Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.     

Urinary proteases degrade epithelial sodium channels

Summary The mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, an-other a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability.Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.     

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.     

Ca2+-dependent, temperature-sensitive regulation of Na+ channels in tight epithelia. A study using membrane vesicles

Na+ fluxes were measured in toad bladder microsomes. Under favorable conditions, 60-90% of the tracer uptake was blocked by amiloride (Ki = 2.3 X 10(-8) M), i.e. mediated by the apical Na+-specific channels. Vesicles derived from cells maintained at 0 degrees C exhibited relatively small amiloride-sensitive fluxes. However, incubating the scraped cells at 25 degrees C prior to homogenization induced a nearly 5-fold increase of the amiloride-blockable flux in vesicles. This activation was fairly slow (t 1/2 = 5-10 min), irreversible, and strongly dependent on the incubation temperature. On the other hand, the Na+-specific apical conductance measured in mounted bladders was only slightly affected by the incubation temperature. The above activation process could be observed only in Ca2+-free EGTA-containing solutions. Adding Ca2+ (1 mM) to the cell suspension and subsequently removing it before homogenization blocked almost completely the amiloride-sensitive tracer uptake in the vesicles. The data are compatible with the model that the epithelial Na+ channels are down-regulated by a Ca2+-dependent reaction. The incubation of scraped, somewhat permeabilized cells in a Ca2+-free solution releases channels from this down-regulation and increases the Na+ conductance in a temperature-dependent process. The regulation of channels appears to involve a cytoplasmic factor which induces a stable modification of the apical membrane, preserved by the isolated vesicle.     

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.     

Sodium-chloride transport in the medullary thick ascending limb of henle's loop: Evidence for a sodium-chloride cotransport system in plasma membrane vesicles

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell.     

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.     

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

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

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

A bicarbonate-dependent process inhibitable by disulfonic stilbenes and a Na+/H+ exchange mediate 22Na+ uptake into cultured bovine corneal endothelium

22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.     

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.     

Methylation increases the open probability of the epithelial sodium channel in A6 epithelia

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.     

Effect of cyclosporin A on aldosterone production by dispersed rabbit adreno-capsular cells

The effect of cyclosporin A on aldosterone production by dispersed adreno-capsular cells from rabbit was examined. Cyclosporin A significantly stimulated aldosterone production at concentrations of 10(-7) M and 10(-6) M. The maximum stimulation of aldosterone production by cyclosporin A (at 10(-6) M) was comparable to that by angiotensin II at 10(-8) M). This stimulating effect of cyclosporin A on aldosterone production was not accompanied by an increase in cyclic AMP production, and was not inhibited by a calcium-channel blocker, nicardipine. These results suggest that the aldosterone-stimulating action of cyclosporin A at these concentrations is not mediated by a known second messenger system such as channel-linked Ca2+ inflow or cyclic AMP.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Aldosterone upregulates purinergic responses in larval amphibian skin epithelium

Bullfrog tadpoles respond to apical application of 100 microM amiloride, acetylcholine (ACh) or ATP with a sharp transient inward (apical to basolateral) cation current. In adult skin, amiloride blockable transepithelial Na+ transport is upregulated by the hormone aldosterone. Tadpoles were treated in vivo with aldosterone and changes in short circuit current (Isc) in response to apical application of ATP were determined. Bullfrog tadpoles were exposed to aldosterone (10(-6) M) for periods ranging from 3 h to 60 h. Skins from 60-h aldosterone-treated animals showed a two- to three-fold increase in apical ATP-activated short circuit current when compared to animals treated with vehicle alone. Sodium replacement with a large, nonpermeable cation resulted in no measurable increase in Isc after exposure to ligand, consistent with ATP activation of an inward cation current and not chloride efflux. Activation/desensitization time courses and treatment with blockers revealed no measurable differences between aldosterone-treated and non-treated skins. Activation by amiloride and ACh gave essentially identical results. Studies with RT/PCR showed significant increases over controls of levels of mRNA associated with P2X channels. Given these data, our working hypothesis is that all three ligands activate the same process that exhibits both purinergic and cholinergic characteristics. These data are consistent with aldosterone upregulation of ATP gated channels expressed in the apical membrane of larval frog skin.     

Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl- Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M- 1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.     

Oral amiloride treatment decreases taste sensitivity to sodium salts in C57BL/6J and DBA/2J mice

Sodium taste transduction is thought to occur via an amiloride-sensitive, sodium-selective pathway and an amiloride-insensitive, cation nonselective, anion-dependent pathway(s). It has been shown by others that amiloride, an epithelial sodium channel (ENaC) blocker, significantly reduces the chorda tympani nerve response to lingually applied NaCl in C57BL/6 (B6) mice but not in DBA/2 (D2) mice, suggesting that the latter strain might not possess functional ENaCs in taste receptor cells. We psychophysically measured and compared taste detection thresholds of NaCl and sodium gluconate (NaGlu) prepared with and without 100 microM amiloride in these two strains (eight/strain). Mice were trained and tested in a two-response operant signal detection procedure conducted in a gustometer. Surprisingly, no strain effect was found for the detection thresholds of both salts (approximately 0.05-0.06 M). Moreover, these thresholds were increased by almost an order of magnitude by amiloride adulteration of the solutions. This marked effect of amiloride on sodium detection thresholds suggests that ENaCs are necessary for normal sensitivity to sodium salts in both strains. In addition, because NaGlu is thought to stimulate primarily the amiloride-sensitive pathway, especially at low concentrations, the similarity of NaCl and NaGlu thresholds (r > 0.81 both strains) suggests that ENaCs are also sufficient to support the detection of sodium in weak solutions by B6 and D2 mice.     

Molecular size of the Na+-H+ antiport in renal brush border membranes, as estimated by radiation inactivation

The radiation inactivation method was applied to brush border membrane vesicles from rat kidney, in order to estimate the molecular size of the Na+-H+ antiporter. Sodium influx (1mM) driven by an acid intravesicular pH was unaffected by the high osmolarity of the cryoprotective solution. Initial rate of influx was estimated by linear regression performed on the first 10 seconds of transport: 0.512 pmol/micrograms protein/s. There was no binding component involved. Incubation performed in the presence of 1 mM amiloride, an inhibitor of the Na+-H+ antiport gave an initial rate of only 0.071 pmol/microgram/s, an 82% inhibition. Membrane vesicles were irradiated at -78 degrees C in a Gammacel Model 220. Sodium influx was reduced, as the dose of radiation increased, but the influx remained linear for the period of time (10s) during which the initial rate was estimated, indicating no alteration of the proton driving force during this time period. Amiloride-insensitive flux remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards sodium was unaffected. The amiloride-sensitive pathway presented a monoexponential profile of inactivation, allowing the molecular size to be estimated at 321 kDa. Based on DCCD-binding studies suggesting the molecular size of the monomer to be around 65 kDa for rat kidney, our results suggest that the functional transporter in the membrane to be a multimer.