Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with ³⁵S or ³²P. When immune complexes were resolved on SDS-PAGE, the -subunit migrated at 85 and 65 kDa, the -subunit at 115 and 100 kDa, and the -subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The -subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of -, -, and -subunits approximately twofold. Exposure to 1 µM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport. epithelial sodium channel; aldosterone; sodium transport     

Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

The significance of changes in thermodynamic affinity induced by aldosterone in sodium-transporting epithelia

Summary The energetics of sodium transport were examined in toad (and occasionally frog) skin, with particular emphasis on the effect of aldosterone.Thermodynamic affinity was computed according to Essig and Caplan. Following treatment with antidiuretic hormone or drugs believed to affect only the apical membrane barrier, no change in thermodynamic affinity was observed either acutely (after one to two hours) or chronically (after 18-odd hours).By contrast, following treatment with aldosterone overnight, thermodynamic affinity was considerably increased, whether or not incubation was conducted in the presence of sodium in the outer solution; addition of glucose at the end of incubation, whereby sodium transport was stimulated further, failed to influence affinity as measured. The stoichiometry between sodium transport and oxygen consumption was, however, unchanged by aldosterone treatment in short-circuit conditions, neither was that fraction of aerobic metabolism unrelated to sodium transport influenced.It is concluded that the change observed with aldosterone can be directly ascribed to the hormone, as it is independent of glucose availability and of sodium transport. Aldosterone action, at least following prolonged incubation, therefore does not involve only an increase in apical conductance for sodium.     

Effects of aldosterone on (Na+ + K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture

Stimulation by aldosterone of sodium reabsorption can be reproduced on a cell line, A6, derived from the renal tissue of Xenopus laevis. These cells organize themselves as a polarized epithelium carrying out unidirectional sodium transport, reflected by the short-circuit current (Isc). Isc response to aldosterone starts to be apparent after a latency period of 2-3 h; the full hormonal effect takes much longer. On the other hand, (Na+ + K+)-ATPase activity and density in ouabain binding sites did not increase before several hours of treatment. At that stage, while Isc more than trebled, Na+ pump activity and density went up by less than 50%. A significant influence of aldosterone on the way the Na+ pump operates is considered unlikely, since cell interaction with ouabain remained unchanged (Kd approximately 18 nM). Furthermore, the close correspondence of hormonal effect, in relative terms, on (Na+ + K+)-ATPase activity vs density, argues against a significant degree of recruitment of spare pump units. Thus aldosterone effect on Na+ pump probably results from increased biosynthesis of the enzyme. The aldosterone dependent Na+ pump stimulation is apparently unrelated to sodium available for transport. The hormone seems to act on Na+ pump directly.     

The role of mitochondria-rich cells in the chloride current conductance across toad skin

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt-acclimated toads (Bufo viridis) and was correlated with the number of mitochondria-rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short-circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride-dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = -100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP-mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.     

An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.     

Role of adrenal hormones in regulating distal nephron structure and ion transport

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.     

Methylation of cytosolic proteins may be a possible biochemical pathway of early aldosterone action in cultured renal collecting duct cells

The common model of aldosterone-dependent sodium transport is that the hormone increases sodium transport during the "early" and "late" response phases by inducing specific proteins (AIPs). However, in actual biochemical studies, AIPs were mostly detected 6-24 h after aldosterone application. Regarding the physiological early response phase, this implies temporal dissociation of the physiological and biochemical events. The discrepancy raises the question as to whether other biochemical events, such as protein modifications, may be involved in addition to the novo protein synthesis. Labelling of cultured renal collecting duct epithelia for 1-5 h with a radioactive methylgroup donor, S-adenosyl methionine (SAM), following tissue fractionation, resulted in progressive methylations of specific cytosolic proteins. Aldosterone-dependent methylations increased consistently with time, and accounted for a 60% increase in total cytosolic protein content as compared to controls after 5 h labelling. The different methylated proteins showed a molecular weight of 220, 97 and 75 kd and comprised groups of proteins with an isoelectric point of 5.1-5.7 and 6.0-7.5. Methylation of identical proteins was obtained by incubation of the epithelia with unlabelled SAM instead of aldosterone. SAM-induced as well as aldosterone-induced methylation of proteins with an isoelectric point of 6.0-7.5 could be inhibited by the methylation inhibitor S-adenosylhomocysteine. The results indicate that aldosterone may influence the SAM cycle in cultured collecting-duct epithelia during increase of the Na+-transport.     

Control of aldosterone secretion in zona glomerulosa cell suspensions and in the perfused adrenal gland of the rat

The secretion of aldosterone and its responses to stimulation have been studied in rat adrenal zona glomerulosa tissue incubated as intact capsules or as collagenase-dispersed cell suspensions, and in intact perfused rat adrenal glands. Several differences are apparent in the functions of the various preparations. Aldosterone secretion rates are similar in incubated intact capsules and in the perfused gland. Relative to corticosterone, lower yields of aldosterone are obtained in dispersed glomerulosa cell in vitro. This may be related to the loss in the dispersed cells of a pool of tissue steroid (aldosterone or a precursor) which is revealed only in intact tissue incubations by trypsin stimulation of aldosterone secretion. Trypsin-released aldosterone is increased by prior dietary sodium restriction. In addition, differences occur in the responses of dispersed cells and perfused glands to stimulation. Perfused glands from animals on a normal diet are less sensitive to stimulation by ACTH or alpha-MSH, but more sensitive than dispersed cells to angiotensin II amide. In the perfused gland, sensitivity of response (lowest effective concentration) to all three stimulants is increased by prior dietary sodium restriction, in contrast to dispersed cells in which increased sensitivity has been reported only to alpha-MSH. The perfused gland is particularly sensitive to angiotensin II amide, and a bolus administration of 1 amol gives significant stimulation in glands from animals on low sodium intake. Electrical (field) stimulation or dopamine administration at 10(-6) mol/l (which is ineffective in dispersed cells) both depress aldosterone secretion by the perfused gland. The data suggest that the sequestered pool of steroid is utilized in the perfused gland for aldosterone secretion. They furthermore suggest that in the intact gland there are mechanisms, which involve neural components, for intraglandular regulation of aldosterone secretion, which are lost in dispersed cells in vitro. Such mechanisms may be involved in sensitivity increases in sodium depletion.     

Aldosterone-stimulated sodium uptake by apical membrane vesicles from A6 cells

Sodium fluxes in plasma membrane vesicles prepared from the cultured toad kidney epithelial cell line A6 are studied. The vesicles are enriched 7-10 times in apical membrane markers. Sodium uptake is osmotically sensitive and inhibited by low concentrations of amiloride (K0.5 = 7 X 10(-8) M at 1 mM NaCl). Vesicles prepared from aldosterone-treated cells (4.5 h at 10(-7) M aldosterone) show a 2-fold enhancement of amiloride-sensitive sodium flux relative to appropriate controls. The above observations are in good agreement with studies of sodium transport across the apical membrane of intact A6 epithelia. Thus, the amiloride-sensitive sodium transporter in the apical membrane of these cells is preserved in the vesicle preparation, making it possible to study the effects of aldosterone in the absence of nonmembrane-related phenomena.     

In situ demonstration of angiotensin-dependent and independent pathways for hyperaldosteronism during chronic extracellular fluid volume depletion.

In wild-type mice, 2-wk administration of losartan, an angiotensin (Ang) II type 1 (AT1) receptor antagonist, along with dietary sodium restriction, resulted in an elevation of plasma aldosterone greater than that seen with sodium restriction alone (2.75 +/- 0.35 vs. 1.38 +/- 0.16 ng/ml, P < 0.01). Plasma potassium increased in sodium-restricted, losartan-treated mice (6.0 +/- 0.2 mEq/liter), while potassium remained unchanged in mice with sodium restriction alone. To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 +/- 0.27 ng/ml) and potassium remained on average at 4.5 +/- 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 +/- 0.1 mEq/liter) and aldosterone (1.84 +/- 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.     

Aldosterone induces chloride transport in the cortical collecting tubule

The ontogenetic differentiation of transepithelial chloride transport was evaluated in the cortical collecting tubule of the rabbit kidney. Tubules from four control groups (I-IV) were studied during in vitro perfusion. I: body weight 150-280 g; II: 330-480 g; III: 530-880 g; IV: 980-1610 g. In each group, aldosterone (100 micrograms/100 g body weight/day) was given subcutaneously in three doses daily, for 6 days (IA-IVA). Transepithelial net chloride flux (pmol cm2 s1) increased by a factor of almost 3 from group I to group IV (p less than 0.01). Aldosterone induces net chloride flux by 103% (P = 0.03) in IA and by 78% (P = 0.01) in IIA; changes in groups III (21%) and IV (27%) were small. Therefore, the mineralocorticoid induces transepithelial chloride transport in cortical collecting tubule during early transport differentiation. The inducing action decreases with natural differentiation. Moreover, aldosterone alone suffices to induce the complete expression of transepithelial chloride transport in the cortical collecting tubule.     

Effects of transport and racing on ionic changes in thoroughbred race horses.

1. Packed cell volume (PCV), blood glucose, total plasma proteins (TPP) and plasma electrolytes, osmolality, cortisol and aldosterone alterations produced by transport and racing, were investigated in race horses. 2. Plasma cortisol, sodium and blood glucose, found after transport, were higher, while aldosterone was lower than control levels. 3. After racing, PCV, blood glucose, TPP and plasma cortisol, sodium and osmolality were higher than control, while chloride diminished and aldosterone returned to control values. 4. These results demonstrate that transport and racing are different kinds of stressors, suggesting that the sympathetic system and hypophysis-suprarenal cortex axis have a dissimilar contribution to the physiological response.     

Electrical parameters of the toad skin: effects of forskolin.

Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration-dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase.

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca2+ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

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.