Adrenocorticoid receptors in C6 glioma cells: Effects on cell growth

Rat C6 glioma cells contain two receptors for adrenocorticoids—the predominant glucocorticoid receptor and low densities of the Type I corticosteroid (mineralocorticoid) receptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosteceptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosterone, which fully occupy Type I receptors, produced a slight stimulatory effect on C6 cell growth in serum-free media. However, spironolactone, a Type I receptor antagonist, and pregnenolone, which does not bind to Type I receptors, had similar effects. Therefore, the slight growth stimulation produced by low steroid concentrations is not mediated by Type I or glucocorticoid receptors, but may be due to an effect on cell membrane properties or other receptor-independent action. Occupation of glucocorticoid receptors by higher concentrations of corticosteroids inhibited C6 cell growth.     

Inhibition by glucocorticoids of phosphate transport in primary cultured renal cells

The regulation by glucocorticoids of phosphate transport in primary cultured chick renal cells was examined. Dexamethasone inhibited the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The mineralocorticoid aldosterone did not alter phosphate uptake. The inhibition of Na+-dependent phosphate uptake by dexamethasone was concentration-dependent, exhibited an induction period, was blocked by inhibitors of RNA and protein synthesis, and was rapidly reversed when the steroid was removed. Following reversal, the cells could respond a second time to the glucocorticoid. However, this time the response was rapid, could be evoked at least for 24 h after glucocorticoid withdrawal, and might be prevented by actinomycin D and cycloheximide. These findings demonstrate that glucocorticoids act on renal cells to modulate phosphate transport and suggest that the renal cell system provides an attractive model to examine the mechanism by which glucocorticoids control gene expression and regulate plasma membrane transport function.     

D-Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D-glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0-40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time-dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Differential effects of harmaline and ouabain on intestinal glucose transport in the pigeon

Effects of harmaline and ouabain on intestinal transport in vitro of D-glucose in the pigeon are investigated. Harmaline inhibits glucose influx and affects intestinal Na+-K+-ATPase activity though the substrate uptake is more sensitive than the enzyme activity. Low concentration of harmaline while drastically inhibiting glucose uptake, does not affect intracellular concentration of Na+ and K+. In contrast, ouabain, though has no significant effect on glucose uptake, alters substantially the ionic balance of cells. Harmaline also affects that component of nutrient influx which is left unaffected by ouabain. Mucosal-serosal flux of glucose is reduced by harmaline when it is present only on the mucosal side of everted intestinal sacs. On the contrary, similar effect is produced by ouabain when it is placed only on the serosal side. It appears that harmaline possibly inhibits glucose transport in the pigeon intestine by two ways: first, by irreversible binding Na+-K+-ATPase - a feature shared by ouabain, and second, by reversible binding Na+-binding sites of enterocyte membrane - an effect not shared by ouabain.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

Effect of thallium ions on sodium and potassium transport in frog skin

It has been shown that Tl+ accumulated in the frog skin cells (Rana temporaria) inhibits irreversibly the unidirectional transport of Na+ estimated by the short circuit current (SCC). The inhibiting effect of Tl+ cannot be attributed to a decrease of Na+ penetration through the apical membranes. The influx of 22Na+ from mucosal bathing solution into the skin poisoned with Tl+ was about 50% of that observed in the intact skin, while the SCC was completely inhibited. The activity of the ouabain-sensitive Na+/K+ pumps located in the basolateral cell membranes was estimated by studying the uptake of 86Rb+ as a tracer for K+. This activity was high enough to maintain the ion composition of epithelial cells in spite of their ability to accomplish the undirectional transport of Na+. Tl+ seems to inhibit the production of respiration energy utilized in the undirectional Na+ transport, while the ion homeostasis of epithelial cells may be supported by the Na+/K+ pumps consuming energy of glycolytic reactions.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

Effects of spironolactone on glucose transport and interleukin-6 secretion in adipose cells of women.

Adipose tissue inflammation and insulin resistance are central to the pathogenesis of the metabolic syndrome. Spironolactone, an antagonist of mineralocorticoid receptor, glucocorticoid receptor and androgen receptor, and agonist of progesterone receptor, has anti-inflammatory activity. Blockade of the renin-angiotensin-aldosterone system has been shown to improve glucose metabolism. We have investigated whether spironolactone has direct effects on glucose uptake and interleukin-6 secretion in human adipocytes. Spironolactone, but not its active metabolite canrenoic acid, significantly increased basal and insulin-stimulated glucose uptake in cultured IN VITRO-differentiated adipocytes of women, without affecting insulin sensitivity. The effect was not due to changes in abundance of glucose transporters 1 or 4 or in degree of cell differentiation. Spironolactone, but not canrenoic acid, significantly reduced basal interleukin-6 secretion by cultured stromal-vascular cells. These effects of spironolactone were not mediated by ligand-dependent antagonism of the mineralocorticoid, glucocorticoid, or androgen receptors. Spironolactone may have a novel role in increasing glucose uptake into adipose cells and attenuating adipose tissue inflammation, with implications for management of metabolic syndrome.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.     

Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Glucose reduces both Rb+ influx and efflux in pancreatic islet cells

Microdissected, beta-cell-rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D-Glucose (20 mM) induced a biphasic reduction in 86Rb+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose-dependently reduced by ouabain with maximum inhibition at 1 mM. D-Glucose (20 mM) did not affect the ouabain-sensitive 86Rb+ influx but markedly reduced (48%) the ouabain-resistant isotope influx. The results suggest that D-glucose does not affect the Na+/K+ pump in pancreatic beta-cells and that the glucose-sensitive K+-transporting modalities (K+ channels) in the beta-cells can mediate both inward and outward K+ flux.     

Arterial effects of aldosterone and antimineralocorticoid compounds mechanism of action

The aim of our work was to study the mechanism of action of aldosterone and antialdosterone compounds on Na+ and K+ fluxes in vascular smooth muscle. In the long term, regulation of salt metabolism depends on aldosterone effects on Na+, K+, H+ and H2O transport by the renal tubules. Furthermore, it has been shown that aldosterone modifies several epithelial transports, inducing a positive sodium balance. The chronic in vivo administration of aldosterone modifies transmembrane ionic fluxes in vascular smooth muscle. Garwitz and Jones suggested that aldosterone may enhance net Na+ transport through the stimulation of the sodium pump. The results obtained in our laboratory indicate that aldosterone has a direct stimulatory action on ouabain-dependent and on ouabain-independent Na efflux. Furthermore, the mineralocorticoid enhances passive K permeability, as well as the Na pump dependent K influx. Both effects are blocked by antimineralocorticoid compounds. Recent experiments have shown that vasopressin potentiates some of the in vivo effects of aldosterone.     

Characteristics of Sorbitol Uptake in Rat Glial Primary Cultures

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.     

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.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.