High gene flow due to pelagic larval dispersal among South Pacific archipelagos in two amphidromous gastropods (Neritomorpha: Neritidae).

The freshwater stream fauna of tropical oceanic islands is dominated by amphidromous species, whose larvae are transported to the ocean and develop in the plankton before recruiting back to freshwater habitat as juveniles. Because stream habitat is relatively scarce and unstable on oceanic islands, this life history would seem to favor either the retention of larvae to their natal streams, or the ability to delay metamorphosis until new habitat is encountered. To distinguish between these hypotheses, we used population genetic methods to estimate larval dispersal among five South Pacific archipelagos in two amphidromous species of Neritid gastropod (Neritina canalis and Neripteron dilatatus). Sequence data from mitochondrial cytochrome oxidase I (COI) revealed that neither species is genetically structured throughout the Western Pacific, suggesting that their larvae have a pelagic larval duration (PLD) of at least 8 weeks, longer than many marine species. In addition, the two species have recently colonized isolated Central Pacific archipelagos in three independent events. Since colonization, there has been little or no gene flow between the Western and Central Pacific archipelagos in N. canalis, and high levels of gene flow across the same region in N. dilatatus. Both species show departures from neutrality and recent dates for colonization of the Central Pacific archipelagos, which is consistent with frequent extinction and recolonization of stream populations in this area. Similar results from other amphidromous species suggest that unstable freshwater habitats promote long-distance dispersal capabilities.

Phenotype-dependent synthesis of transferrin receptor in rat alveolar epithelial cell monolayers.

The iron carrier protein transferrin plays a prominent antioxidant and anti-bacterial role in the lower respiratory tract and is present at elevated concentrations in lung epithelial lining fluid relative to plasma. The level of transferrin receptor synthesis in primary cultures of rat alveolar epithelial cells (AECs) was investigated. Transferrin receptor was found to be synthesized early in AEC cultures with the alveolar type II cell-like phenotype. Cell-surface receptor localization was attenuated upon apparent transdifferentiation to the alveolar type I cell-like phenotype later in culture. Binding of (125)I-labeled transferrin to the receptor indicated that surface and total cellular transferrin receptor levels were decreased in the type I-like cells. Inclusion of keratinocyte growth factor (KGF) in culture media (10 ng/ml) resulted in retention of transferrin receptor localized to the basolateral surface. Transferrin-receptor-specific internalization of (59)Fe-transferrin was also limited to the basolateral surface of KGF-treated monolayers. These data suggest that alveolar type II (but not type I) cells express functional transferrin receptor in adult rat alveolar epithelium.

Vesicular stomatitis virus G-pseudotyped lentivirus vectors mediate efficient apical transduction of polarized quiescent primary alveolar epithelial cells.

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.

Re-evaluating the Na(+) conductance of adult rat alveolar type II pneumocytes: evidence for the involvement of cGMP-activated cation channels.

1. Alveolar epithelial type II pneumocytes were isolated and purified from adult rat lung by elastase digestion and differential adhesion, and cultured in serum-free medium for approximately 2 days on glass coverslips for subsequent patch-clamp studies employing symmetrical sodium isethionate solutions. 2. Whole-cell Na(+) currents exhibited essentially linear current-voltage relationships which were mildly inhibited (by approximately 25 %) by 10 microM amiloride. In contrast, 1 mM Zn(2+) inhibited the currents by approximately 55 % with an IC(50) of approximately 134 microM and maximal blockade achieved between 5 and 10 mM. The effects of Zn(2+) and amiloride were additive, and independent of the order of blocker addition. 3. Gd(2+), Zn(2+) and La(3+) at 10 mM were all effective at rapidly, reversibly and significantly blocking the amiloride-insensitive currents by approximately 60%. in contrast, Ni(2+) was a very weak inhibitor (30 % inhibition at 10 mM). 4. Pimozide (10 microM) caused inhibition of whole-cell cation conductance by approximately 55 %. The inhibitory effect of pimozide was concentration dependent with an IC(50) of approximately 1 microM and was maximally effective between 10 and 30 microM. Sequential addition of Zn(2+) and pimozide, in either order, revealed no overlapping inhibitory effect on the amiloride-insensitive conductance, and supported the notion that the Zn(2+)- and pimozide-sensitive currents are identical. 5. The amiloride-insensitive, Zn(2+)-blockable conductance was characterised by a Na(+)/K(+) permeability ratio (P(Na)/P(K)) of 0.73 +/- 0.02. 6. 8Br-cGMP (100 microM), a membrane-permeable analogue of cGMP, evoked a robust activation of whole-cell cation conductance to 220 % of control. This activation was apparent in either the absence or the presence of 10 microM amiloride, but was completely abolished in the presence of Zn(2+). 7. These data support the in vivo and in situ observations of a substantial amiloride-resistant Na(+) conductance, demonstrate directly that cyclic nucleotide-gated non-selective cation channels are functionally expressed in alveolar epithelial type II cells, and suggest that these channels may contribute to the fluid-reabsorptive driving force in adult lung.

Differential regulation of rat aquaporin-5 promoter/enhancer activities in lung and salivary epithelial cells.

Aquaporin-5 (AQP5) is a water channel protein that is selectively expressed in respiratory, salivary, and lacrimal tissues. In order to establish the tissue-specific transcriptional programs that underlie its lung- and salivary-specific expression, a 4.5-kilobase pair DNA fragment encompassing the 5'-flanking region of the rat AQP5 gene has been characterized in detail. A major transcription start site utilized in lung and salivary glands has been localized downstream of a TATAA-like motif. Transient transfection assays of -4.3- and -1.7-AQP5-luciferase constructs in AQP5-expressing lung (MLE-15) and salivary (Pa-4) cells and nonexpressing fibroblast (NIH3T3) and epithelial (HeLa) cells demonstrate preferential transcriptional enhancement of reporter activities in MLE-15 and Pa-4 cells. Transient transfection assays of a series of 5' --> 3' deletion constructs of -4.3-AQP5-luciferase suggest that a common salivary and lung enhancer is located between nucleotides -274 and -139, and a lung-specific enhancer is located between nucleotides -894 and -710. There is one putative lung-specific repressor located in the region of nucleotides -1003/-894 and a common lung and salivary repressor located at nucleotides -503/-385. Moreover, 3' --> 5' deletions up to -171 and -127 base pairs almost abolish transcriptional activation in salivary and lung cells, respectively. Together, our findings indicate that the combination of enhancer/repressor elements within the proximal 5'-flanking region of rat AQP5 gene dictates its restricted expression in both lung and salivary cells.

Rates of protein transport across rat alveolar epithelial cell monolayers.

The transport of model proteins, ranging from 12,300 to 150,000 Da, across tight rat alveolar epithelial cell monolayers (> 2000omegacm2) grown on polycarbonate filters, was studied. Model proteins were 14C-cytochrome c, 14C-ovalbumin, granulocyte-colony stimulating factor (G-CSF), 14C-bovine serum albumin (BSA), 125I-transferrin, and 14C-immunoglobulin G. Cytochrome c was extensively metabolized, as indicated by < 10% of the dose being translocated in intact form. This contrasts with 20-80% for the other model proteins studied. The flux of cytochrome c and G-CSF was symmetric in the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. By contrast, the flux of intact ovalbumin, BSA, transferrin and immunoglobulin G showed asymmetry, with the ab flux being higher by 2-5 times. There was no relationship between ab or ba fluxes and the molecular weights of these four model proteins. Since some of the proteins were translocated at much greater rates than are consistent with restricted diffusion or pinocytosis, receptor-mediated or adsorptive transcytosis may be involved.

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels.

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha-, beta- and gamma-subunits of rat epithelial Na(+) channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (P(Na(+))/P(K(+)) approximately 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells.

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both alpha1 and beta1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha1- and beta1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha1- and beta1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+ transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.

Modulation of t1alpha expression with alveolar epithelial cell phenotype in vitro.

T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1alpha expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1 (DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1alpha expression was quantified by Northern and Western blotting, respectively. Expression of T1alpha progressively increases in AEC grown in MDSF +/- BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1alpha expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1alpha on days 4 and 8. T1alpha expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1alpha expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.

We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.e., the time of plating) or Day 4 or 6 through Day 8 in culture. AECs monolayers express AQP5 only on their apical surfaces as determined by cell surface biotinylation studies. Control AECs grown in the absence of KGF through Day 8 express increasing levels of AQP5, consistent with transition toward the AT1 cell phenotype. Exposure of AECs to KGF from Day 0 results in decreased AQP5 expression, retention of a cuboidal morphology, and greater numbers of lamellar bodies relative to control on Day 8 in culture. AECs treated with KGF from Day 4 or 6 exhibit a decrease in AQP5 expression through subsequent days in culture, as well as an increase in expression of surfactant apoproteins. These data, showing that KGF both prevents and reverses the increase in AQP5 (and decrease in surfactant apoprotein) expression that accompanies progression of the AT2 toward the AT1 cell phenotype, support the concepts that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible and that KGF may play a major role in modulating AEC phenotype.

Size-dependent dextran transport across rat alveolar epithelial cell monolayers.

The transport of dextrans (approximately 4 to approximately 150 kDa) across an in vitro model of the alveolar epithelial barrier was studied to determine the effects of molecular size on pulmonary absorption of macromolecular drugs. Fluorescein isothiocyanate (FITC)-labeled dextrans (FDs) with average molecular weights (all in kDa) of 3.86 (FD4), 9 (FD10), 19.8 (FD20), 40.5 (FD40), 71.6 (FD70), and 156.9 (FD150) were utilized as model macromolecular drugs. Unidirectional fluxes of FDs at 37 and 4 degrees C were measured from the appearance rates of FD in the receiver fluid of open-circuited monolayers (>2000 omega-cm2) of rat alveolar epithelial cells. Apparent permeability coefficients (P(app)) were estimated from the observed flux and the corresponding concentration gradient of FD. Results showed that FD fluxes were the same in both apical-to-basolateral (AB) and opposite (BA) directions at each molecular weight studied. The P(app) was not significantly different at 0.5 and 1.0 mg/mL FD40 donor concentrations. The FD P(app) (x 10(-8)cm/s) decreased gradually from 1.35 for FD4 to 0.32 for FD40, indicating an apparent inverse relationship between P(app) and molecular weight of FD. By contrast, P(app) was about the same at 0.13 for both FD70 and FD150. When experimental temperature was lowered to 4 degrees C, P(app) decreased by approximately 40% for FDs of 4 through 40 kDa, whereas the decrease in P(app) was by approximately 80% for larger FDs of both 70 and 150 kDa. Moreover, these FDs were found to be relatively intact (approximately 90%) in either receiver fluid after 5-h flux experiments without detectable levels of metabolites in the respective donor fluid, suggesting that alveolar epithelial cells allow translocation of FDs intact across the barrier. Equivalent pore analysis, assuming restricted diffusion of FDs of 4-40 kDa via cylindrical, water-filled pores across the cell monolayer revealed a population of large equivalent pores with approximately 5.6 nm radius. These data suggest that smaller macromolecules (radius <5 nm) traverse the alveolar epithelial barrier via paracellular pathways, and that larger (i.e., radius > or = 6 nm) macromolecules likely cross the barrier via other pathways (e.g., pinocytosis).

Effects of Mycobacterium tuberculosis on the bioelectric properties of the alveolar epithelium.

To investigate the hypothesis that Mycobacterium tuberculosis penetrates the alveolar epithelium by downregulating its barrier properties, we evaluated the interactions between M. tuberculosis and rat alveolar epithelial cell monolayers that are believed to share electrophysiologic properties of the human alveolar epithelium. Nonproteinaceous components of M. tuberculosis caused marked declines in electrical resistance and equivalent short-circuit current of the alveolar epithelial cell monolayers, indicating a reduction in the capacity to maintain tight intercellular junctions and to actively reabsorb sodium. M. tuberculosis elicited production of TNF-alpha mRNA and protein by alveolar epithelial cells, and the effects of recombinant TNF-alpha on the bioelectric properties of the alveolar epithelial paralleled those of M. tuberculosis. Furthermore, the effects of M. tuberculosis on alveolar epithelial resistance were abrogated by neutralizing anti-TNF-alpha antibodies. These results indicate that M. tuberculosis elicits production of TNF-alpha, which in turn reduces the bioelectric barrier properties of the alveolar epithelium. These findings provide insight into potential mechanisms by which M. tuberculosis establishes infection and disease in the lung.

Horseradish peroxidase transport across rat alveolar epithelial cell monolayers.

PURPOSE: To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm2) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters. METHODS: Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was uses to quantify HRP activity in the bathing fluid and cells. RESULTS: Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 microM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4 degrees C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T1/2) of approximately 5 min, reaching a plateau at approximately 67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T1/2 of approximately 20 min, attaining a steady-state at approximately 13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T1/2 = approximately 5 min) reaching a steady-state at approximately 35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T1/2 = approximately 70 min), leveling off at 50% of the initial cell-associated HRP. CONCLUSIONS: HRP appears to be transported relatively intact (approximately 50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm2/h.

Effects of EGF on alveolar epithelial junctional permeability and active sodium transport.

We evaluated the effects of epidermal growth factor (EGF) on transepithelial resistance (Rt) and active ion transport by alveolar epithelial cell (AEC) monolayers on tissue culture-treated polycarbonate filters. Rat type II cells were cultured in completely defined serum-free medium (MDSF) or MDSF supplemented with EGF. The addition of EGF from either day 0 (chronic) or day 4 (subacute) resulted in significant increases in Rt and short-circuit current (ISC) on day 5. After subacute exposure, these effects were delayed in onset by 6-12 h and sustained for > 24 h. Basolateral (but not apical) EGF was responsible for these effects, which were prevented by preincubation with tyrphostin RG-50864, a reversible specific inhibitor of the EGF receptor tyrosine kinase. ISC decreased, with a sensitivity to apical inhibitors of sodium transport in the order benzamil > amiloride > 5-(N-ethyl-N-isopropyl) amiloride in MDSF +/- EGF, and was completely inhibited by the addition of basolateral ouabain. Net sodium flux and Na+, K+ -ATPase activity both increased approximately 50% in the presence of EGF. These results indicate that 1) EGF decreases tight junctional permeability and increases active sodium transport by AEC monolayers via basolaterally located EGF receptors, and 2) the pathways for AEC sodium entry and exit (+/- EGF) are apical high amiloride affinity sodium channels and basolateral sodium pumps.

Reversible transdifferentiation of alveolar epithelial cells.

Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

Basolateral localization of Na(+)-HCO3- cotransporter activity in alveolar epithelial cells.

We investigated the polarized distribution of Na(+)- and HCO3(-)-dependent recovery from intracellular acidification in alveolar epithelial cell monolayers. Rat alveolar type II cells were grown in primary culture on detachable tissue culture-treated Nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer. Cells were loaded with the pH-sensitive dye BCECF and intracellular pH (pHi) measured spectrofluorometrically. Monolayers were studied at ambient temperature on days 3-4 in culture, coincident with the development of high tissue resistance (RT > or = 1000 omega.cm2). After the cells were acidified by NH3 prepulse, pHi recovered to baseline when Na+ was present in the basolateral fluid, but did not recover when Na+ was present only in the apical fluid. This basolateral Na(+)-dependent pHi recovery in the presence of HCO3-/CO2 was reduced, but present, in experiments where dimethylamiloride (DMA, 100 microM) or the stilbene derivative DIDS (500 microM) was in basolateral fluid. However, recovery was completely inhibited when both DMA and DIDS were present basolaterally. pHi recovery was not inhibited under Cl(-)-free conditions, indicating that cytoplasmic realkalinization was not effected by Na(+)-dependent Cl-HCO3- exchange. These data indicate that alveolar epithelial cells express a basolateral Na(+)- and HCO3(-)-dependent, DIDS-sensitive, Cl(-)-independent pHi recovery process that probably represents Na(+)-HCO3(-)-cotransport (symport). Basolateral Na(+)-HCO3- cotransport modulates pHi in alveolar epithelial cells, may contribute to regulation of intracellular volume and osmolarity, and may participate in signal transduction by hormones and growth factors.

Cl(-)-HCO3- exchanger isoform AE2 is restricted to the basolateral surface of alveolar epithelial cell monolayers.

We investigated the polarized distribution and isoform specificity of anion exchange (Cl(-)-HCO3- exchange) in alveolar epithelial cell monolayers. Rat alveolar type II epithelial cell monolayers were grown in primary culture on detachable tissue culture-treated nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer, the cells loaded with pH-sensitive dye, and intracellular pH (pHi) measured spectrofluorometrically. To assay for Cl(-)-HCO3- exchange, monolayers were incubated in medium containing 24 mM HCO3-/5% CO2 and 140 mM NaCl at pH 7.4 and acutely alkalinized by replacement of the fluid by HCO3(-)-free buffer containing Hepes (6 mM) at pH 7.4. Monolayers exhibited basolateral (but not apical) Cl(-)-dependent, Na(+)-independent recovery from an alkaline load that was abolished when Cl- was substituted by equimolar gluconate in the basolateral fluid, or if DIDS (500 microM) was present basolaterally. Substitution of gluconate for Cl- in the basolateral fluid, but not the apical fluid, resulted in a rise in steady-state pHi that was reversible on replacement of the basolateral fluid with Cl(-)-containing buffer, which occurred in HCO3(-)- but not Hepes-buffered medium. These data indicate that alveolar epithelial cells express basolateral membrane domain of these cells. Northern analysis of alveolar epithelial cell mRNA using anion exchanger (AE) isoform-specific cDNA probes indicates that alveolar epithelial cells express the AE2 isoform predominantly, if not exclusively, and do not express detectable AE1 (i.e., band-3 protein) or AE3.(ABSTRACT TRUNCATED AT 250 WORDS)