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.     

Interactions of cardiac glycosides with cells and membranes. IV. Effects of ouabain and bumetanide on 86Rb+ influx in cultured cardiac myocytes from neonatal rats

Ouabain at nanomolar concentrations stimulates total Rb+ influx by 20 +/- 2% in monolayer cultures of myocytes which were either in physiologic ionic steady-state conditions ('control') or 'loaded with Na+' following exposure to K+-free medium. The ouabain-stimulated Rb+ influx was completely abolished by 0.1 mM bumetanide both in 'control' and in 'Na+-loaded' myocytes. Thus, addition of nanomolar concentrations of ouabain to myocytes markedly stimulate the bumetanide-sensitive Rb+ influx. This influx was increased up to 3- and 4-fold in 'control' and 'Na+-loaded' myocytes, respectively. Ouabain at nanomolar concentrations had no significant effect on the component of 86Rb+ influx which is inhibited by millimolar concentrations of ouabain (the so called 'ouabain-sensitive' or 'pump-mediated' Rb+ influx) in 'control' and 'Na+-loaded' cells. It is proposed that the increased rates of bumetanide-sensitive Rb+ influx are accompanied by an increased bumetanide-sensitive Na+ influx through the Na+/K+ cotransporter and thus to a transient increase in intracellular Na+ concentrations [Na+]i. The increase in [Na+]i, subsequently causes a transient elevation in [Ca2+]i via the Na+/Ca2+ exchanger and may be involved in the regulation of cardiac cells' contractility.     

Energy requirements for the Na+ gradient in the oxygenated isolated heart: effect of changing the free energy of ATP hydrolysis

This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (Delta GATP) below a threshold value will inhibit Na+-K+-ATPase (Na+ pump) activity and result in an increase of intracellular Na+ concentration ([Na+]i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the Delta GATP of the LowBu hearts. 31P, 23Na, and 87Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na+]i, and Rb+ uptake. Rb+ uptake was used to estimate Na+ pump activity. To measure [Na+]i using a shift reagent for cations, extracellular Ca2+ was reduced to 0.85 mM, which eliminated work demand Delta GATP reductions. Increasing extracellular Na+ (Nae+) to 200 mM restored work demand Delta GATP reductions. In response to higher [Na+]e, [Na+]i increased equally in LowBu and HighBu hearts to approximately 8.6 mM, but Delta GATP decreased only in LowBu hearts. At lowest work demand the LowBu heart Delta GATP was -53 kJ/mol, Rb+ uptake was similar to that of HighBu hearts, and [Na+]i was constant. At highest work demand the LowBu heart Delta GATP decreased to -48 kJ/mol, the [Na+]i increased to 25 mM, and Rb+ uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart Delta GATP was -54 kJ/mol and [Na+]i increased only approximately 10%. We conclude that a Delta GATP below -50 kJ/mol limits the Na+ pump and prevents maintenance of [Na+]i homeostasis.     

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.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts

The effects of hyperthermia (41-43 degrees C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 degrees C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37 degrees C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+:H+ exchange. Shifting temperature to 41 degrees C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43 degrees C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41 degrees C only in the presence of ouabain. At 43 degrees C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43 degrees C, 0.25 mM amiloride inhibited heat-stimulated Na+ uptake. Na+ efflux was enhanced at 41 degrees C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41 degrees C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures greater than or equal to 43 degrees C, the Na+ pump is inhibited.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway.

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.     

CSF-1 stimulates Na+K+-ATPase mediated 86Rb+ uptake in mouse bone marrow-derived macrophages

86Rb+ was used as an isotopic tracer for the measurement of K+-uptake into quiescent murine bone marrow-derived macrophages. 86Rb+ uptake was inhibited by ouabain indicating a Na+K+-ATPase is being measured. In support of this finding, increased sensitivity to ouabain inhibition was seen when the K+ content of the medium was reduced. A purified colony stimulating factor (CSF-1) was shown to stimulate the ouabain-sensitive 86Rb+ uptake in a dose-dependent manner. Such colony stimulating factor stimulation of 86Rb+ (K+) influx was rapid, with a maximal effect seen 10 minutes after growth factor addition followed by a gradual decrease. Thus increased Na+K+-ATPase activity was an early response of macrophages to the colony stimulating factor.     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Model for the regulation of platelet volume and responsiveness by the trans-membrane Na+/K(+)-pump.

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.     

Influence of chronic alterations of salt intake and aging on the kinetic of red cell Na+ and K+ transport in Sprague-Dawley rats

The kinetics of Na+ and K+ (Rb)+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system (assessed as a function of Rb+o and Na+i) as well as the magnitude of cation leaks were determined in red cells of young male rats subjected to chronic salt deprivation or salt loading (0.1% and 8% NaCl diet). These salt intake alterations induced moderate kinetic changes of the Na(+)-K+ pump which did not result in significant changes of ouabain-sensitive (OS) Rb+ uptake or Na+ net extrusion at in vivo Na+i and K+o concentrations because a decreased affinity for Na+i in salt-loaded animals was compensated by an increased maximal transport rate. High furosemide-sensitive (FS) Rb+ uptake in red cells of salt-deprived rats was caused by an increase of both the maximal transport rate and the affinity for Rb+o. Cation leaks were also higher in salt-deprived than in salt-loaded rats. In three age groups of rats fed a 1% NaCl diet FS Rb+ uptake (but not FS Na+ net uptake) rose with age due to an increasing maximal transport rate whereas the affinity of the cotransport system for Rbo+ did not change. The age-dependent changes in the kinetics of the Na(+)-K+ pump resulted in a slight decrease of OS Rb+ uptake with age that was not paralleled by corresponding Na+ net extrusion. No major age-related changes of cation leaks were found. Thus some intrinsic properties of red cell transport systems can be altered by salt intake and aging.