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.     

Effect of glucose intake on human leucocyte 86Rb influx and [3H]-ouabain binding

86Rb influx and [3H] ouabain binding by human leucocytes were measured in eight normal nonobese fasting subjects before and after a challenge with 75 g glucose orally. The mean ouabain-sensitive 86Rb influx increased significantly from 194 to 283 mmol/kg protein/h (P less than .01), and [3H]-ouabain binding increased from 236 to 403 fmol/mg protein. The mean plasma potassium concentration fell from 4.2 to 3.9 mmol/L (P less than .05). Following intravenous glucose infusion, the median 86Rb transport increased from 186 to 267 mmol/kg protein/h, while median plasma potassium concentration fell from 4.3 to 3.9 mmol/L. Therefore, glucose intake acutely increases Na-K ATPase units, stimulates potassium (Rb) transport, and causes a concomitant fall in plasma potassium concentrations. Nutritional intake is probably an important determinant of Na-K ATPase units and activity in the human leucocyte.     

Diminished [3H] ouabain binding and 86Rb influx by leukocytes in anorexia nervosa

Leukocyte Na-K ATPase was assessed in ten patients with anorexia nervosa. The indices of Na-K ATPase measured were 86Rb influx and [3H]-ouabain binding. Both 86Rb influx and [3H]-ouabain binding were significantly lower in patients with anorexia nervosa than in controls. Following weight gain (4.1 to 11.9 kg) in seven patients both 86Rb influx and [3H]-ouabain binding increased in each patient to levels similar to those in controls, although the patients still remained underweight. Acute oral glucose challenge (75 g) also resulted in an increase in 86Rb influx and [3H]-ouabain binding in each of the eight patients tested. There was a significant correlation between 86Rb influx and [3H]-ouabain binding. We conclude that the leukocytes of patients with anorexia nervosa have a significantly diminished number of Na-K ATPase units with a parallel decrease in 86Rb influx. Weight gain and acute glucose challenge result in an increase in both indices of Na-K ATPase. Nutrition appears to play an important role in the modulation of this enzyme.     

86Rb(K) influx and [3H]ouabain binding by human platelets: evidence for beta-adrenergic stimulation of Na-K ATPase activity

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [3H]ouabain binding by the human platelet. This 86Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.     

Increased leucocyte Na-K ATPase in obesity: reversal following weight loss

Ouabain-sensitive 86Rb influx and [3H] ouabain binding capacity were investigated in the leucocytes of 17 obese patients and 15 control subjects. Both were significantly increased in the obese when compared with controls. Following dietary restriction and a 4% to 5% weight reduction in the obese over 2 weeks, [3H] ouabain binding and ouabain-sensitive 86Rb influx (a model for K+ influx) decreased to levels similar to those in controls. This shows that the number of Na-K ATPase sites on leucocyte membranes of the obese are significantly increased and that this is associated with accelerated 86Rb transport. Since both of these indices decreased following 4% to 5% reduction in body weight while the patients were still obese, increased Na-K ATPase is neither a marker of nor cardinal to the pathogenesis of obesity. We conclude that (1) increase in Na-K ATPase units and 86Rb influx are not characteristic of obesity itself and (2) dietary restriction over the short-term with limited weight reduction restores Na-K ATPase units and 86Rb influx to normal.     

Lymphocyte Na,K-ATPase is reduced in aged people

Na,K-ATPase-dependent 86Rb uptake, maximum velocity (Vmax), Michaelis constant (Km) of the uptake, and [3H]-ouabain binding were investigated in the lymphocytes of 10 elderly subjects (age greater than 60 years), and in 10 middle-aged (41 to 60 years) and 10 young controls (age less than or equal to 40 years). 86Rb uptake was reduced in elderly versus both middle-aged and young subjects (20.14 +/- 3.30 v 35.60 +/- 2.67, P = .002, and v 36.53 +/- 4.49 nmol, P = .012), as was the number of [3H]-ouabain binding sites per cell (32,662 +/- 2,215 v 40,420 +/- 1,184, P = .011, and v 40,596 +/- 1,349, P = .014). Vmax was reduced in elderly v young subjects (1.20 +/- 0.10 v 1.64 +/- 0.13, P = .034), but not versus the middle-age group (1.20 +/- 0.10 v 1.54 +/- 0.12 nmol.min-1, NS). Km was no different among the three groups. No differences were found between middle-aged and young subjects. Significant correlations were observed between age and Na,K-ATPase-dependent 86Rb uptake (r = -.620, P = .00009), Vmax (r = -.439, P = .024), and [3H]-ouabain binding sites (r = -.648, P = .002). Moreover, the site number was positively correlated with both uptake (r = .635, P = .002) and Vmax (r = .554, P = .011). These differences were observed both in women and men. We conclude that there is an age-dependent reduction in lymphocyte Na,K-ATPase activity, which is fully manifested over 60 years, and that this alteration is probably due to the reduced number of functional units of Na,K-ATPase in advancing age.     

Serum rapidly stimulates ouabain-sensitive 86-RB+ influx in quiescent 3T3 cells.

Serum causes a 4-fold increase in 86Rb+ (a K+ tracer) influx in quiescent 3T3 mouse fibroblast cells. It is one of the earliest changes caused by serum, being seen in 2 min and reaching a maximum in 10 min. Removal of serum causes rapid reversal of this effect. Serum acts mainly by increasing the maximum velocity, Vmax, of entry. Ouabain inhibits entry of 86Rb+ (82-90%) both in the presence and absence of serum, but does not alter exit. The rapid increase in cation influx is unaffected by cycloheximide and by changes in cyclic AMP and GMP. Low concentrations of insulin, epidermal growth factor, and prostaglandins (E1 and F2alpha) produced a smaller (80%) activation of 86Rb+ entry. Ouabain, at a level that inhibits cation influx, also prevents the onset of DNA synthesis following serum addition; this is reversible effect dependent on the concentration of K+ in the medium. This suggests that cation pumping activity may be required for initiation of DNA synthesis.     

Phencyclidine in nanomolar concentrations binds to synaptosomes and blocks certain potassium channels.

Phencyclidine [1-(phenylcyclohexyl)piperidine; PCP], in low dose (approximately equal to 0.1-0.2 mg/kg of body weight), induces a schizophrenia-like behavioral syndrome in man; this effect has been attributed to block of neuronal K channels. We used a K-stimulated 86Rb efflux assay to demonstrate that low concentrations of PCP (10-50 nM) block a class of depolarization-activated K channels in rat brain synaptosomes--pinched-off presynaptic nerve terminals. The dose-response curve is biphasic, and much higher PCP concentrations (greater than 10 microM) are required to block the remainder of the K-stimulated 86Rb efflux. The [3H]PCP binding curve for synaptosomes is also biphasic: PCP binds to some components with high affinity (Kd approximately equal to 6.0 X 10(-8) M), and to other components with much lower affinity (Kd approximately equal to 1.15 X 10(4) M). PCP can be photoactivated with UV light to form covalent bonds: after UV irradiation, previously-bound [3H]PCP is no longer displaceable by a large excess of unlabeled PCP. Preliminary data from NaDodSO4/polyacrylamide gel electrophoresis studies after covalent binding of [3H]PCP to synaptosomes, suggest that the high-affinity binding site may be on a large protein (Mr approximately equal to 220,000). We conclude that the high-affinity PCP binding protein is associated with the K channels that are blocked by nanomolar concentrations of PCP. Block of these channels could, by prolonging action-potential duration in presynaptic nerve terminals, enhance calcium entry and neurotransmitter release, thereby altering transmission at central synapses involved in behavioral expression.     

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.     

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.     

Aging: effects on sodium- and potassium-activated adenosine triphosphatase activity and ouabain binding sites in rat brain

Sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) activity and [3H]-ouabain binding were examined in homogenates of cerebral cortex, striatum, and hypothalamus of 3-, 8-, and 26-month-old rats to determine if aging-related alterations in energy utilization demonstrated in brain slices and homogenates are potentially associated with alterations in Na,K-ATPase. There were no consistent age-related changes seen in Na,K-ATPase activity, the number of [3H]-ouabain binding sites, or their affinity for ouabain. Moreover, enzyme activities of the two molecular forms of Na,K-ATPase and their inhibition by strophanthidin did not appear to be different in partially purified enzyme preparations obtained from whole brain of 3- and 26-month-old rats. In contrast, the concentration of [3H]-ouabain binding sites was lower in cardiac muscle of senescent rats indicating a reduction in the number of active Na,K-ATPase units. It appears unlikely that aging-related central nervous system changes are associated with alterations in the Na,K-ATPase enzyme system.     

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.     

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+,K+-ATPase activity.

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 degrees C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding, whereas incubation with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[3H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[3H]amphetamine binding, suggesting the involvement of Na+,K+-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na+,K+-ATPase activity and the number of specific "high-affinity" binding sites for [3H]ouabain, and a good correlation was observed (r = 0.89; P less than 0.02) between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. Similar increases in (+)-[3H]amphetamine binding, [3H]ouabain binding, and Na+,K+-ATPase activity were observed in the hypothalamus after parenteral administration of glucose to rats. The administration of anorectic doses of amphetamine (0.1-5.0 mg/kg of body weight) also increased Na+,K+-ATPase activity in the hypothalamus. These data suggest that the (+)-[3H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na+,K+-ATPase activity, and the latter may be involved in the "glucostatic" regulation of appetite.     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Effect of dopamine agonists and antagonists on the binding of [3H]estradiol to its receptors in the anterior pituitary gland of male rats

The synthesis of PRL and DNA in PRL cells is regulated by estrogens and dopamine. To investigate a possible relationship between these two components, we studied the influence of dopamine agonists and antagonists on the binding of [3H]estradiol ([3H]E2) to its receptors in the anterior pituitary gland of estrogenized male rats. The administration of sulpiride (dopamine antagonist) or bromocriptine (dopamine agonist) decreased the binding of [3H]E2 to cytosolic receptors when the concentration of [3H]E2 in the assay mixture was 1 nM. Both drugs also diminished the binding of [3H]E2 when they were added in vitro to the incubation media, apparently in a competitive way. Dopamine and alpha-methyltyrosine also inhibited competitively the binding of [3H]E2 to cytosolic receptors. The inhibition constants were determined by the Lineweaver-Burk plot. To overcome the competitive inhibition of dopamine agonists and antagonists, the concentration of the titrated steroid in the incubation mixture was increased to 16 nM. This concentration was established by saturation analysis. The administration of alpha-methyltyrosine increased the binding of [3H]E2 to nuclear receptors without modifying the binding to cytosolic receptors. This increase paralleled an increment in the levels of plasma PRL. Bromocriptine prevented the increase in [3H]E2 binding produced by alpha-methyltyrosine and had no effect on the binding when administered to nontreated rats. These results suggest that dopamine can regulate the biological effects of estradiol in the anterior pituitary gland by decreasing the binding of this hormone to its receptors.     

Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells.

Vanadyl sulfate and sodium orthovanadate in the concentration range between 5 and 50 microM are shown to be mitogenic for quiescent cultures of Swiss mouse 3T3 and 3T6 cells. The compounds caused a striking shift in the dose-response for the effect of serum on [3H]thymidine incorporation and DNA synthesis. In the absence of serum the effect of vanadium was greatly potentiated by insulin. Vanadium ions produced no more than additive increases in [3H]thymidine incorporation when combined with epidermal growth factor, cholera toxin, or phorbol 12-myristate 13-acetate. Both vanadium compounds stimulated ouabain-inhibitable 86Rb+ uptake, indicating that the vanadium ions increase, rather than inhibit, Na+/K+ pump activity in the intact cell. Neither vanadium compound had any effect on cellular cAMP under a variety of different conditions. The mitogenic effect of the vanadium compounds was similar to that of colchicine. Taxol, which stabilizes cytoplasmic microtubules, prevented the stimulation of DNA synthesis by vanadium.     

Increased sodium permeability and transport as primary events in the hypertensive response to deoxycorticosterone acetate (DOCA) in the rat

We report here that 18-24 h after subcutaneous administration of DOCA to the rat, plasma [Na] was significantly increased and [K] reduced without change in cell Na concentration, [Na]i, or cell K concentration, [K]i, as measured in rapidly excised tail arteries. The large increase in the transmembrane K gradient, operationally [K]i/[K]o, was associated with a significant decline of about 7 mV in the transmembrane membrane potential (Em) measured in small mesenteric arteries in situ, and a small but significant rise in blood pressure. We conclude that the permeability of the membrane to Na relative to K was increased at this time but enhanced transport of both ions was sufficient to maintain near-normal cell concentrations. These were primary events in relation to the hypertension induced by DOCA, as the rise in plasma [Na] and fall in [K] were already apparent at 6 h.     

Erythrocyte cation fluxes in the normotensive and hypertensive clients of a health screening clinic

Erythrocyte cation transport was measured in vitro using 22Na+ and 86Rb+ uptake techniques in Caucasian men with newly-detected hypertension and in male control groups. The Na+, K+ cotransport [determined by ouabain-resistant frusemide-sensitive (ORFS) components of Na+ or Rb+ influx], sodium pump activity (determined by ouabain-sensitive Rb+ influx) and erythrocyte Na+ and K+ concentrations were not significantly altered in hypertensive men. The total Na+ influx in hypertensives (n = 59) was significantly greater (P less than 0.001) than in controls. The difference was mainly attributable to an increase in the ouabain-resistant frusemide-resistant component of this flux. The total Rb+ influx in hypertensives (n = 39) was also greater (P less than 0.005) than in controls. Overall, both total Na+ influx and total Rb+ influx were positively correlated (P less than 0.01) with diastolic blood pressure and with habitual dietary intake of alcohol. Multivariate analyses after controlling for the effect of blood pressure showed that mean corpuscular volume (MCV) and alcohol intake were statistically significant predictor variables for total Rb+ influx, although not for total Na+ influx. The results are compatible with increased diffusion of cations across the erythrocyte membrane in hypertension, but raise the question of a possible role of alcohol intake in mediating this effect.     

The relationship between some beta-adrenergic mediated responses and plasma concentrations of adrenaline and cyclic AMP in man

To test the hypothesis that increments in plasma cyclic AMP during beta-adrenergic stimulation reflect integrated second messenger function of the tissues activated by the agonist, graded adrenaline infusion resulting in plasma adrenaline concentrations within the physiological range was performed in 8 healthy subjects with and without concomitant beta-adrenoceptor blockade by iv propranolol. A significant correlation was found between increments in plasma adrenaline and plasma cyclic AMP in the experiments without beta-blockade; during concomitant beta-blockade the increase in plasma cyclic AMP concentrations at low adrenaline infusion rates was prevented, whereas a small increase in cyclic AMP was found at high adrenaline infusion rates, probably owing to incomplete beta-receptor blockade. Likewise, the adrenaline-induced increments in blood substrates (glucose, lactate, glycerol and beta hydroxybutyric acid) were significantly reduced but not completely prevented by beta-blockade. We conclude that an altered relationship between beta-agonist concentrations and plasma cyclic AMP may provide evidence for the existence of differences in beta-adrenergic sensitivity in man.     

Thyroidal inhibition of growth hormone secretion in fowl: tri-iodothyronine-induced down-regulation of thyrotrophin-releasing hormone-binding sites on pituitary membranes

The number, but not affinity, of binding sites for [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) on chicken adenohypophysial plasma membranes was increased in chickens made hypothyroid by goitrogen (methimazole) treatment (50 mg/kg per day for 7 days), which also increased circulating GH concentrations. Daily i.p. injection of thyroxine (T4; 100 micrograms/kg for 7 days) had no effect on [3H]Me-TRH binding to pituitary membranes, although it suppressed endogenous GH secretion. Binding of [3H]Me-TRH to pituitary caudal lobe membranes was, however, suppressed by tri-iodothyronine (T3) injected chronically (100 micrograms/kg per day, i.p., for 7 days) or acutely (100 micrograms/kg, 2 h before being killed). The suppression of [3H]Me-TRH binding and inhibition of GH secretion following T3 administration was dose related. Binding of [3H]Me-TRH to caudal lobe membranes was also suppressed following the incubation of pituitary glands with T3 in vitro, and the response was both dose and time related. These results suggest that T3 inhibits GH secretion in fowl by a down-regulation of pituitary TRH receptors. However, other mechanisms are involved in thyroidal inhibition of GH release in birds, since T4 had no effects on [3H]Me-TRH binding yet suppressed GH secretion in vivo.