Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production.

The control of Na+/K+ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/86Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (IC50, approximately 80 microM), suggesting the role of a single type of Na+/K+ pump (alpha-isoenzyme) in 86Rb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of 86Rb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive 86Rb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na+ concentration is sufficient for maximal activity of the Na+/K+ pump. Angiotensin-II (AII) inhibited the ouabain-sensitive 86Rb uptake by glomerulosa cells. The effect of AII was abolished by the selective antagonist of the AT1 type of AII receptors (DuP 753), while PD 123177, an AT2 antagonist was ineffective. AT1 receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca2+ signal. The inhibitory effect of AII was dependent on the extracellular Ca2+ concentration, but an elevation of cytoplasmic Ca2+ by Ca2+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AII on the Na+/K+pump. Pharmacological activation of protein kinase-C by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII.     

Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Two ionophores (monensin and gramicidin) that carry Na+ into 3T3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing 86Rb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 86Rb+ uptake and cell Na+ and K+ over a range of monensin concentrations show that the activity of the Na+,K+ pump in 3T3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum rapidly enhances the rate of 22Na+ uptake and net Na+ entry when Na+ exit is inhibited by ouabain. At 0.3 microgram/ml, monensin increases the rate of net Na+ entry and activates the Na+,K+ pump by the same degree as serum. The stimulation of 86Rb+ uptake by serum or the ionophores has an absolute requirement for external Na+. Thus, serum appears to stimulate the Na+,K+ pump in quiescent 3T3 cells by increasing its supply of Na+.     

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide

The activity of the beta-cell Na+/K+ pump was studied by using ouabain-sensitive (1mM ouabain) 86Rb+ influx in beta-cell-rich islets of Ume?-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0 mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx during the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect showed its apparent maximum at 0.5 microM. Pre-treatment (60 min) of islets with 1 microM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1 microM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.     

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Ume?-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Ume?-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.     

Na+, K(+)-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells.

Vascular smooth muscle cell hypertrophy is a normal compensatory state that may play a pathogenic role in hypertension. Angiotensin II stimulates a hypertrophic response in cultured vascular smooth muscle cells. As part of the growth response, angiotensin II rapidly activates the Na(+)-H+ exchanger, increasing Na+ influx. Because Na+, K(+)-ATPase is the major cellular mechanism for regulating intracellular Na+, we studied the effects of angiotensin II-induced hypertrophy on Na+, K(+)-ATPase expression and activity. Angiotensin II caused rapid increases in both steady-state Na+, K(+)-ATPase activity (ouabain-sensitive 86Rb uptake) and intracellular [Na+]. Angiotensin II also caused a sustained increase in Na+, K(+)-ATPase at 24 hours with a 73% increase in maximal 86Rb uptake per milligram protein and a fourfold increase in Na+, K(+)-ATPase alpha-1 messenger RNA levels. Thus, angiotensin II hypertrophy was associated with rapid increases in Na+, K(+)-ATPase activity due to increased Na+ entry and sustained increases due to a specific increase in Na+, K(+)-ATPase expression. These data demonstrate dynamic regulation of Na+, K(+)-ATPase at the functional and molecular level and suggest that similar compensatory mechanisms should be present in vivo. Alterations in such compensatory pathways may be fundamental to the pathogenesis of hypertension.     

Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio: possible implication in vascular remodelling

OBJECTIVES : Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. METHODS : DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. RESULTS : Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. CONCLUSIONS : Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.     

Effect of endotoxic shock on basal and insulin-mediated Na+/K(+)-pump activity in rat soleus muscle.

This study evaluated basal and insulin-mediated Na+/K+ transport in skeletal muscle during endotoxic shock. Fasted male Holtzman rats (80-100 g) were killed 5 hr after the i.v. injection of saline (control) or 20 mg/kg Salmonella enteritidis endotoxin. 86Rb+ uptake into the cellular compartment of soleus muscle was determined in the presence and absence of 100 mU/ml insulin. The rate coefficient and rate of 22Na+ efflux and muscle intracellular 22Na+ content (in percentage of total muscle 22Na+) were determined in the presence and absence of 1 mM ouabain. The rate of basal cellular 86Rb+ uptake (cpm x 10(3).g wet weight-1.min-1) was not significantly different between control (77.7 +/- 2.9) and endotoxic soleus muscles (76.1 +/- 3.9). Insulin increased the rate of cellular 86Rb+ uptake by 19% in control (92.4 +/- 3.7) and 23% in endotoxic soleus muscles (93.8 +/- 4.1). Cellular 22Na+ content was 40% greater in endotoxic muscle (16.8 +/- 1.4) as compared to control muscles (12.0 +/- 0.9). The rate coefficient for ouabain-sensitive 22Na+ efflux in endotoxic muscles (0.028 min-1) was 1.7-fold greater than that in control muscles (0.017 min-1). The rate of ouabain-sensitive 22Na+ efflux was more than doubled in endotoxic muscles (0.470%/min) compared with control muscles (0.204%/min). Endotoxic shock did not alter insulin's ability to stimulate Na+/K+ transport in muscle. An increase in the electrogenicity of the Na+/K+ pump is consistent with an increased rate coefficient for 22Na+ efflux, with no change in the rate of 86Rb+ uptake in endotoxic soleus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased membrane permeability of skin fibroblasts from the spontaneously hypertensive rat

Recently, we have demonstrated several abnormalities in Na+ and K+ homeostasis in cultured vascular smooth muscle cells derived from spontaneously hypertensive rats (SHR). To study whether similar defects can be identified in other cells of this rat strain, 86Rb and 22Na flux experiments as well as measurements of intracellular Na+ and K+ levels were performed in cultured skin fibroblasts of SHR and normotensive Wistar-Kyoto rats (WKY). The efflux rate constant (ke) for Rb+ (K+ analogue) was higher (p less than 0.001) in fibroblasts of SHR than in those of WKY (2.11 +/- 0.03 and 1.66 +/- 0.02 X 10-2/min; mean +/- SEM). The ouabain-insensitive influx rate constant (ki) for Rb+ was also higher (p less than 0.001) in fibroblasts of SHR than in those of WKY (13.26 +/- 0.41 and 10.71 +/- 0.27 X 10-2/min. On the other hand, the activity of the Na+-K+ pump of cells of SHR (44.81 +/- 0.81 X 10-2/min) was not different from that of cells of WKY (44.72 +/- 0.47 X 10-2/min). This parameter was obtained by calculating the ouabain-sensitive Rb+ influx rate constant. There was also no difference in the Na+ uptake (in the presence of ouabain) between cells of the two rat strains. Although there was no statistically significant difference in the measured intracellular total K+ levels between the two groups, on the basis of equilibrium distribution of 86Rb+, we calculated a significantly lower (p less than 0.001) level of exchangeable intracellular K+ in fibroblasts of SHR (98.2 +/- 1.2 mEq/L) as compared with cells of WKY (115.3 +/- 1.5 mEq/L). These findings indicate increased membrane permeability to K+ in fibroblasts of SHR and that this defect is likely to be innate to their membrane structure.     

Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells.

Diacylglycerol and phorbol esters activate protein kinase C in intact cells. We report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na+/K+ pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na+ influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt2) also enhanced ouabain-sensitive 86Rb+ uptake and amiloride-sensitive 22Na+ influx. Prolonged treatment (40 hr) of 3T3 cells with PBt2 at a saturating dose, which reduces the number of PBt2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt2. Appropriate controls using acid "loads" and the Na+ ionophore monensin showed that the function of the Na+/H+ antiport system and of the Na+/K+ pump was not impaired in the PBt2-desensitized cells. We suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na+/H+ antiport activity, which, in turn, leads to Na+ influx, intracellular pH modulation, and stimulation of the Na+/K+ pump.     

Serum factors affect Na+ pump activity and DNA synthesis in cultured cerebellar neural cells

In neurone-enriched cultures derived from early postnatal rat cerebellum and maintained in serum-free medium, addition of serum (10% FCS) evoked a rapid increase in Na+ pump activity (as measured by ouabain-sensitive 86Rb accumulation) by activation of a Na+/H+ exchanger. This effect did not occur with cultured cerebellar astrocytes. In contrast, exposure to serum increased DNA synthesis ([3H]thymidine incorporation) in both cultured cerebellar astrocytes and in the neurone-enriched cultures. However, in the latter cultures this effect was shown by autoradiography to be due to contaminating astrocytes. Thus, in cultured cerebellar neural cells an enhancement of intracellular Na+ accumulation by serum factors may not be linked to initiation of DNA synthesis. Furthermore, raising intracellular Na+ by ouabain exposure actually decreased neural cellular DNA synthesis.     

Na and K dependence of the Na/K pump in cystic fibrosis fibroblasts.

The Na and K dependence of the Na/K pump was measured in skin fibroblasts from patients with cystic fibrosis and age/sex-matched controls. Under basal conditions, there was no difference between control and cystic fibrosis cells in protein per cell, intracellular Na and K content, or Na/K pump activity (measured as ouabain-sensitive 86Rb uptake). There was no difference in the Na dependence of the Na/K pump between cystic fibrosis cells and control cells. In cells from patients with cystic fibrosis, the Na/K pump had a significantly lower affinity for K (Km = 1.6 mM) when compared to normals (Km = 0.9 mM). This difference was demonstrated by using two independent experimental designs.     

Activation of the sodium pump blocks the growth hormone-induced increase in cytosolic free calcium in rat adipocytes

GH promptly increases cytosolic free calcium ([Ca2+]i) in freshly isolated rat adipocytes. Adipocytes deprived of GH for 3 h or longer are incapable of increasing [Ca2+]i in response to GH, but instead respond in an insulin-like manner. Insulin blocks the GH-induced increase in [Ca2+]i in GH-replete cells and stimulates the sodium pump (i.e. Na+/K+-ATPase), thereby hyperpolarizing the cell membrane. Blockade of the Na+/K+-ATPase with 100 microM ouabain reversed these effects of insulin and enabled GH to increase [Ca2+]i in GH-deprived adipocytes. Both insulin and GH activated the sodium pump in GH-deprived adipocytes, as indicated by increased uptake of 86Rb+. Decreasing availability of intracellular Na+ by blockade of Na+/K+/ 2Cl- symporters or Na+/H+ antiporters abolished the effects of both hormones on 86Rb+ uptake and enabled both GH and insulin to increase [Ca2+]i in GH-deprived adipocytes. The data suggest that hormonal stimulation of Na+/K+-ATPase activity interferes with activation of voltage-sensitive calcium channels by either membrane hyperpolarization or some unknown interaction between the sodium pump and calcium channels.     

Increases in Na+,K+-ATPase activity of erythrocytes and skeletal muscle after chronic ethanol consumption: evidence for reduced efficiency of the enzyme.

Increased Na+,K+-ATPase activity observed after chronic ethanol consumption has been examined to determine whether the increase is due to changes in the kinetic properties of the enzyme or increases in the amount of enzyme in the membranes examined. In skeletal muscle and erythrocyte ghosts from rat, as well as from humans, increased Na+,K+-ATPase activity in ethanol-consuming individuals was not accompanied by an increase in the number of ouabain binding sites. In studies with intact human erythrocytes, similar ouabain-sensitive 22Na+ and 86Rb+ pumping rates were observed between normal and ethanol-consuming individuals and the Na+ to Rb+ pumping ratio was found to be 1.5 in all cases. However, ouabain-sensitive lactate plus Pi formation was increased in cells from alcoholic individuals. Thus these data suggest that increased enzyme activity may be due to a kinetic alteration of the Na+,K+-ATPase and that the enzyme may be less efficient in coupling ion pumping to ATP hydrolysis than the enzyme in normal cells.     

Effect of short term triiodothyronine administration on human leukocyte Rb(K) influx and Na efflux

The effect of short term T3 administration on leukocyte ouabain-sensitive 86Rb(K) influx and Na efflux in normal subjects was investigated. At a dose of 60 micrograms daily for 7 days, T3 induced a significant increase in leukocyte 86Rb(K) influx and a significant fall in plasma K concentrations. Plasma and intracellular Na concentrations did not change. [3H]Ouabain binding, a measure of Na-K ATPase units, did not change. A week after T3 administration, 86Rb(K) influx, Na efflux, and plasma K concentrations were normal. In a series of five hyperthyroid patients, both ouabain-sensitive 86Rb influx and [3H]ouabain binding were significantly greater than in normal subjects. We conclude that T3 stimulates 86Rb(K) influx and Na efflux by leukocytes in vivo independently of [3H]ouabain binding and that this increase is rapidly reversible. However, in hyperthyroid patients both 86Rb influx and [3H]ouabain binding are increased, probably due to prolonged exposure to thyroid hormone excess.     

Hypertensive dog plasma inhibits the Na+-K+ pump of cultured vascular smooth muscle

We investigated the effects of plasma from dogs with perinephritic hypertension on the Na+-K+ pump of cultured dog vascular smooth muscle cells. We also measured [3H]ouabain binding by myocardium and vascular tissue. Fresh, unprocessed plasma from healthy dogs during the first 6 weeks of benign one-kidney, one wrapped hypertension and from paired normotensive control dogs was layered over confluent primary cultured puppy aortic smooth muscle cells that had been sodium-loaded with monensin. In 26 paired assays of plasma from four pairs of dogs, cells incubated in the presence of plasma from hypertensive dogs had significantly reduced total (p less than 0.01) and ouabain-sensitive (p less than 0.001) 86Rb+ uptakes, but their intracellular sodium content did not differ from cells incubated in paired normotensive plasma. We no longer detected these uptake differences when passaged cells or cells cocultured with bovine endothelial cells were used for assay or when plasma was treated with protease inhibitors or boiled. However, boiled plasma increased the sodium content of the assay cells, suggesting an ionophorelike effect. Levels of pump inhibitory activity in plasma appeared to remain constant during Weeks 1 to 6 of hypertension. We found no evidence for altered numbers of pump sites in cardiovascular tissues from these hypertensive dogs. These findings support the hypothesis that plasma factors inhibit the membrane Na+-K+ pump in vascular smooth muscle cells in this form of hypertension. These plasma inhibitory factors apparently do not induce pump molecules.     

Phorbol ester inhibits furosemide-sensitive potassium transport in BALB/c 3T3 preadipose cells.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) rapidly decreased the rate of 86Rb+ uptake into BALB/c 3T3 preadipose cells. The component of total 86Rb+ influx affected by PMA is insensitive to ouabain but sensitive to the diuretic furosemide. Experiments designed to investigate the characteristics of the K+ transport system sensitive to PMA revealed that: (i) 86Rb+ uptake is highly dependent on external Na+, (ii) 86Rb+ uptake is highly dependent on external Cl-, (iii) 22Na+ uptake is dependent on external K+, and (iv) a major component of 86Rb+ efflux that is sensitive to PMA and furosemide is not dependent on extracellular K+. These features strongly implicate a Na+K+/Cl- cotransport system as the target of PMA and furosemide in these experiments. PMA caused a net intracellular accumulation of K+ within 15 min in these cells, presumably via its inhibitory effect on furosemide-sensitive K+ transport. Within 30 min after PMA treatment, the mean cell volume was significantly reduced in treated compared to control cells, with a maximum decrease of 21% attained at 4 hr after PMA. The significance of these findings for biologic changes induced by PMA is discussed.     

Myocardial sodium pump activity in endotoxin shock

The effect of endotoxin administration on the integrity of sodium pump was studied in canine heart myocardium. Sodium pump activity was determined based on the 86Rb+ uptake by ventricular muscle strips. The results show that 4 hr following endotoxin administration the ouabain-sensitive 86Rb+ uptake was inhibited by 44-70%, and the inhibition was noncompetitive with 86Rb+ ions and was independent of Na+ concentrations. The sensitivity of sodium pump toward verapamil inhibition was unaffected by endotoxin administration, suggesting that the endotoxin-induced decrease in sodium pump activity is not mediated through alteration in either calcium or slow sodium channel. The sensitivity of 86Rb+ uptake to ouabain inhibition was decreased approximately sixfold in endotoxin-injected dogs. These data suggest that sodium pump is greatly impaired in dog hearts 4 hr after endotoxin administration. The impairment in sodium pump might have physiological significance in the development of myocardial dysfunction during endotoxic shock.     

Liver angiotensin II receptors in the rat: binding properties and regulation by dietary Na+ and angiotensin II

Recent evidence suggests that angiotensin II (AII) acts on the liver via specific receptors. The aims of this study were to examine the general binding properties of these receptors in the rat and to determine the role of dietary Na+ and AII in the regulation of AII receptors. Binding of 125I-labelled Ile5-AII to liver membranes was saturable and behaved as a single class of sites with an affinity (Ka) of 2.9 l/nmol and a Hill coefficient of 0.99. Kinetic analysis of AII binding gave estimates for the rates of association and dissociation of 42 l/mumol per min and 1.5 X 10(-2)/min respectively. The binding of analogues exhibited the following order of potency: Val5-AII greater than Ile5-AII greater than AIII greater than Sar1-Ala8-AII greater than Sar1-Gly8-AII greater than AI greater than des-Asp-AI greater than C4-C8 pentapeptide greater than Phe1-Tyr8-AII greater than neurotensin greater than luteinizing hormone-releasing hormone. Binding was enhanced by Mg2+ and Ca2+ and inhibited by EDTA and the reducing agent dithiothreitol. Low dietary Na+ affected in a biphasic manner both the Ka and concentration (Ro) of liver AII receptors. Initially, Ro decreased from 25.9 +/- 3.8 (control) to 16 +/- 1.9 (S.E.M.) pmol/g tissue by 1.5 days but thereafter it rapidly increased to 47.3 +/- 8.7 pmol/g tissue (3.5 days) and remained elevated to the end of the experiment, 8.5 days later. The Ka initially increased from 2.7 +/- 0.3 (control) to 4.3 +/- 0.5 l/nmol (1.5 days) and then decreased steadily to 1.2 +/- 0.1 l/mol (8.5 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/H+ antiport in Swiss 3T3 cells: mitogenic stimulation leads to cytoplasmic alkalinization.

Addition of Na+ to Na+-depleted Swiss 3T3 cells causes a rapid and dramatic increase in intracellular pH, as monitored by uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione. The effect of Na+ is concentration dependent (half-maximal effect at 38 mM); this cation can be replaced by Li+ but not by K+ or the choline ion. Amiloride prevents the Na+-induced increase in intracellular pH and also blocks the entry of Na+ into 3T3 cells; the half-maximal concentrations of amiloride for inhibiting the two processes are similar (40 microM). Increase in extracellular pH caused an increase in the initial rate of Na+ influx that was of sufficient magnitude to stimulate the activity of the Na+/K+ pump in quiescent 3T3 cells. Taken together, these findings suggest the presence of a functional Na+/H+ antiport in Swiss 3T3 cells. Addition of the potent mitogenic combination platelet-derived growth factor, vasopressin, and insulin to quiescent Swiss 3T3 cells increased the intracellular pH from 7.21 +/- 0.07 to 7.36 +/- 0.09 in 10 independent experiments (P less than 0.001). This combination of growth factors also stimulated Na+ entry and ouabain-sensitive Rb+ uptake. The data support the hypothesis that early changes in ion fluxes play a role in signaling mitogenesis in 3T3 cells.