TLC. These in vitro findings are consistent with disassembly of adherens junctions and increased junctional transport data from whole lung studies that demonstrate total loss permeability. Thus caveolin-1, dynamin, and intersectins may of barrier function at 100% TLC, as determined by high pulmo- play a crucial role in the mechanism of transcellular-paracel- nary permeability to macromolecule tracers (9, 10). These find- lular cross talk such that decreased transcellular permeability ings, in conclusion, make important contributions to our under- of albumin results in increased paracellular permeability. This standing of the etiology of barrier dysfunction induced by me- is necessary to maintain appropriate tissue oncotic pressure and chanical ventilation at high tidal volumes. 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 CROSS TALK BETWEEN TRANSCELLULAR AND supply causing a reduction in caveolae-mediated transcytosis). PARACELLULAR ENDOTHELIAL PERMEABILITY PATHWAYS Also, the signaling mechanisms in mediating cross talk be- Since endothelial barrier function is a tightly regulated tween these pathways are unknown. Their understanding is process requiring the signaling machinery for both transcellular likely to be important in delineating how transcytosis inversely and paracellular permeability pathways, the suggestion has regulates the paracellular junctional pathway and thereby ex- been made that there may be cross talk between these two quisitely controls transendothelial permeability and lung fluid routes of solute transport in the endothelial barrier (20). There balance.

AJP-Lung Cell Mol Physiol • VOL 293 • SEPTEMBER 2007 • www.ajplung.org L524 EB2007 SYMPOSIUM REPORT

SUMMARY AND FUTURE DIRECTIONS 9. Egan EA. Lung inflation, lung solute permeability, and alveolar edema. J Appl Physiol 53: 121–125, 1982. This symposium was designed to investigate cross talk 10. Egan EA, Nelson RM, Olver RE. Lung inflation and alveolar perme- between the transcellular and paracellular pathways in lung ability to non-electrolytes in the adult sheep in vivo. J Physiol 260: 409–424, 1976. epithelia and endothelia. Before addressing this topic, we had ϩ ϩ a primer in the use of noise analysis to determine membrane 11. Fisher JL, Margulies SS. Na -K -ATPase activity in alveolar epithelial cells increases with cyclic stretch. Am J Physiol Lung Cell Mol Physiol ion channel regulation and impedance analysis using morpho- 283: L737–L746, 2002. logically based models to determine the permeability and 12. Ghanem E, Lovdahl C, Dare E, Ledent C, Fredholm BB, Boeynaems surface area of the apical and basolateral membranes, the JM, Van Driessche W, Beauwens R. Luminal adenosine stimulates permeability of the paracellular pathway, and dimensions of chloride secretion through A1 receptor in mouse jejunum. Am J Physiol LIS. Impedance analysis was then used to address cross talk in Gastrointest Liver Physiol 288: G972–G977, 2005. 13. Gray TE, Guzman K, Davis CW, Abdullah LH, Nettesheim P. Muco- both bronchial and alveolar epithelia. Cell biological, physio- ciliary differentiation of serially passaged normal human tracheobronchial logical, and biochemical techniques were also used to investi- epithelial cells. Am J Respir Cell Mol Biol 14: 104–112, 1996. gate transcellular and paracellular cross talk in endothelia and 14. Hammond TG, Verroust PJ, Majewski RR, Muse KE, Oberley TD. alveolar epithelia. In the future, combining modern cell bio- Heavy endosomes isolated from the rat renal cortex show attributes of logical techniques [e.g., knockdown/knockout/knock-in of spe- intermicrovillar clefts. Am J Physiol Renal Fluid Electrolyte Physiol 267: cific gene(s) responsible for transport of water, ions, and F516–F527, 1994. 15. Hillyard SD, Cantiello HF, Van Driessche W. Kϩ transport and capac- solutes including macromolecules] and noise and impedance itance of the basolateral membrane of the larval frog skin. Am J Physiol analyses should allow a dissection of the mechanisms under- Cell Physiol 273: C1995–C2001, 1997. lying such cross talk(s) in lung epithelial and endothelial 16. Kim KJ, Borok Z, Ehrhardt C, Willis BC, Lehr CM, Crandall ED. barriers under diverse experimental conditions (e.g., inflamma- Estimation of paracellular conductance of primary rat alveolar epithelial tion, injury/repair) and in specific diseases (e.g., cystic fibro- cell monolayers. J Appl Physiol 98: 138–143, 2005. sis). 17. Kim KJ, Zhang LQ, Bertrand CA, Van Driessche W, Borok Z, Crandall ED. Impedance analysis of rat alveolar epithelium in vitro ACKNOWLEDGMENTS (Abstract). FASEB J 20: A1440, 2006. 18. Kreindler JL, Jackson AD, Kemp PA, Bridges RJ, Danahay H. J. L. Kreindler acknowledges the contribution of Dr. Henry Danahay to the Inhibition of chloride secretion in human bronchial epithelial cells by work presented herein on airway epithelial transport studies and also thanks cigarette smoke extract. Am J Physiol Lung Cell Mol Physiol 288: Dr. Robert Bridges for continued support and encouragement for the study of L894–L902, 2005. transepithelial ion transport physiology. 19. Maniatis NA, Brovkovych V, Allen SE, John TA, Shajahan AN, GRANTS Tiruppathi C, Vogel SM, Skidgel RA, Malik AB, Minshall RD. Novel mechanism of endothelial nitric oxide synthase activation mediated by This work was supported in part by the Hastings Foundation and by caveolae internalization in endothelial cells. Circ Res 99: 870–877, 2006. National Heart, Lung, and Blood Institute Grants K08-HL-081080 (to J. L. 20. Mehta D, Malik AB. Signaling mechanisms regulating endothelial per- Kreindler), HL-07829, HL-07239, HL-45638, HL-46350, HL-60678, HL- meability. Physiol Rev 86: 279–367, 2006. 64573, and HL-77806 (to A. B. Malik), HL-57204 (to S. Margulies), and 21. Miyawaki-Shimizu K, Predescu D, Shimizu J, Broman M, Predescu S, HL-38658 and HL-64365 (to K.-J. Kim). Malik AB. siRNA-induced caveolin-1 knockdown in mice increases lung REFERENCES vascular permeability via the junctional pathway. Am J Physiol Lung Cell Mol Physiol 290: L405–L413, 2006. 1. Adler KB, Cheng PW, Kim KC. Characterization of guinea pig tracheal 22. Patel AS, Reigada D, Mitchell CH, Bates SR, Margulies SS, Koval M. epithelial cells maintained in biphasic organotypic culture: cellular com- Paracrine stimulation of surfactant secretion by extracellular ATP in position and biochemical analysis of released glycoconjugates. Am J response to mechanical deformation. Am J Physiol Lung Cell Mol Physiol Respir Cell Mol Biol 2: 145–154, 1990. 289: L489–L496, 2005. 2. Cavanaugh KJ, Cohen TS, Margulies SS. Stretch increases alveolar 23. Predescu D, Predescu S, Shimizu J, Miyawaki-Shimizu K, Malik AB. epithelial permeability to uncharged micromolecules. Am J Physiol Cell Constitutive eNOS-derived nitric oxide is a determinant of endothelial Physiol 290: C1179–C1188, 2006. junctional integrity. Am J Physiol Lung Cell Mol Physiol 289: L371–L381, 3. Cavanaugh KJ Jr, Oswari J, Margulies SS. Role of stretch on tight 2005. junction structure in alveolar epithelial cells. Am J Respir Cell Mol Biol 24. Predescu SA, Predescu DN, Timblin BK, Stan RV, Malik AB. Inter- 25: 584–591, 2001. sectin regulates fission and internalization of caveolae in endothelial cells. 4. Chibalin AV, Pedemonte CH, Katz AI, Feraille E, Berggren PO, Mol Biol Cell 14: 4997–5010, 2003. Bertorello AM. Phosphorylation of the catalytic alpha-subunit constitutes 25. Singh AK, Singh S, Devor DC, Frizzell RA, Van Driessche W, Bridges a triggering signal for Naϩ,Kϩ-ATPase endocytosis. J Biol Chem 273: RJ. Transepithelial impedance analysis of chloride secretion. Methods 8814–8819, 1998. Mol Med 70: 129–142, 2002. 5. Clausen C, Lewis SA, Diamond JM. Impedance analysis of a tight Tschumperlin DJ, Margulies SS. epithelium using a distributed resistance model. Biophys J 26: 291–317, 26. Alveolar epithelial surface area-vol- 1979. ume relationship in isolated rat lungs. J Appl Physiol 86: 2026–2033, 6. Clerici C, Friedlander G, Amiel C. Impairment of sodium-coupled 1999. uptakes by hydrogen peroxide in alveolar type II cells: protective effect of 27. Tschumperlin DJ, Margulies SS. Equibiaxial deformation-induced in- D-alpha-tocopherol. Am J Physiol Lung Cell Mol Physiol 262: L542–L548, jury of alveolar epithelial cells in vitro. Am J Physiol Lung Cell Mol 1992. Physiol 275: L1173–L1183, 1998. 7. Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, 28. Zeissig S, Bojarski C, Buergel N, Mankertz J, Zeitz M, Fromm M, Bridges RJ. Membrane capacitance and conductance changes parallel Schulzke JD. Downregulation of epithelial apoptosis and barrier repair in mucin secretion in the human airway epithelium. Am J Physiol Lung Cell active Crohn’s disease by tumour necrosis factor alpha antibody treatment. Mol Physiol 290: L558–L569, 2006. Gut 53: 1295–1302, 2004. 8. Devor DC, Bridges RJ, Pilewski JM. Pharmacological modulation of ion 29. Zhang LQ, Borok Z, Crandall ED, Kim KJ. Impedance analysis of transport across wild-type and ⌬F508 CFTR-expressing human bronchial EGTA-treated primary rat alveolar epithelial cell monolayers (RAECM) epithelia. Am J Physiol Cell Physiol 279: C461–C479, 2000. (Abstract). FASEB J 21: A554, 2007.

AJP-Lung Cell Mol Physiol • VOL 293 • SEPTEMBER 2007 • www.ajplung.org