Ras activates the epithelial Na(+) channel through phosphoinositide 3-OH kinase signaling

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na(+) channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect K-Ras actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85(PI3K). The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone-sensitive Na(+) reabsorption.

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na(+) transport. Polyclonal antibodies raised against X. laevis pcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na(+) transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na(+) reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na(+) reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na(+) transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na(+) transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells ∼3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPβS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110α) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.

The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.     

AS160 Modulates Aldosterone-stimulated Epithelial Sodium Channel Forward Trafficking

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I_sc). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I_sc 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I_sc under basal conditions to ∼80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, β and ε. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3β and ε and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.     

Role of Sgk1 in salt and potassium homeostasis

Aldosterone plays a pivotal role in NaCl and K(+) homeostasis by stimulation of Na(+) reabsorption and K(+) secretion in the aldosterone-sensitive distal nephron (ASDN). Recent studies demonstrated that the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by aldosterone in the ASDN and that polymorphisms of the kinase associate with arterial blood pressure in normotensive subjects. This review discusses the role of Sgk1 in NaCl and K(+) homeostasis as evidenced by in vivo studies, including those in Sgk1-deficient mice. The studies indicate that Sgk1 is not absolutely required for Na(+) reabsorption and K(+) secretion in the ASDN. On a standard NaCl and K(+) diet, modestly enhanced plasma aldosterone concentrations appear sufficient to establish a compensated phenotype in the absence of Sgk1. The kinase is necessary, however, for upregulation of transcellular Na(+) reabsorption in the ASDN. This may involve Sgk1-mediated stimulation of basolateral Na(+)-K(+)-ATPase as well as retention of epithelial Na(+) channel, ENaC, in the apical membrane. Such an upregulation is a prerequisite for adequate adaptation of 1) renal NaCl reabsorption during restricted dietary NaCl intake, as well as 2) K(+) secretion in response to enhanced K(+) intake. Thus gain-of-function mutations of Sgk1 are expected to result in renal NaCl retention and enhanced K(+) secretion. Further studies are required to elucidate renal and nonrenal aldosterone-induced effects of Sgk1, the role of other Sgk1 activators, as well as the link of Sgk1 polymorphisms to arterial hypertension in humans.     

Regulation of epithelial Na+ channels (ENaC) by methylation: a novel methyltransferase stimulates ENaC activity

Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.     

Mechanisms of Aldosterone's Action on Epithelial Na + Transport

Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.     

Rapid translocation and insertion of the epithelial Na+ channel in response to RhoA signaling

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epithelia. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. Two members of the Ras superfamily of small GTPases, K-Ras and RhoA, activate ENaC. K-Ras activates ENaC via a signaling pathway involving phosphatidylinositol 3-kinase and production of phosphatidylinositol 3,4,5-trisphosphate with the phospholipid directly interacting with the channel to increase open probability. How RhoA increases ENaC activity is less clear. Here we report that RhoA and K-Ras activate ENaC through independent signaling pathways and final mechanisms of action. Activation of RhoA signaling rapidly increases the membrane levels of ENaC likely by promoting channel insertion. This process dramatically increases functional ENaC current, resulting in tight spatial-temporal control of these channels. RhoA signals to ENaC via a transduction pathway, including the downstream effectors Rho kinase and phosphatidylinositol-4-phosphate 5-kinase. Phosphatidylinositol 4,5-biphosphate produced by activated phosphatidylinositol 4-phosphate 5-kinase may play a role in targeting vesicles containing ENaC to the plasma membrane.     

Aldosterone modulates sodium kinetics of Na,K-ATPase containing an alpha 1 subunit in A6 kidney cell epithelia.

Short-term aldosterone (10(-6) M, 2.5 h) induces in A6-C1 cell epithelia an increase in Na transport, which is due to the in situ activation of the apical Na channel and, presumably, the basolateral Na pump (Na,K-ATPase). We have now directly measured the effect of aldosterone on the transport activity of endogenous Na pumps and hybrid Na pumps containing an exogenous alpha 1 subunit by measuring the pump current (Ip) across epithelia apically permeabilized with amphotericin B. Aldosterone (2.5 h) had no significant early effect on the maximal Ip, nor on the Na concentration required for half-maximal activation. In contrast, it increased the Ip at physiological intracellular Na concentrations (1.7-fold at 5 mM Na). This effect was blocked by the protein synthesis inhibitor cycloheximide. Hybrid pumps containing the transfected cardiotonic steroid-resistant alpha 1 subunit of Bufo marinus were also stimulated by aldosterone (2.5 h). A long aldosterone treatment (4 days) increased the maximal Ip produced by the endogenous pumps 1.5 to 2.1-fold. In conclusion, aldosterone acts on Na pumps containing an alpha 1 subunit in two ways. During its early phase of action it stimulates their transport activity by increasing their apparent Na affinity at physiological intracellular Na concentrations. In the long term it produces an increase in the maximal transport capacity, which corresponds to the known increase in the number of Na pumps.     

Rapid responses to aldosterone in human distal colon.

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K+ channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K+ channel (K(ATP)) and inhibits a Ca2+ -dependent K+ channel (K(Ca)). In Ussing chamber experiments, we have shown that the K(ATP) channel is required to drive sodium absorption, whereas the K(Ca) channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K(ATP), K(Ca) channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K(ATP) channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K(Ca) channels involved in secretion through crypt cells.     

Aldosterone-mediated regulation of Na+, K(+)-ATPase gene expression in adult and neonatal rat cardiocytes.

By altering the Na+/K+ electrochemical gradient, Na+,K(+)-ATPase activity profoundly influences cardiac cell excitability and contractility. The recent finding of mineralocorticoid hormone receptors in the heart implies that Na+,K(+)-ATPase gene expression, and hence cardiac function, is regulated by aldosterone, a corticosteroid hormone associated with certain forms of hypertension and classically involved in regulating Na+,K(+)-ATPase gene expression and transepithelial Na+ transport in tissues such as the kidney. The regulation by aldosterone of the major cardiac Na+,K(+)-ATPase isoform genes, alpha-1 and beta-1, were studied in adult and neonatal rat ventricular cardiocytes grown in defined serum-free media. In both cell types, aldosterone-induced a rapid and sustained 3-fold induction in alpha-1 mRNA accumulation within 6 h. beta-1 mRNA was similarly induced. alpha-1 mRNA induction occurred over the physiological range with an EC50 of 1-2 nM, consistent with binding of aldosterone to the high affinity mineralocorticoid hormone receptor. In adult cardiocytes, this was associated with a 36% increase in alpha subunit protein accumulation and an increase in Na(+)-K(+)-ATPase transport activity. Aldosterone did not alter the 3-h half-life of alpha-1 mRNA, indicating an induction of alpha-1 mRNA synthesis. Aldosterone-dependent alpha-1 mRNA accumulation was not blocked by the protein synthesis inhibitor cycloheximide, whereas amiloride inhibited both an aldosterone-dependent increase in intracellular Na+ [Na+]i) and alpha-1 mRNA accumulation. This demonstrates that aldosterone directly stimulates Na+,K(+)-ATPase alpha-1 subunit mRNA synthesis and protein accumulation in cardiac cells throughout development and suggests that the heart is a mineralocorticoid-responsive organ. An early increase in [Na+]i may be a proximal event in the mediation of the hormone effect.     

Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon

Studies from our laboratory have demonstrated rapid ( < 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC activity and a PKC-dependent Ca(2+) entry through L-type Ca(2+) channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of the PKC alpha isoform (but not PKC delta, PKC epsilon and PKC zeta) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na(+)-H(+) exchanger. In isolated colonic crypts, aldosterone produced a PKC alpha sensitive intracellular alkalinisation within 1 min of hormone addition. Intracellular alkalinisation upregulates an ATP-dependent K(+) channel, which is involved in K(+) recycling to maintain the electrical driving force for Na(+) absorption, while inhibiting a Ca(2+) -dependent K(+) channel, which generates the charge balance for Cl(-) secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (< 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC alpha activity, and a PKC delta- and PKA-dependent Ca(2+) entry through di-hydropyridine-blockable Ca(2+) channels specifically by 17beta-estradiol in distal colon. These rapid effects are female gender specific and are insensitive to inhibitors of the classical estrogen receptor (ER). 17 beta-Estradiol directly stimulated the activity of both PKC delta and PKC alpha (but not PKC epsilon or PKC zeta) in a cell-free assay system. E2 rapidly inhibited basolateral K(Ca) channel activity which would be expected to result in an acute inhibition of Cl(-) secretion. Physiological concentrations of E2 (0.1-10 nM) reduced both basal and secretagogue-induced Cl(-) secretion. This anti-secretory effect of E2 is sensitive to PKC inhibition, intracellular Ca(2+) chelation, and is female gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific physiological effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and estradiol is to shift the balance from net secretion to net absorption in a pluripotential epithelium.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Aldosterone stimulates vacuolar H(+)-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(αq) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(αq) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.