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• W2052571887 abstract "The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 ± 3.2 and 22.5 ± 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 μm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 ± 0.26 to 0.82 ± 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (O2.¯) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that O2.¯ and NO signaling plays an important role in maintaining lung fluid balance. The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 ± 3.2 and 22.5 ± 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 μm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 ± 0.26 to 0.82 ± 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (O2.¯) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that O2.¯ and NO signaling plays an important role in maintaining lung fluid balance. The alveolar epithelium is responsible for maintaining effective gas exchange and is composed of two morphologically distinct types of cells referred to as type 1 (AT1) 2The abbreviations used are:AT1alveolar type 1AT2alveolar type 2ENaCepithelial sodium channelECLE. crista-galli LectinpSpicosiemensCFTRcystic fibrosis transmembrane conductance regulatorPBSphosphatebuffered salineDHEdihydroethidiumHSChighly selective channelsNSCnon-selective channels. 2The abbreviations used are:AT1alveolar type 1AT2alveolar type 2ENaCepithelial sodium channelECLE. crista-galli LectinpSpicosiemensCFTRcystic fibrosis transmembrane conductance regulatorPBSphosphatebuffered salineDHEdihydroethidiumHSChighly selective channelsNSCnon-selective channels. and type 2 (AT2) cells. AT1 cells are believed to be terminally differentiated squamous cells and cover >95% of the alveolar surface with extensive flattened processes. In the past type 1 cells have been thought to be predominantly responsible for gas exchange and possibly water permeability. Our research efforts, however, reveal that type 1 cells play a more complex role in the alveoli. Type 2 cells are easily discernable from the long, flask-shaped type 1 cells in the alveolar epithelium because they tend to be located at the corners of the alveoli (covering only 2-5% of the alveolar surface) and are cuboidally shaped. In addition to contributing to net fluid transport, type 2 cells are responsible for surfactant synthesis (14Fehrenbach H. Respir. Res. 2001; 2: 33-46Crossref PubMed Scopus (531) Google Scholar) and are considered to be progenitors of AT1 cells (3Cheek J.M. Evans M.J. Crandall E.D. Exp. Cell Res. 1989; 184: 375-387Crossref PubMed Scopus (114) Google Scholar, 6Danto S.I. Shannon J.M. Borok Z. Zabski S.M. Crandall E.D. Am. J. Respir. Cell Mol. Biol. 1995; 12: 497-502Crossref PubMed Scopus (149) Google Scholar, 7Danto S.I. Zabski S.M. Crandall E.D. Am. J. Respir. Cell Mol. Biol. 1992; 6: 296-306Crossref PubMed Scopus (111) Google Scholar, 11Dobbs L.G. Williams M.C. Gonzalez R. Biochim. Biophys. Acta. 1988; 970: 146-156Crossref PubMed Scopus (214) Google Scholar). To date the biophysical properties of type 2 cells have only been studied in cultured cell models. Furthermore, the mechanisms by which Na+ transport is regulated when both cell types are present in the same preparation is unclear. alveolar type 1 alveolar type 2 epithelial sodium channel E. crista-galli Lectin picosiemens cystic fibrosis transmembrane conductance regulator phosphatebuffered saline dihydroethidium highly selective channels non-selective channels. alveolar type 1 alveolar type 2 epithelial sodium channel E. crista-galli Lectin picosiemens cystic fibrosis transmembrane conductance regulator phosphatebuffered saline dihydroethidium highly selective channels non-selective channels. Together the two cell types (AT1 and AT2 cells) provide an effective barrier against leakage of water and solutes into the airspace by maintaining a thin fluid layer on the luminal surface of the alveoli, promoting proper gas exchange. The thickness of the fluid layer must be maintained within very narrow limits to allow for proper oxygenation of the blood. We and others have shown that epithelial sodium channels play a critical role in maintaining lung fluid clearance under both normal conditions and in the event of lung injury. For instance, α-ENaC subunit knock-out mice die within 40 h of birth due to an inability to clear lung fluid (21Hummler E. Barker P. Gatzy J. Beermann F. Verdumo C. Schmidt A. Boucher R. Rossier B.C. Nat. Genet. 1996; 12: 325-328Crossref PubMed Scopus (762) Google Scholar), highlighting the importance of normal transepithelial Na+ transport in the neonatal lung. Moreover, we have shown that therapeutic agents, such as dopamine (18Helms M.N. Chen X.J. Ramosevac S. Eaton D.C. Jain L. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 290: 710-722Crossref PubMed Scopus (72) Google Scholar, 19Helms M.N. Self J. Bao H.F. Job L.C. Jain L. Eaton D.C. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: 610-618Crossref PubMed Scopus (51) Google Scholar), nitric oxide (22Jain L. Chen X.J. Brown L.A. Eaton D.C. Am. J. Physiol. 1998; 274: L475-L484PubMed Google Scholar), and β-adrenergic agonists (5Chen X.J. Eaton D.C. Jain L. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; 282: 609-620Crossref PubMed Scopus (52) Google Scholar) (all commonly used to treat respiratory illnesses) have significant effects on lung ENaC function. Hence, a better understanding of lung ENaC regulation will lead to a greater understanding of how the alveoli maintain a clear breathing space. In addition to actively transporting salt, AT1 and AT2 cells must be able to sense and immediately respond to changes in oxygen tension as well as harmful pollutants such as reactive oxygen and nitrogen species in inspired air. Therefore, it is particularly important to understand redox signaling in the alveoli, especially as it relates to normal ENaC function. Several studies have indeed suggested that ENaC activity may be regulated by changes in oxygen tension or by the associated increase in the production of reactive oxygen species (such as the superoxide anion, O2.¯) after an increase in oxidative metabolism and mitochondrial activity. For example, at birth an increase in O2 tension contributes to increased Na+ reabsorption, which helps clear the newborn lung of excess fluid (for review, see Ref. 12Elias N. O’Brodovich H. NeoReviews. 2006; 7: e88-e94Crossref Google Scholar)). O’Brodovich and co-workers (29Rafii B. Tanswell A.K. Otulakowski G. Pitkanen O. Belcastro-Taylor R. O’Brodovich H. Am. J. Physiol. 1998; 275: L764-L770PubMed Google Scholar) and Matalon co-workers (31Thome U.H. Davis I.C. Nguyen S.V. Shelton B.J. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003; 284: 376-385Crossref PubMed Scopus (49) Google Scholar) have shown experimentally that maintaining rat fetal distal lung cells in high (20%) O2 concentrations increased ENaC mRNA (29Rafii B. Tanswell A.K. Otulakowski G. Pitkanen O. Belcastro-Taylor R. O’Brodovich H. Am. J. Physiol. 1998; 275: L764-L770PubMed Google Scholar) and protein (31Thome U.H. Davis I.C. Nguyen S.V. Shelton B.J. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003; 284: 376-385Crossref PubMed Scopus (49) Google Scholar) expression. In fetal lung cells, a switch from low PO2 to high PO2 environments increases amiloride-sensitive short-circuit current and also increases total ENaC protein expression (1Baines D.L. Ramminger S.J. Collett A. Haddad J.J. Best O.G. Land S.C. Olver R.E. Wilson S.M. J. Physiol. (Lond.). 2001; 532: 105-113Crossref Scopus (55) Google Scholar, 29Rafii B. Tanswell A.K. Otulakowski G. Pitkanen O. Belcastro-Taylor R. O’Brodovich H. Am. J. Physiol. 1998; 275: L764-L770PubMed Google Scholar, 31Thome U.H. Davis I.C. Nguyen S.V. Shelton B.J. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003; 284: 376-385Crossref PubMed Scopus (49) Google Scholar). Conversely, in adult rat alveolar type 2 cells, hypoxic culture conditions decreases amiloride-sensitive 22Na+ influx as well as α,β- and γ-ENaC mRNA expression and α-ENaC protein levels (28Planes C. Escoubet B. Blot-Chabaud M. Friedlander G. Farman N. Clerici C. Am. J. Respir. Cell Mol. Biol. 1997; 17: 508-518Crossref PubMed Scopus (125) Google Scholar). Thus, oxygen signaling is important in ENaC regulation; however, it remains unclear whether the regulation results from direct changes in PO2 or the associated increases in O2.¯ production after elevated oxygen tension. In our present study we successfully made single channel recordings from alveolar type 2 cells from live lung tissue (as we have recently done in type 1 cells) using cell-attached patch clamp to further investigate redox signaling in the alveoli. In addition to allowing us to make comparisons between ENaC regulatory processes in AT1 and AT2 cells, this novel approach also allows us to validate the standard use of cultured cells as models for type 1 and 2 cell transport properties. By examining the individual transport properties of each cell type as would naturally occur in tissue slices, we gain a better understanding of how type 1 and 2 cells work together in the alveoli to regulate net ion transport and balance lung fluid levels. Our results indicate that type 1 and 2 cells share similar biophysical properties yet display distinctly different cellular responses to reactive oxygen and nitrogen species. Lung Tissue Preparation—We have recently recorded single channel activity from live rat lung tissue (19Helms M.N. Self J. Bao H.F. Job L.C. Jain L. Eaton D.C. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: 610-618Crossref PubMed Scopus (51) Google Scholar). Using similar methodologies, we have characterized the single channel properties and redox response of alveolar type 1 and 2 cells in slices of rat lung tissue. Briefly, we housed male Sprague-Dawley rats with access to standard rat diet and water ad libitum. Between weeks 8 and 12, animals were anesthetized and killed for experimentation in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. All animal protocols conform to National Institutes of Health animal care and use guidelines and were approved by Emory University IACUC. After lung perfusion via the pulmonary artery with 75 ml of PBS, 2% low melting point agarose in PBS (kept warm in a 35 °C water bath) was intratracheally instilled into the lungs to expand airspaces and to provide support for the tissue during the slicing process. Excised lungs were removed en bloc and iced to solidify the agarose before mounting onto a vibratory microtome (model VT1000S, Leica Microsystems). The Vibratome blade was set to high frequency, slow forward advancement, and 250-300 μm for lung slice preparations. Immediately after the lung slice procedure, tissue for patch clamp recordings was placed in 50:50 ice-cold Dulbecco’s modified Eagle’s medium/F-12 (containing 10% fetal bovine serum, 2 mm l-glutamine, 1 μm dexamethasone, 84 μm gentamicin, and 20 units/ml penicillin-streptomycin). Lung slices were treated and patched within 6 h of the initial tissue preparation. Single-channel Patch Clamp Analysis of Na+ Channels in Alveolar Cells—Lung slices were thoroughly washed in patch clamp solution containing 140 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.4. Alveolar epithelial cells on the top surface of lung tissue slices were brought into focus using a Nikon inverted microscope with Hoffman modulation contrast under a 40× objective. Gigohm seals were formed on type 1 or 2 cells using a fire-polished glass microelectrode back-filled with patch solution, and lung slices were kept immersed in the same patch solution. An Axopatch 1-D (Molecular Devices) amplifier interfaced through an analog-to-digital board to a personal computer collected single-channel data. Channel currents were recorded at 5 kHz and filtered at 1 kHz with a low-pass Bessel filter. For cell attached patches, voltages are given as the negative of the patch pipette potential (-Vp), which is the displacement of the patch potential from the resting potential. Positive potentials represent depolarizations, and negative potentials represent hyperpolarization of the cell membrane away from the resting potential. We used the product of the number of channels (N) and the single-channel open probability (Po) as a measure of ENaC activity within a patch as described in Yu et al. (33Yu L. Bao H.F. Self J.L. Eaton D.C. Helms M.N. Am. J. Physiol. Renal Physiol. 2007; 293: 1666-1677Crossref PubMed Scopus (52) Google Scholar). NPo was calculated using FETCHAN and Clampfit 10.1 software (Molecular Devices, Sunnyvale, CA). Chemicals Used in Patch Clamp Analysis—The nitric oxide donor, l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) rapidly releases 2 m NO per mol of parent compound and is available commercially from Caymen Chemical (Ann Arbor, MI). Local concentrations of O2.¯ were increased in alveolar type 2 cells with 1 μg/ml Ethiolat, a cell-permeable superoxide dismutase inhibitor purchased from Sigma Aldrich. Fluorescent Microscopy—Alveolar type 2 cells were positively identified by labeling lung slice preparations either with surfactant protein A-specific antibody (purchased from Chemicon, Billerica, MA), rat type II antibody developed by Dr. Leland Dobbs (10Dobbs L.G. Pian M.S. Maglio M. Dumars S. Allen L. Am. J. Physiol. Lung Cell. Mol. Physiol. 1997; 273: 347-354Crossref PubMed Google Scholar), or LysoTracker Red (available commercially from Invitrogen), which recognizes the lamellar bodies in type 2 cells that are not present in alveolar type 1 cells (as described in Refs. (13Fang X. Song Y. Zemans R. Hirsch J. Matthay M.A. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 287: 104-110Crossref PubMed Scopus (33) Google Scholar, 17Haller T. Ortmayr J. Friedrich F. Volkl H. Dietl P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1579-1584Crossref PubMed Scopus (125) Google Scholar, 32Wang P.M. Fujita E. Bhattacharya J. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; 282: 912-916Crossref PubMed Scopus (21) Google Scholar)). When used to label AT2 cells for single-channel recording purposes, Lysotracker Red was diluted 1:1000 directly into the patch solution in which the lung tissue was immersed. For immunolocalization of type 2 cells, lung slices were fixed with 4% paraformaldehyde (10 min at room temperature) and then labeled with a 1:1000 dilution (in PBS-bovine serum albumin with 3% goat serum) of surfactant protein A or 1:100 dilution of rat type 2 antibody. Lung slices were then incubated in goat anti-rabbit secondary antibody conjugated to Alexa 568 (1:20,000 dilution; Molecular Probes) for 30 min. Fluorescein-labeled lectin from the crybaby tree Erythrina crista-galli Lectin (ECL; Vector Laboratories, Burlingame, CA) was used to label AT1 cells in lung slice preparations as well as transdifferentiated “AT1-like” cells. Detailed methods for labeling of type 1 cells using ECL can be found in Helms et al. (19Helms M.N. Self J. Bao H.F. Job L.C. Jain L. Eaton D.C. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: 610-618Crossref PubMed Scopus (51) Google Scholar). All tissue samples were mounted onto a glass slide with ProLong antifade reagent (Molecular Probes) and imaged using a laser scanning confocal microscope (model 510 NLO META, Carl Zeiss, Maple Grove, MN). Measurement of O2.¯ Release—Dihydroethidium (DHE; purchased from Invitrogen-Molecular Probes) is a fluorescent probe that intercalates into DNA and has an excitation wavelength of 520 nm and an emission of 610 nm. We have recently established that 2-hydroxyethidium production from DHE can be used as a quantitative measure of O2.¯ production in epithelial monolayers (33Yu L. Bao H.F. Self J.L. Eaton D.C. Helms M.N. Am. J. Physiol. Renal Physiol. 2007; 293: 1666-1677Crossref PubMed Scopus (52) Google Scholar). Additionally, other groups have shown that DHE measurements can be used to measure changes in intracellular O2.¯ levels in cultured cells and frozen tissue sections (8Dikalov S. Griendling K.K. Harrison D.G. Hypertension. 2007; 49: 717-727Crossref PubMed Scopus (393) Google Scholar, 27Munzel T. Afanas’ev I.B. Kleschyov A.L. Harrison D.G. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1761-1768Crossref PubMed Scopus (229) Google Scholar). In this study alveolar type 2 cells were isolated using methodologies described in Dobbs et al. (9Dobbs L.G. Gonzalez R. Williams M.C. Am. Rev. Respir. Dis. 1986; 134: 141-145PubMed Google Scholar) and Jain et al. (24Jain L. Chen X.J. Ramosevac S. Brown L.A. Eaton D.C. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 280: 646-658Crossref PubMed Google Scholar) and maintained in culture for up to 7 days in Dulbecco’s modified Eagle’s medium/F-12-50/50 media with 10% fetal bovine serum, 2 mm l-glutamine, 20 units/ml penicillin-streptomycin, 84 μm gentamycin, 1 μm dexamethasone, and 10 ng/ml keratinocyte growth factor/fibroblast growth factor-7 growth factors for O2.¯ measurements. Before O2.¯ levels were measured, cells were rinsed with PBS and then incubated with 2 μm DHE in PBS solution for 30 min in a light-protected humidified 5% CO2 chamber maintained at 37 °C. After DHE labeling, cells were fixed in 4% paraformaldehyde and sealed between a glass slide and coverslip with Vectashield (Vector Laboratories) mounting medium. DHE fluorescence was detected and quantified with a Zeiss LSM 510 NLO META laser scanning confocal microscope and compatible LSM 5 Image Browser software. Western Blot Analysis—Standard Western blot analysis was used to demonstrate that alveolar type 2 cells cultured on plastic begin to express proteins that are specific for type 1 cells as early as 72 h in culture. Briefly, protein lysate was electrophoresed on a 7.5% acrylamide gel under denaturing conditions and then transferred to Protran nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked in TBST buffer (10 mm Tris, pH 7.5, 70 mm NaCl, and 0.1% Tween) with 5% dry milk and then incubated with rat type I-40 specific antibody (obtained from Dr. Leland Dobbs (11Dobbs L.G. Williams M.C. Gonzalez R. Biochim. Biophys. Acta. 1988; 970: 146-156Crossref PubMed Scopus (214) Google Scholar) and used at 1:100-fold dilution) for 1 h at room temperature. IgG-alkaline phosphatase-labeled secondary antibody (KPL, Gaithersburg, MD) was added at a concentration of 1 μg/10 ml of TBST and incubated for another 1 h at room temperature. After being washed thoroughly, alkaline phosphatase (AP) signal was detected using CDP-Star chemiluminescent substrate for AP (Tropix, Bedford, MA). Blots were analyzed on a Kodak Image Station 2000MM (Carestream Molecular Imaging, New Haven, CT) and compatible Kodak MI software. Methods for Statistical Analysis—ENaC Po values were examined before and after the redox state of the cell had been pharmacologically altered in either alveolar type 1 or type 2 cells. Therefore, the same patch clamp recording before drug treatment could be used as its own control, and statistical significance could be determined by paired t test analysis, with p values <0.05 considered significant. Statistical significance for O2.¯ release was determined using one-way analysis of variance and the post hoc Holm-Sidak test, with p ≤ 0.05 assumed to be statistically significant. Immunolocalization of Alveolar Cells for Patch Clamp Analysis—We have recently reported epithelial Na+ transport properties of alveolar type 1 cells in lung tissue slices, which represent a novel and physiologically relevant model for studying the ion transport mechanism responsible for lung fluid balance. Here, we report for the first time ion transport properties of type 2 cells in the same model. We positively identified type 2 cells in lung slices by labeling type 2 cells with either polyclonal antibodies (Fig. 1, A and C) or vital cell-permeable dyes. Our immunohistochemical data identified the cuboidal cells, located at the junction of type 1 cells, for patch clamp analysis. Fig. 1A shows that the cells identified are indeed secreting surfactant protein A, an essential protein for lung compliance produced only by type 2 cells. LysoTracker Red (Fig. 1, B, 40× magnification, and D, 100× magnification) is a vital dye that can permeate the cell membrane and binds to spherical organelles with low internal pH or surfactant-producing lamellar bodies in type 2 cells (not found in type 1 cells). In Fig. 1C, we additionally verified that type 2 cells were indeed immunoreactive with specific rat type II-70 (RTII-70) antibody, which has been extensively characterized by others as a type 2-specific marker. Alongside each panel of labeled alveolar type 2 cells (using either LysoTracker Red, surfactant protein A, or rat type II-70 antibodies in 1A-C), we also counter-labeled alveolar type 1 cells with ECL to contrast the distinct morphology and localization of type 1 and type 2 cells. Single Channel Activity in Alveolar Type 2 Cells in Lung Slices—We have measured for the first time single channel activity from type 2 cells in situ and found that type 2 cells indeed have functional epithelial sodium channels with highly selective and non-selective transport properties when accessed from live lung tissue. A representative single channel trace is shown in Fig. 2A at −60 through +60 mV (-Vp) holding potentials. Figs. 2, B and C, shows current-voltage relationships that are typical of highly selective channels (HSC) and non-selective channels (NSC), respectively. For clarity, we enlarged a portion of the recording taken at −80 mV (-Vp) in Fig. 2D highlighting both the smaller conducting HSC as well as the larger NSC, found within the same patch of cell surface membrane. Additionally, in Fig. 2E we generated an all-points histogram (and fitted the data to a Gaussian function) from the representative trace in Fig. 2D to show that, indeed, HSCs and NSCs are present in alveolar type 2 cells patch-clamped from our lung slice model, and on occasion are both opened simultaneously. We also observed channels that have the electrophysiological properties commonly associated with the cystic fibrosis transmembrane conductance regulator (CFTR) channels in our cell-attached recordings. Fig. 2F shows a representative patch clamp recording of anion channels in type 2 cells (using the same conditions used to examine Na+ current). In this trace the downward deflections likely represent Cl- movement across the cellular membrane. The gating of this channel, with quick bursts of activity interrupted by brief flickering closures, is not different from the normal channel characteristics of CFTR that have been described by others (23Yue H. Devidas S. Guggino W.B. J. Biol. Chem. 2000; 275: 10030-10034Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 30Sheppard D.N. Gray M.A. Gong X. Sohma Y. Kogan I. Benos D.J. Scott-Ward T.S. Chen J.H. Li H. Cai Z. Gupta J. Li C. Ramjeesingh M. Berdiev B.K. Ismailov I.I. Bear C.E. Hwang T.C. Linsdell P. Hug M.J. J. Cyst. Fibros. 2004; 3: 101-108Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The conductance of this anion channel is shown in Fig. 2G; there are three open levels fit by gaussian distributions, with a calculated conductance of 10 pS that is also characteristic of CFTR. We estimate that these channels occur in less than 5% of total cell recordings. Distribution of Conductances Recorded from Lung Cells—To compare the distribution of conductances between AT1 and AT2 cells, we generated a histogram of conductances in the two cell types using the same intervals and then fitted the histograms to two Gaussian functions. In Fig. 3 the normal distribution of Na+ channel conductances obtained from type 2 cells is compared with the conductances of cation channels found in type 1 cells (19Helms M.N. Self J. Bao H.F. Job L.C. Jain L. Eaton D.C. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: 610-618Crossref PubMed Scopus (51) Google Scholar). The conductances were calculated from the linear portion of the I-V curves (between −20 and −80 mV) measured in type 1 and 2 cells under control conditions. The fits show that both type 1 and type 2 cells have HSC and NSC channels with similar conductances in lung slices. The ratios of HSC:NSC are 1.5 ± 0.16 and 0.95 ± 0.19 for type 2 and 1 cells, respectively. Seemingly, HSCs occur more frequently in type 2 cells, and both types of channels are present with equal frequency in alveolar type 1 cells. In a recent review we describe amiloride-sensitive channels with high, moderate, or no selectivity for Na+ over K+ in the apical membranes of epithelial cells (26Matalon S. Lazrak A. Jain L. Eaton D.C. J. Appl. Physiol. 2002; 93: 1852-1859Crossref PubMed Scopus (112) Google Scholar). Hence, reports of NSCs and HSCs in cultured and primary cell models previously reported are not different from what we currently describe in alveolar type 1 and type 2 cells patch clamped from lung tissue. Reactive Nitrogen Signaling in the Alveolar Epithelium—Our previous research has identified nitric oxide as a negative regulator of Na+ channel function in cultured cells (20Helms M.N. Yu L. Malik B. Kleinhenz D.J. Hart C.M Eaton D.C. Am. J. Physiol. Cell Physiol. 2005; 289: 717-726Crossref PubMed Scopus (56) Google Scholar, 22Jain L. Chen X.J. Brown L.A. Eaton D.C. Am. J. Physiol. 1998; 274: L475-L484PubMed Google Scholar, 33Yu L. Bao H.F. Self J.L. Eaton D.C. Helms M.N. Am. J. Physiol. Renal Physiol. 2007; 293: 1666-1677Crossref PubMed Scopus (52) Google Scholar). However, nitric oxide signaling in type 1 cells has not been examined at a single channel level. It is important to test the effect of nitric oxide on sodium transport properties of all cells that make up the alveolar epithelium in live tissue slices, as inhibition of ENaC may lead to undesirable effects related to lung edema clearance. This is also particularly important since inhaled NO is currently being used to treat pulmonary hypertension in newborns and older patients. Fig. 4, A and B, shows that PAPA-NONOate significantly decreased sodium transport in AT2 cells patched from lung slices but failed to alter ENaC NPo in AT1 cells. In 18 independent patch clamp studies of type 2 cells accessed in situ, 1.5 μm PAPANONOate significantly decreased ENaC NPo from control values of 1.38 ± 0.26 to 0.82 ± 0.16 (p < 0.05). Higher concentrations of the NO donor (500 μm) further decreased ENaC NPo values to 0.37 ± 0.22 in type 2 cells. In type 1 cells, however, ENaC NPo values did not differ from control values of 0.63 ± 0.12 when either 1.5 or 500 μm nitric oxide donor was applied to the bath media. We show segments of the single channel recordings from more than 20 min of recording after nitric oxide treatment in AT2 and AT1 cells in Figs. 5 and 6, respectively. Fig. 5 shows that before 1.5 μm nitric oxide application to the patch clamp bath, the probability of seeing HSC and NSC channels in AT2 cells is greater than after NO treatment. Approximately 20 min after the initial application of 1.5 μm nitric oxide treatment, ENaC activity was still detectable albeit significantly lower than untreated levels in several AT2 cell recordings (data not shown). Therefore, we increased the concentration of nitric oxide releasing compound to 500 μm ∼22 min into the recording time (where indicated in Fig. 5). Increasing the concentration of nitric oxide treatment by 100-fold led to a further significant decrease in ENaC activity in type 2 cells. Interestingly, however, neither 1.5 nor 500 μm nitric oxide treatment changed ENaC NPo in alveolar type 1 cells (shown in Fig. 6). Together, these results indicate that nitric oxide signaling mechanisms in alveolar type 1 and type 2 cells differ considerably.FIGURE 5AT2; nitric oxide decreases lung ENaC activity in alveolar type 2 cells patched from lung slice preparation. A, after a control recording period o" @default.
• W2052571887 created "2016-06-24" @default.
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• W2052571887 date "2008-08-01" @default.
• W2052571887 modified "2023-10-15" @default.
• W2052571887 title "Redox Regulation of Epithelial Sodium Channels Examined in Alveolar Type 1 and 2 Cells Patch-clamped in Lung Slice Tissue" @default.
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