Renal Epithelial A6 Cells Research Articles

Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model

Background/Aims: Epithelial Na₊ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na₊ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na₊ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na₊ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. Methods: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. Results: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na₊ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. Conclusion: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (k_I), and the recycling rate (k_R) of ENaCs, but increased degradation rate (k_D) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (k_I) and the recycling rate (k_R) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (k_E) in EGFR and PDGFR-related PTKs-mediated pathways.

Raman micro-spectroscopy as a viable tool to monitor and estimate the ionic transport in epithelial cells

The typical response to the lowering of plasma Na+ concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na+ concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na+ current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone.

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model

Background/Aim: Epithelial Na⁺ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na⁺ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. Methods: We simulated the ENaC-mediated Na⁺ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. Results: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na⁺ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na⁺ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na⁺ transport via an increase in recycling of ENaC to the apical membrane. Conclusion: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na⁺ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.

Prolactin stimulates sodium and chloride ion channels in A6 renal epithelial cells.

Many hormonal pathways contribute to the regulation of renal epithelial sodium channel (ENaC) function, a key process for maintaining blood volume and controlling blood pressure. In the present study, we examined whether the peptide hormone prolactin (PRL) regulates ENaC function in renal epithelial cells (A6). Basolateral application of several different concentrations of PRL dramatically stimulated the transepithelial current in A6 cells, increasing both amiloride-sensitive (ENaC) and amiloride-insensitive currents. Using cell-attached patch clamp, we determined that PRL increased both the number (N) and open probability (Po) of ENaC present in the apical membrane. Inhibition of PKA with H-89 abolished the effect of PRL on amiloride-sensitive and insensitive transepithelial currents and eliminated the increase in ENaC NPo with PRL exposure. PRL also increased cAMP in A6 cells, consistent with signaling through the cAMP-dependent PKA pathway. We also identified that PRL induced activity of a 2-pS anion channel with outward rectification, electrophysiological properties consistent with ClC4 or ClC5. RT-PCR only detected ClC4, but not ClC5 transcripts. Here, we show for the first time that PRL activates sodium and chloride transport in renal epithelial cells via ENaC and ClC4.

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.

A physiological role of p38 on aldosterone‐dependent regulation of ENaC endocytosis in renal epithelial A6 cells

Aldosterone (ALD) is a crucial hormone to regulate ENaC‐mediated Na+ reabsorption in the kidney for controlling ECF volume and blood pressure. So far, we found ALD activated p38 and pretreatment with SB202190 (a specific p38 inhibitor: SB) abolished ALD‐stimulated benzamil‐sensitive Isc (INa ) by reducing benzamil‐sensitive conductance (GNa). In this study, we investigated a physiological role of p38 in the surface expression of ENaC through exocytic and endocytic pathways in ALD‐treated renal epithelial A6 cells by using MG132 (a proteasome inhibitor) and brefeldin A (an inhibitor of protein translocation). Pretreatment with MG132 mostly recovered the SB‐induced reduction of INa, whreas brefeldin A showed further an inhibitory effect on the SB‐decreased INa. Moreover, SB decreased both phosphorylated SGK1 and Nedd4‐2. These suggest that the p38‐ mediated signal pathway suppresses the endocytic/proteasome‐dependent ENaC degradation contributing to the stimulation of Na+ reabsorption by ALD. Supported by JSPS (19590212, 20390060), SSRF (0837, 1035) and FFPR.

Analysis of Blocker-labeled Channels Reveals the Dependence of Recycling Rates of ENaC on the Total Amount of Recycled Channels

Trafficking is one of the primary mechanisms of epithelial Na⁺ channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 µg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na⁺ transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

The epithelial Na(+) channel (ENaC) contributes to control of blood pressure by reabsorbing Na(+) in the cortical collecting duct of the kidney. The luminal Cl(-) concentration in the duct varies under physiological conditions. As the body Na(+) content is lower, the luminal Cl(-) concentration in the duct becomes lower. Thus, we hypothesized that the extracellular Cl(-) elevates ENaC activity in AVT-stimulated renal epithelial A6 cells (a model cell line of the cortical collecting duct) leading to recovery from a low body Na(+) content. To clarify this point, we studied effects of extracellular Cl(-) concentration on ENaC activity using cell-attached patch clamp technique. We found that ENaC had a single-channel conductance of 4.6 +/- 0.1 pS (mean +/- SE) and channel activity (open probability, Po) of 0.30 +/- 0.02 at a pipette potential of 60 mV. Lowering pipette Cl(-) concentration diminished Po to 0.23 +/- 0.02 associated with a significant decrease in open time from 0.78 +/- 0.03 to 0.61 +/- 0.02 s with no significant change in closed time, and shifted the current-voltage relationship leftward. These results suggest that the extracellular Cl(-) regulates the ENaC-mediated Na(+) reabsorption by affecting ENaC properties in AVT-stimulated renal epithelial cells.

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of SGK1 and ENaC in renal epithelial A6 cells

In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na(+) transport. Hypotonic action on Na(+) transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na(+) transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial Na(+) channel (ENaC), increasing Na(+) transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca(2+) in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca(2+) concentration in A6 cells, we hypothesized that Ca(2+)-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca(2+)-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na(+) transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca(2+) plays a role as the second messenger in hypotonic stress-induced Na(+) transport by stimulating transcription of SGK1 and ENaC subunits.

Involvement of p38 MAPK in hypotonic stress-induced stimulation of β- and γ-ENaC expression in renal epithelium

We investigated a role of p38 MAPK in the regulation of transepithelial Na + reabsorption by chronic application (20–24 h) of hypotonicity (hypotonic stress) in renal epithelial A6 cells. Pretreatment with a specific p38 MAPK inhibitor (SB202190) significantly reduced the chronic hypotonicity-stimulated transepithelial Na + reabsorption by diminishing the Na + entry through epithelial Na + channel (ENaC) in the apical membrane and the Na + extrusion via the Na +/K + ATPase (pump), although the rate limiting step was still the Na + entry step. We further examined whether the inhibitory effects of SB202190 on the transepithelial Na + reabsorption is caused through suppression of mRNA expression of ENaC participating in the transepithelial Na + reabsorption as the Na + entry pathway. The chronic hypotonicity increased the mRNA expression of α-, β-, and γ-subunits of ENaC. Moreover, we found that inhibition of p38 MAPK by SB202190 diminished the mRNA expression of β- and γ-ENaC but not α-ENaC. Based on these observations, it is suggested that the chronic hypotonicity stimulates the renal transepithelial Na + reabsorption by upregulating the mRNA expression of β- and γ-ENaC via a p38 MAPK-dependent pathway.

Hypotonicity stimulates renal epithelial sodium transport by activating JNK via receptor tyrosine kinases

We previously reported that hypotonic stress stimulated transepithelial Na(+) transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63-77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of Na(+) transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of Na(+) transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK was diminished by coapplication of AG1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of Na(+) transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced Na(+) transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated Na(+) transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of Na(+) transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of Na(+) transport.

Genomic effect of hypotonic stress on Na + reabsorption through Ca 2+ /calmodulin‐dependent SGK1 induction in renal epithelial A6 cells

Genomic effect of hypotonic stress on Na ⁺ reabsorption through Ca ²⁺ /calmodulin‐dependent SGK1 induction in renal epithelial A6 cells

Receptor tyrosine kinases‐mediated mechanism in hypotonicity‐provoked Na + reabsorption in renal epithelial A6 cells

In the amphibian kidney epithelium, A6, hypotonicity increases transepithelial Na+ transport via translocation of intracellular epithelial Na+ channel (ENaC) to the apical surface in a protein tyrosine kinase (PTK)-dependent manner. Evaluating effects of inhibitors on hypotonicity-induced amiloride-sensitive short-circuit current and conductance to determine what type of PTK is involved in hypotonic action, we identified that receptor tyrosine kinases (RTKs) were involved in the stimulatory action of hypotonic stress on ENaC translocation. Further, we found that the downstream of this RTKs-mediated signaling pathway was not mediated via PI3 kinase-SGK1 signaling pathway, which participates in hypotonicity-induced ENaC translocation. Interestingly, our experimental data also disclosed that these two hypotonicity-activated signaling pathways had the completely different effects on hypotonicity-induced Na+ reabsorption. In addition to the above, we found an interesting possibility that JNK was located in the downstream of hypotonicity-activated RTKs to activation of ENaC translocation. These data supports the concept of novel RTKs-mediated mechanism in Na+ reabsorption in response to the hypo-osmotic stress. Supported by Grants-in-Aids from JSPS (17390057 and 17590191).

Synergic action of insulin and genistein on Na+/K+/2Cl‐ cotransporter (NKCC) in renal epithelium

We studied the effect of insulin and quercetin on Cl- secretion in renal epithelial A6 cells by estimating Cl- channel activity and Cl− secretion by measuring NPPB-sensitive transepithelial conductance (Gt) and short-circuit current (Isc). Pretreatment with 1mM insulin for 24h had no effects on Gt or Isc, but increased the Isc with no effects on Gt in A6 cells treated with MG132 (100mM for 2h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space. Genistein (100mM) induced sustained increases in Gt and Isc, which were diminished by brefeldin A. Co-application of insulin and genistein synergically stimulated the Isc without any effects on Gt. These observations suggest that: 1) insertion and endocytosis of NKCC are stimulated by insulin, 2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, 3) genistein stimulates both insertion of CFTR Cl− channel into the apical membrane and insertion of NKCC into the basolateral membrane, and 4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane. Supported by JSPS (17390057, 17590191).

The role of intracellular Cl‐ concentration in cell proliferation

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. Our recent study indicates that intracellular Cl− concentration ([Cl−]i) could act as a signal to regulate ion channel mRNA expression in renal epithelial A6 cells (AJP 287:F932-9, 2004). This result suggests that the changes of [Cl−]i is one of the important candidates to cell proliferation. In the present study, we studied whether [Cl−]i regulates cell proliferation in A6 cells. Treatment with low Cl− (about 15 mM) medium (replacement of NaCl by NaNO3) for 72 h induce a significant decrease of [Cl−]i associated with reduction of cell growth rate compared with control (normal Cl− medium). Using flow cytometrical analysis, we found that cell cycle arrest occurred at the G0/G1 phase in low Cl−-treated cells. These data indicate that [Cl−]i plays important roles in cell proliferation and cell cycle transition through the G1/S checkpoint. This work was supported by Grants-in-Aid from JSPS (17390057, 17590191, 17790154).

Quercetin and NPPB‐induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transpithelial Cl− secretion due to activation of the Na+/K+/2Cl− cotransporter (NKCC), and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC) and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity and alpha-ENaC mRNA expression was diminished by 24 h-pretreatment with quercetin (an activator of NKCC) or NPPB (a blocker of Cl− channel), while 24 h-pretreatment with bumetanide (a blocker of NKCC) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effects on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of NKCC, and that modification of activity of Cl− transporter/channel participating in the transepithelial Cl− secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport). Supported by Grants-in-Aid from JSPS (17390057, 17590191).

Hypotonicity stimulates Na+ reabsorption through activation of src kinase by decreasing cytosolic Cl‐ concentration

We have previously indicated that in renal epithelial A6 cells hypotonicity stimulates Na+ reabsorption by activating a PTK-dependent pathway and that hypotonicity causes a decrease in cytosolic Cl- concentration ([Cl−]c) through regulatory volume decrease (RVD). In this study, we found that hypotonicity increased tyrosine phosphorylation of src kinase at pY416 (an active form of src kinase) in a manner dependent on RVD-induced [Cl−]c decrease. We further found that decreasing [Cl−]c caused a significant increase in tyrosine phosphorylation of src kinase at pY416 under an isotonic condition without any effect on tyrosine phosphorylation state of src kinase at pY527. Furthermore, pretreatment with PP2 (a specific inhibitor of src kinase) abolished hypotonicity-induced stimulation of Na+ rebasorption and alpha-subunit of epithelial Na+ channel (ENaC) mRNA expression. Taken together these results, it is suggested that hypotonicity stimuates Na+ rebasorption through induction of alpha-ENaC gene expression by activating src kinase through RVD-dependent decrease in [Cl−]c. Supported by Grants-in-Aids from JSPS (17590191 and 17390057).

The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Change in intracellular chloride concentration caused by regulatory volume decrease is the primary hypotonic signal in renal epithelial A6 cells

Change in intracellular chloride concentration caused by regulatory volume decrease is the primary hypotonic signal in renal epithelial A6 cells