Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation ofprotein kinase C with phorbol 12-myristate 13-acetate (PMA) causedcomplex transient perturbations of amiloride-sensitive short-circuitNa⁺ currents(I_Na) in A6epithelia and frog skins that were tissue and concentration dependent.A noninvasive channel blocker pulse method of noise analysis (18) wasused to investigate how PMA caused time-dependent changes of apicalmembrane epithelial Na⁺ channel(ENaC) single-channel currents, channel open probabilities (P_o), andchannel densities(N_T). In A6epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependentsustained decreases ofP_o (~55% belowcontrol, 50 nM) and rapid compensatory transient increases ofN_T within 7 min(~220% above control, 50 nM), resulting in either small transientincreases of I_Naat 5 nM PMA or small biphasic decreases ofI_Na at 50 nM PMA.In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin causedafter a delay of at least 10 min transient increases ofN_T to~60-70% above control at 30-60 min. Unlike A6 epithelia,P_o was increased~15% above control within 7 min and remained within±10-15% of control for the duration of the 2-h experiments.Despite differences in the time courses of secondary inhibition oftransport in A6 epithelia and frog skin, the delayed downregulation oftransport was due to time-dependent decreases ofN_T from theirpreelevated levels in both tissues. WhereasP_o is decreasedwithin minutes in A6 epithelia as measured by noise analysis or bypatch clamp (8), the discrepancy in regulation ofN_T in A6epithelia as measured by noise analysis and patch clamp is most likelyexplained by the inability of on-cell patches formed before treatmentof tissues with PMA to respond to regulation of their channeldensities.

     

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

Weak channel blocker-induced noise analysis wasused to determine the way in which the steroids aldosterone andcorticosterone stimulated apical membraneNa⁺ entry into the cells oftissue-cultured A6 epithelia. Among groups of tissues grown on avariety of substrates, in a variety of growth media, and with cells atpassages 73-112, the steroidsstimulated both amiloride-sensitive and amiloride-insensitiveNa⁺ transport as measured byshort-circuit currents in chambers perfused with either growth mediumor a Ringer solution. From baseline rates of blocker-sensitiveshort-circuit current between 2 and 7 µA/cm², transport was stimulatedabout threefold in all groups of experiments. Single channel currentsaveraged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and weredecreased 6-20% from controls by steroid due to the expecteddecreases of fractional transcellular resistance. Irrespective ofbaseline transport rates, the steroids in all groups of tissuesstimulated transport by increase of the density of blocker-sensitiveepithelial Na⁺ channels (ENaCs).Channel open probability was the same in control and stimulatedtissues, averaging ~0.3 in all groups of tissues. Accordingly,steroid-mediated increases of open channel density responsible forstimulation of Na⁺ transport aredue to increases of the apical membrane pool of functional channels andnot their open probability.

     

Hormonal regulation of ENaCs: insulin and aldosterone

Although a variety of hormones and other agents modulate renalNa⁺ transport acting by way of theepithelial Na⁺ channel (ENaC), themode(s), pathways, and their interrelationships in regulation of thechannel remain largely unknown. It is likely that several hormones maybe present concurrently in vivo, and it is, therefore, important tounderstand potential interactions among the various regulatory factorsas they interact with the Na⁺transport pathway to effect modulation ofNa⁺ reabsorption in distal tubulesand other native tissues. This study represents specifically adetermination of the interaction between two hormones, namely,aldosterone and insulin, which stimulate Na⁺ transport by entirelydifferent mechanisms. We have used a noninvasive pulse protocol ofblocker-induced noise analysis to determine changes in single-channelcurrent (i_Na),channel open probability (P_o), andfunctional channel density(N_T) ofamiloride-sensitive ENaCs at various time points following treatmentwith insulin for 3 h of unstimulated control and aldosterone-pretreatedA6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs(N_T) involvedin transport. The very early (10 min) increases of channel density wereaccompanied by relatively small decreases ofi_Na(10-20%) and decreases ofP_o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes ofi_Na,P_o, andN_T weretransient, returning very slowly over 3 h toward their respectivecontrol values at the time of addition of insulin. We conclude thataldosterone and insulin act independently to stimulate apicalNa⁺ entry into the cells of A6epithelia by increase of channel density.

     

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.     

Identification of aldosterone metabolites formed by A6 cells

The A6 cell line of the toad kidney is well known to form an Na+ transporting tight epithelium in culture and is often used as an experimental model for Na+ transport systems. Although it has been shown that A6 cells can convert aldosterone to polar metabolites, these metabolites have not been identified. Therefore, in this study, we tried to identify the metabolites of aldosterone formed by A6 cells in culture. A6 cells at confluence were incubated with serum-free culture media containing [3H]aldosterone. When radioactive compounds in incubation media were separated by reversed phase high-pressure liquid chromatography (HPLC), four fractions (fractions A-D) were obtained. Fraction A, a mixture of two components, comprised the majority of metabolites formed. The more polar material (fraction A-1) and the less polar material (fraction A-2) of fraction A contained 47-71 and 9-19% of total radioactivity, respectively. When incubated in cell-free media, fraction A-2 was found to be unstable and partially converted to fraction A-1. Fraction B, 0.7-1.5% of total radioactivity, and fraction C, 8-21% of total radioactivity, cochromatographed with iso-aldosterone and D-aldosterone, respectively. Fraction D, 4-8% of total radioactivity, was a mixture of two components, which cochromatographed with 3 beta,5 beta-tetrahydroaldosterone and 5 alpha-dihydroaldosterone, respectively. In order to identify fraction A-2 material, large-scale cultures were performed and fraction A-2 was separated and purified by reversed phase HPLC. The purified material was analyzed by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. These two procedures unambiguously revealed that this material was 6 beta-hydroxyaldosterone. These results demonstrate that aldosterone can be converted to at least four metabolites by the incubation with A6 cells, and that major metabolites are polar compounds, a portion of which is 6 beta-hydroxyaldosterone.     

cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocalmicroscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na⁺ channels (ENaCs) at apical membranes of A6epithelia. After forskolin stimulation of Na⁺ transport andlabeling apical membranes with the fluorescent dyeN-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl)hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities(N_T) decreased exponentially (time constant~20 min) from mean values of 320 to 98 channels/cell within 55 minduring washout of forskolin. Two populations of apical membrane-labeledvesicles appeared in the cytosol within 55 min, reaching mean valuesnear 18 vesicles/cell, compared with five vesicles per cell in control,unstimulated tissues. The majority of cAMP-dependent endocytosedvesicles remained within a few micrometers of the apical membranes forthe duration of the experiments. A minority of vesicles migrated to >5µm below the apical membrane. Because steady states require identicalrates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at theapical membranes, but also principally to increase exocytic rates.These observations are consistent with and support, but do not prove,that vesicle trafficking is a mechanism for cAMP-mediated regulation ofapical membrane channel densities in A6 epithelia.

     

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.

     

Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells

The effects of aldosterone and arginine vasotocin (AVT) on transepithelial Na+ transport of cultured A6 cells were investigated. All experiments were performed with cells grown on Millicell TM culture-plate inserts for a period of 2-4 weeks in defined, serum-free medium. Omitting fetal bovine serum 2 days after seeding the cells on filters did not influence potential difference (PD) development or the hormonal responses tested. The cell layers were placed in an Ussing chamber for short-circuit current (ISC) and transepithelial conductance (G) measurements. Base-line values were (n = 93): PD, 51.0 +/- 0.2 mV (apical side negative); ISC, 14.55 +/- 0.06 microA/cm2; G, 0.306 +/- 0.001 mS/cm2. ISC and G were higher in cells pretreated with 10(-7) M aldosterone for 24 h in the incubator, when compared to controls (ISC, 28 +/- 2 vs. 16 +/- 2 microA/cm2, G, 0.41 +/- 0.04 vs. 0.26 +/- 0.01 mS/cm2, n = 5) and both remained stable for at least 6 h. In cells not treated with aldosterone, 10(-7) M AVT increased ISC within 1 min after addition, producing a maximum ISC within 15 min which then declined to baseline levels over the next 5 h. Addition of AVT to aldosterone-pretreated cells resulted in a significantly greater peak increase in ISC than in non-pretreated cells (change in ISC compared to controls: 8.1 +/- 0.4 vs. 4.9 +/- 0.4 microA/cm2, n = 5, P less than 0.001), indicating a synergistic effect. A dose-response curve for amiloride obtained in the presence of AVT showed that amiloride completely inhibits ISC. Pretreatment of the A6 cells with aldosterone for 24 h shifted the amiloride dose-response curve to the right, as expressed in a doubling of the apparent Ki value (from 0.17 +/- 0.02 to 0.33 +/- 0.04 microM). In conclusion, A6 cells grown in defined, serum-free medium express a greater than additive synergism between aldosterone and AVT in stimulating transepithelial Na+ transport.     

Evidence for a cold-induced aldosterone stimulation in the rat

This study was undertaken to determine the secretion of aldosterone by male Long-Evans rats acclimated for six weeks to moderate cold (15 C), in comparison with rats maintained at thermo-neutral temperature (28 C). The following determinations were made: corticosteroids in plasma and adrenals, PRA, and hydromineral balance. Cold acclimation highly increased the plasma and adrenal levels of aldosterone and corticosterone. The cold stimulation of aldosterone was induced neither by the renin-angiotensin system, nor by alterations of hydromineral balance: PRA, plasma sodium and potassium concentrations, blood hematocrit, and hydromineral balance at 15 C and 28 C did not differ. Moreover this stimulation was induced neither by ACTH, nor by any other hypophyseal factors, since plasma aldosterone levels remained high in hypophysectomized rats. This study provides evidence of an aldosterone stimulation which appeared during moderate cold acclimation; the origin of this stimulation must be investigated.     

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations

Effects of basolateral monovalent cation replacements(Na⁺ byLi⁺,K⁺,Cs⁺, methylammonium, andguanidinium) on permeability to⁸⁶Rb of volume-sensitive cationchannels (VSCC) in the basolateral membrane and on regulatory volumedecrease (RVD), elicited by a hyposmotic shock, were studied in A6epithelia in the absence of apicalNa⁺ uptake. A complete and quickRVD occurred only when the cells were perfused withNa⁺ orLi⁺ saline. With both cations,hypotonicity increased basolateral ⁸⁶Rb release(R^bl_Rb), which reached a maximum after 15 min and declined back to control level. When the major cation wasK⁺,Cs⁺, methylammonium, orguanidinium, the RVD was abolished. Methylammonium induced a biphasictime course of cell thickness(T_c), with an initial decline ofT_c followed by a gradual increase.With K⁺,Cs⁺, or guanidinium,T_c increased monotonously afterthe rapid initial rise evoked by the hypotonic challenge. In thepresence of K⁺,Cs⁺, or methylammonium,R^bl_Rb remained high during most of thehypotonic period, whereas with guanidinium blockage of R^bl_Rb was initiated after 6 min ofhypotonicity, suggesting an intracellular location of the site ofaction. With all cations, 0.5 mM basolateralGd³⁺ completely blocked RVD andfully abolished the R^bl_Rb increaseinduced by the hypotonic shock. The lanthanide also blocked theadditional volume increase induced byCs⁺,K⁺, guanidinium, ormethylammonium. When pH was lowered from 7.4 to 6.0, RVD andR^bl_Rb were markedly inhibited. This studydemonstrates that the VSCCs in the basolateral membrane of A6 cells arepermeable to K⁺,Rb⁺,Cs⁺, methylammonium, andguanidinium, whereas a marked inhibitory effect is exerted byGd³⁺, protons, and possiblyintracellular guanidinium.