Regulation and function of the two-pore-domain (K2P) potassium channel Trek-1 in alveolar epithelial cells

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

Hypoxia and beta 2-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of beta(2)-agonists. To investigate the mechanism of beta(2)-agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O(2) for 24 h. Hypoxia reduced transepithelial sodium current and amiloride-sensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially beta and gamma) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of beta-and gamma-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxia-induced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that beta(2)-agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.     

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.     

Expression and function of the C5a receptor in rat alveolar epithelial cells

Although alveolar epithelial cells (AEC) form an important barrier for host defenses in the lung, there is limited information about ways in which AEC can directly participate in the lung inflammatory response. In the current studies, primary cultures of rat AEC (RAEC) have been shown to specifically bind recombinant rat C5a at high affinity and in a saturable manner. This binding was enhanced in a time-dependent manner by pre-exposure of RAEC to LPS, IL-6, or TNF-alpha, the increased binding of C5a being associated with increased levels of mRNA for the C5a receptor (C5aR). Exposure of RAEC to C5a also caused increased expression of mRNA for C5aR. As compared with exposure of RAEC to LPS or to C5a alone, exposure to the combination caused enhanced production of TNF-alpha, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1beta. These data indicate that RAEC, when activated, have enhanced binding of C5a in association with increased mRNA for C5aR. The functional outcome is enhanced release of proinflammatory mediators. These data underscore the phlogistic potential of RAEC and the ability of C5a to enhance the phlogistic responses of RAEC.     

Mechanism of K+ channel block by verapamil and related compounds in rat alveolar epithelial cells

The mechanism by which the phenylalkylamines, verapamil and D600, and related compounds, block inactivating delayed rectifier K+ currents in rat alveolar epithelial cells, was investigated using whole-cell tight- seal recording. Block by phenylalkylamines added to the bath resembles state-dependent block of squid K+ channels by internally applied quarternary ammonium ions (Armstrong, C.M. 1971. Journal of General Physiology. 58:413-437): open channels are blocked preferentially, increased [K+]o accelerates recovery from block, and recovery occurs mainly through the open state. Slow recovery from block is attributed to the existence of a blocked-inactivated state, because recovery was faster in three situations where recovery from inactivation is faster: (a) at high [K+]o, (b) at more negative potentials, and (c) in cells with type l K+ channels, which recover rapidly from inactivation. The block rate was used as a bioassay to reveal the effective concentration of drug at the block site. When external pH, pHo, was varied, block was much faster at pHo 10 than pHo 7.4, and very slow at pHo 4.5. The block rate was directly proportional to the concentration of neutral drug in the bath, suggesting that externally applied drug must enter the membrane in neutral form to reach the block site. High internal pH (pHi 10) reduced the apparent potency of externally applied phenylalkylamines, suggesting that the cationic form of these drugs blocks K+ channels at an internal site. The permanently charged analogue D890 blocked more potently when added to the pipette than to the bath. However, lowering pHi to 5.5 did not enhance block by external drug, and tertiary phenylalkylamines added to the pipette solution blocked weakly. This result can be explained if drug diffuses out of the cell faster than it is delivered from the pipette, the block site is reached preferentially via hydrophobic pathways, or both. Together, the data indicate the neutral membrane-bound drug blocks K+ channels more potently than intracellular cationic drug. Neutral drug has rapid access to the receptor, where block is stabilized by protonation of the drug from the internal solution. In summary, externally applied phenylalkylamines block open or inactivated K+ channels by partitioning into the cell membrane in neutral form and are stabilized at the block site by protonation.     

Importance of glycosylation on function of a potassium channel in neuroblastoma cells

The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type), partially glycosylated (N220Q and N229Q), and unglycosylated (N220Q/N229Q) Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.     

Evidence of a functional CFTR Cl(-) channel in adult alveolar epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the fetal lung, but during lung development it gradually disappears in cells of future alveolar spaces. Recent studies have implicated the CFTR in fluid transport by the adult alveolar epithelium, but its presence has not been demonstrated directly. This study re-evaluated CFTR expression and activity in the adult pulmonary epithelium by using freshly isolated rat alveolar type II (ATII) cells. CFTR mRNA was detected by semiquantitative polymerase chain reaction on the day of cell isolation but was rapidly reduced by 60% after 24 h of cell culture. This was paralleled by a similar decrease of surfactant protein A expression and alkaline phosphatase staining, markers of the ATII cell phenotype. CFTR expression increased significantly on day 4 in cells grown on filters at the air-liquid interface compared with cells submerged or grown on plastic. Significantly higher CFTR expression was detected in distal lung tissue compared with the trachea. The CFTR was also found at the protein level in Western blot experiments employing lysates of freshly isolated alveolar cells. Whole cell patch-clamp experiments revealed cAMP-stimulated, 5-nitro-2-(3-phenylpropylamino)-benzoate-sensitive Cl(-) conductance with a linear current-voltage relationship. In cell-attached membrane patches with 100 microM amiloride in pipette solution, forskolin stimulated channels of approximately 4 pS conductance. Our results indicate that 50-250 of functional CFTR Cl(-) channels occur in adult alveolar cells and could contribute to alveolar liquid homeostasis.     

Chloride transport in rabbit esophageal epithelial cells

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).     

Deformation-induced lipid trafficking in alveolar epithelial cells

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.     

Molecular diversity and function of voltage-gated (Kv) potassium channels in epithelial cells

Voltage-gated K+ channels belonging to Kv1-9 subfamilies are widely expressed in excitable cells where they play an essential role in membrane hyperpolarization during an action potential and in the propagation of action potentials along the plasma membrane. Early patch clamp studies on epithelial cells revealed the presence of K+ currents with biophysical and pharmacologic properties characteristic of Kv channels expressed in excitable cells. More recently, molecular approaches including PCR and the availability of more selective antibodies directed against Kv alpha and auxiliary subunits, have demonstrated that epithelial cells from various organ systems, express a remarkable diversity Kv channel subunits. Unlike neurons and myocytes however, epithelial cells do not typically generate action potentials or exhibit dynamic changes in membrane potential necessary for activation of Kv alpha subunits. Moreover, the fact that many Kv channels expressed in epithelial cells exhibit inactivation suggest that their activities are relatively transient, making it difficult to ascribe a functional role for these channels in transepithelial electrolyte or nutrient transport. Other proposed functions have included (i) cell migration and wound healing, (ii) cell proliferation and cancer, (iii) apoptosis and (iv) O2 sensing. Certain Kv channels, particularly Kv1 and Kv2 subfamily members, have been shown to be involved in the proliferation of prostate, colon, lung and breast carcinomas. In some instances, a significant increase in Kv channel expression has been correlated with tumorogenesis suggesting the possibility of using these proteins as markers for transformation and perhaps reducing the rate of tumor growth by selectively inhibiting their functional activity.     

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.     

Chloride channel function: partial charges and dipolar rods

The regulated flow of ions across biological membranes is a process fundamental to all living organisms. The crystal structures of representative chloride channels recently published in Nature, together with the previously determined structures of a potassium channel, provide a solid basis for understanding the chemical principles that govern selective ion flow.     

Expression of intermediate conductance potassium channel immunoreactivity in neurons and epithelial cells of the rat gastrointestinal tract

Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.     

Gap junction-microtubule associations in rat alveolar epithelial cells

Connexin 43 (Cx43) is a predominant gap junction (GJ) protein expressed by alveolar epithelial cells (AEC) in primary cell culture. Cx43 trafficking, assembly, and turnover are regulated by multiple mechanisms, including those mediated by integrins, by extracellular matrix, and by the cytoskeleton. Immunocytochemical double labeling demonstrates association of microtubules with internalization of Cx43-positive GJ plaques. Antibodies against the alpha 5-integrin subunit block cell-matrix interactions without effect on tubulin expression, whereas inhibition of MAP kinase kinase by PD-98059 reduces tubulin expression, based on both Western blot and immunostaining. To examine direct association of microtubules (MT) with GJ plaques, we treated day 3 AEC for 0.5-24 h with colchicine, an inhibitor of tubulin polymerization. After 60 min, MTs were disassembled, whereas Western blot analysis showed no change in tubulin expression. In parallel, colchicine initiated redistribution of immunopositive Cx43 from the membrane to the cytosol. These observations support the premise that direct association of the cytoskeleton with gap junctions plays a significant role in regulation of Cx43 expression and distribution through integrin-mediated signal transduction pathways.     

Transport of sodium and potassium in intestinal epithelial cells

Transport properties of rabbit small intestinal mucosa were investigated $\text{in vitro}$ to characterize the process by which epithelial cells maintain normal Na and K gradients across the cell membrane. Active transport of Na from the cell proceeded at a faster rate in the presence of K; and active transport of K into the cell was stimulated by the presence of Na. Following preincubation at 0°C to reduce tissue K content, a greater transmural electrical potential difference (PD) and short-circuit current (I_sc) developed as the temperature was raised to 37°C if K was present in the bathing solution. The PD and I_sc, which generally reflect the rate of active Na transport in ileum under control conditions, increased immediately upon raising the K concentration in the serosal solution from 0 to 10 nM.The results present the first direct indication in mammalian intestine of an interdependence of the Na and K active transport processes which regulate the intracellular content of these cations.