Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers

Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% O(2), 5% CO(2)), and resultant changes in bioelectric properties [i.e., short-circuit current (I(sc)) and transepithelial resistance (R(t))] were measured in Ussing chambers. Results showed that I(sc) decreased with duration of exposure to hypoxia, while relatively little change was observed for R(t). In normoxia, amiloride inhibited approximately 70% of I(sc). The amiloride-sensitive portion of I(sc) decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of I(sc) was not affected. Na pump capacity measured after permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% O(2) for 24 h, as did the capacity of amiloride-sensitive Na uptake measured after imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion.     

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers.

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-beta(1) (TGF-beta(1)) decreases overall monolayer resistance (R(te)) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-beta(1) (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5-7 in culture, tight-junctional resistance (R(tj), kOmegacm(2)) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-beta(1) or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/R(te)) and tight-junctional conductance (1/R(tj)) are 0.59 and 0.14 mS/cm(2) for control monolayers in MDSF, 1.59 and 0.38 mS/cm(2) for monolayers exposed to TGF-beta(1), and 0.38 and 0.18 mS/cm(2) for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-beta(1)-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.     

Release of biologically active TGF-beta1 by alveolar epithelial cells results in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal fibrotic lung disease. Transforming growth factor (TGF)-beta1 is present in a biologically active conformation in the epithelial cells lining lesions with advanced IPF. To determine the role of aberrant expression of biologically active TGF-beta1 by alveolar epithelial cells (AECs), the AECs of explanted normal rat lungs were transfected with the TGF-beta1 gene using the retrovirus pMX-L-s223,225-TGF-beta1. In situ hybridization using a digoxigenin-labeled cDNA of the puromycin resistance gene contained in the pMX demonstrated that pMX-L-s233,225-TGF-beta1 was selectively transfected into AECs of the explants. Conditioned media overlying explants obtained 7 days after being treated with pMX-L-s223,225-TGF-beta1 contained 14.5 +/- 3.15 pg/ml of active TGF-beta1. With the use of Masson's trichrome staining of explant sections obtained 14 days after transfection, there were lesions similar to those in IPF, characterized by type II AEC hyperplasia, interstitial thickening, extensive increase in interstitial and subepithelial collagen, an increase in the number of fibroblasts, and areas resembling fibroblast buds. Collagens I, III, IV, and V and fibronectin were increased in explants treated with pMX-L-s223,225-TGF-beta1. The findings in the current study suggest that IPF may be a disorder of epithelial cells and not inflammatory cells.     

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

We evaluated theeffects of acute hyperoxic exposure on alveolar epithelial cell (AEC)active ion transport and on expression ofNa⁺ pump(Na⁺-K⁺-ATPase)and rat epithelial Na⁺ channelsubunits. Rat AEC were cultivated in minimal defined serum-free medium(MDSF) on polycarbonate filters. Beginning on day5, confluent monolayers were exposedto either 95% air-5% CO₂(normoxia) or 95% O₂-5%CO₂ (hyperoxia) for 48 h.Transepithelial resistance(R_t) andshort-circuit current(I_sc) weredetermined before and after exposure.Na⁺ channel -, -, and-subunit andNa⁺-K⁺-ATPase₁- and₁-subunit mRNA levels werequantified by Northern analysis.Na⁺ pump₁- and₁-subunit protein abundance wasquantified by Western blotting. After hyperoxic exposure,I_sc across AECmonolayers decreased by ~60% at 48 h relative to monolayersmaintained under normoxic conditions.Na⁺ channel -subunit mRNAexpression was reduced by hyperoxia, whereas - and -subunit mRNAexpression was unchanged. Na⁺ pump₁-subunit mRNA was unchanged,whereas ₁-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in₁-subunit protein. Becausekeratinocyte growth factor (KGF) has recently been shown to upregulateAEC active ion transport and expression ofNa⁺-K⁺-ATPaseunder normoxic conditions, we assessed the ability of KGF to preventhyperoxia-induced changes in active ion transport by supplementingmedium with KGF (10 ng/ml) from day2. The presence of KGF prevented theeffects of hyperoxia on ion transport (as measured byI_sc) relativeto normoxic controls. Levels of₁ mRNA and protein wererelatively preserved in monolayers maintained in MDSF and KGF comparedwith those cultivated in MDSF alone. These results indicate that AECnet active ion transport is decreased after 48 h of hyperoxia, likelyas a result of a decrease in the number of functionalNa⁺ pumps per cell. KGF largelyprevents this decrease in active ion transport, at least in part, bypreserving Na⁺ pump expression.

     

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP >or= UTP > adenosine >or= 2-methylthioadenosine-5'-diphosphate, with alpha,beta-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATPalphaS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-kappaB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.     

TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.     

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.     

Absorption of intact albumin across rat alveolar epithelial cell monolayers

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral (ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical (ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of approximately 1.6 microM and maximal transport rate of approximately 0.15 fmol.cm(-2).s(-1). At 15 and 4 degrees C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an approximately 60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current- voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on delta pH and not on the absolute pH.     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Regulation of ion transport in hypophysial pars intermedia follicular cell monolayers

We have previously reported that cultured monolayers of folliculo-stellate cells (FC) of adenohypophysial pars tuberalis (PT) and pars distalis (PD) origin express morphological and electrical properties typical of ion and fluid transporting epithelia. The objective of the present study was to examine whether cells expressing similar transport properties exist also in the pars intermedia (PI), an area of the adenohypophysis very poorly vascularized, where a cell type expressing such functions would be expected to play an especially significant role in the local regulation of the interstitial fluid content and circulation. Enzymatically and mechanically dispersed bovine pars intermedia fragments yield monolayers of polygonal, contact inhibited cells which rapidly develop domes. Such cells exhibit morphological features and growth properties very similar or identical to those expressed by cells previously identified af FC cells in PD and PT cultures. Similarly to their counterparts in the PD and PT, the PI FC display a potential difference and a resistance when mounted in Ussing chambers. Isoproterenol, prostaglandin E2, bradykinin and lysine vasopressin are able to stimulate active ion transport across FC monolayers. These data indicate that the PI contains ion transporting FC and suggest important local regulatory functions for these cells.