Rubidium Efflux Research Articles

Specificity of action of the novel antihypertensive agent, BRL 34915, as a potassium channel activator: Comparison with nicorandil

Experiments have been performed to investigate the specificity of the mechanism of action of the novel antihypertensive agent, BRL 34915. BRL 34915 (0.5–100 μM) and nicorandil (10–500 μM) stimulated the efflux of rubidium from preloaded rabbit isolated mesenteric arteries. BRL 34915 also caused an increase in the rubidium efflux rate constant in other vascular smooth muscles. Tetraethylammonium (0.1–30 mM) inhibited BRL 34915 (10 μM), nicorandil (100 μM) and potassium (30 mM) induced stimulations of rubidium efflux, but had no effect on noradrenaline (30 μM) induced efflux. Only noradrenaline induced efflux was inhibited by apamin (3–100 nM). Examination of other second messenger systems demonstrated that BRL 34915 (at concentrations up to 100 μM) did not have any appreciable effect on cGMP accumulation in rabbit mesenteric artery, cAMP or cGMP phosphodiesterase in rat heart, or cAMP and inositol phosphate accumulation in rat brain slices. Nicorandil (100 μM) caused a small increase in cGMP accumulation in rabbit mesenteric artery. Radioligand binding studies showed that BRL 34915 did not interact with dihydropyridine, 5-hydroxytryptamine, dopamine, α 1, α 2 or β adrenoceptor binding sites. [ 3H]-BRL 34915 did not bind specifically to any site in any tissue studied, either in vitro or ex vivo. Thus we have been unable to demonstrate an effect of BRL 34915 other than of increasing potassium efflux in rabbit vascular smooth muscle. This lends support to other evidence suggesting that BRL 34915 relaxes vascular smooth muscle (and hence lowers blood pressure) by a novel, and specific, mechanism involving hyperpolarisation of the smooth muscle cell membrane.

Oxygen free-radical reduction of brain capillary rubidium uptake.

Free radicals are proposed to play a role in the injury following cerebral ischemia in which cerebral edema is a prominent feature. To determine whether free radicals might alter the movement of ions and water across the blood-brain barrier, we examined their effect on brain capillary transport. Rat brain capillaries were isolated, incubated with a system that generates free radicals, and various capillary transport systems were studied. Rubidium uptake was reduced 74% whereas rubidium efflux, glucose transport, and capillary water space were unchanged. The results following the addition of radical scavengers indicated that hydrogen peroxide or a related free radical was the toxic species. These data suggest that free radicals can impair capillary endothelial cell mechanisms that help maintain homeostasis of electrolytes and water in brain.

Effects of antiglucocorticoids on glucocorticoid hypertension in the rat.

The early phase of hypertension induced in rats by a glucocorticoid agonist RU 26988 was studied. Systolic blood pressure increased by 35 mm Hg. Water and sodium urinary excretion increased transiently, and plasma volume decreased. Total and ouabain-sensitive sodium efflux, as well as rubidium efflux, were enhanced by glucocorticoid administration. Low salt intake did not prevent hypertension. Pretreatment with RU 38486, a steroid with antiglucocorticoid properties, largely prevented the rise in blood pressure (+10 mm Hg) and suppressed transient natriuresis and the decrease in plasma volume. Changes in total and ouabain-sensitive sodium efflux were completely prevented, whereas changes in rubidium efflux were only partly reversed. Similarly, administration of progesterone, a steroid with antiglucocorticoid effects, prevented glucocorticoid hypertension (+11 mm Hg) and vascular ionic changes. In contrast administration of RU 28318, an antimineralocorticoid agent, was without effect on glucocorticoid hypertension (+38 mm Hg). Progesterone or RU 38486 administered after glucocorticoid also decreased blood pressure. Present data indicate that glucocorticoid hypertension may be prevented or reversed in its early phase by steroid drugs with antiglucocorticoid properties. These drugs also appeared to prevent the sodium and rubidium flux abnormalities induced by glucocorticoid. We suggest that activation of the vascular glucocorticoid receptors may be involved in the pathophysiology of glucocorticoid hypertension.

Proton-activated rubidium transport catalyzed by the sodium pump.

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.

Effects of alpha‐adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets

Epinephrine, norepinephrine or the more selective alpha-2 adrenoceptor agonist, clonidine, inhibited insulin release from isolated pancreatic islets of lean mice or obese mice homozygous for the gene ob. Clonidine was highly effective at 0.1 mumol/l. In contrast, the preferential alpha-1 adrenoceptor agonist, phenylephrine, had no or only a modest effect at 10 mumol/l. The effects of norepinephrine or clonidine were counteracted by yohimbine, a preferential blocker of alpha-2 receptors, but not by prazosine, an alpha-1 receptor blocker. The glucose-stimulated uptake of 45Ca2+ in the islets was only consistently inhibited by epinephrine. This effect was counteracted by yohimbine. Clonidine had no effect on the release of 86Rb+ from preloaded islets. It is concluded that insulin secretion is suppressed by alpha-2 receptor agonism in the pancreatic beta-cells and that this effect is mediated by mechanisms other than the transmembrane fluxes of calcium or potassium ions.

Cation pump activity and membrane permeability in human senile cataractous lenses

The majority of human senile cataractous lenses show a progressive alteration of electrolyte levels resulting in increased lens sodium and decreased potassium, and a concomitant increase in lens water. These changes are known to be associated with the cortical type of senile cataract. The aim of this study was to investigate the mechanisms responsible for the osmotic imbalance in cortical cataract. Determination of Na,K-ATPase activity gave a wide range of values that were not significantly different from normal controls, but rubidium influx showed a normal accumulation in cataractous lenses demonstrating a normal functioning of the cation pump. Both sodium influx and rubidium efflux are increased in the cataractous lenses and the increase is proportional to the absolute level of lens sodium. These results suggest that the biochemical change responsible for the osmotic imbalance in human senile cataract is not a defect in the cation pump mechanism but rather an increase in lens membrane permeability.

Rubidium efflux from neural cell lines through voltage-dependent potassium channels

Cloned cell lines derived from the rat and mouse nervous systems were screened for the presence of voltage-dependent K+ channels by assaying for an increase in 86Rb+ efflux under depolarizing conditions. Two types of depolarizing stimuli, (a) 100 mM KCl and (b) a combination of veratridine and scorpion venom, produced parallel results in all of 11 excitable cell lines tested; i.e., a cell line either responded to both sets of stimuli (8 examples) or to neither (3 examples). In the case of three cell lines where it was possible to compare the Rb+ efflux results with electrophysiological measurements of K+ currents the two methods gave qualitatively parallel results; i.e., cell lines that exhibited delayed rectification responded positively to depolarization in the Rb+ efflux assay, while cell lines that failed to exhibit K+ currents did not respond in the efflux assay. KCl-induced 86Rb+ efflux from the excitable cell lines did not appear to involve a Ca2+-dependent K+ channel, since efflux was not blocked by lowering extracellular Ca2+ from 2 to 0.1 mM. Efflux also was not blocked by tetraethylammonium or 4-aminopyridine, but could be blocked by scorpion venom. Of 14 cell lines previously classified as glial, 7 failed to show evidence of depolarization-induced 86Rb+ efflux while 7 others responded positively to 100 mM K+. Efflux from the latter 7 lines could be distinguished from efflux from the excitable lines in three ways. (1) Veratridine plus scorpion venom did not stimulate efflux. (2) Scorpion venom did not block KCl-induced efflux. (3) Tetraethylammonium and 4-aminopyridine were effective in blocking KCl-induced efflux from 4 of the 7 lines. These results extend the usefulness of ion flux measurements as a means of assessing the electrical properties of cells and raise some interesting questions concerning the nature of cells that appear to have K+ channels but not Na+ channels.

381 - The erythrocyte membrane electric field and its functional role

Results are demonstrated and discussed stressing that the electric field of the cell membrane is an important regulatory parameter of various functions. The electrostatics of the membrane is due both to transmembrane potential and to surface potentials on both sides of the membrane. The transmembrane potential of human erythrocytes is determined by the ionic conditions of the outside solution as well as by the cell state. The outside surface potential depends on the spatial distribution of fixed charges in the glycocalyx and its dynamics. To some extent, both parameters are calculable and measurable. It is demonstrated here that the shape of human erythrocytes depends on the sign of transmembrane potential. The ouabain-sensitive sodium efflux indicates a minimum under conditions were transmembrane potential is near zero. Rubidium efflux, on the other hand, is determined by the properties of surface charges.

The Increased Passive Efflux of Sodium and Rubidium from Rabbit Erythrocytes by Microwave Radiation

temperature of the samples and the power absorbed were controlled. Arrhenius plots of measurements made in the absence of radiation exposure revealed that both the sodium and rubidium efflux have four separate linear regions with transitions at 8-13, 22.5, and 36?C. The efflux rates with microwave exposure were identical to the control rates, except at the critical temperatures, where irradiation increased the efflux of both cations. This response was examined at the 22.5'C transition at three specific absorption rates (100, 190, and 390 mW/g). At all three levels the cation efflux was statistically greater than one would predict from a strictly thermal response. The response does not increase monotonically as a function of absorbed power. Similar increased cation efflux was also observed near the 36'C and 8-13'C transitions. Possible mechanisms for this effect are discussed.

9-aminoacridine- and tetraethylammonium -induced reduction of the potassium permeability in pancreatic B-cells: Effects on insulin release and electrical properties

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05–1 mmol/1) and tetraethylammonium (2–40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca 2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb + efflux from the islets. Both drugs also slightly reduced 86Rb + uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.

The calcium requirement for stimulation of rubidium efflux from isolated rat parotid acinar cells by carbachol

Stimulation of 86Rb + efflux from isolated parotid acinar cells by carbchol was biphasic. The phases of stimulated 86Rb + efflux were separated on the basis of their relative requirements for extracellular Ca 2+. If the isolated cells were incubated in Ca 2+ free buffer containing 1.0 mM ethylene glycol bis (β-aminoethyl ether) N, N 1 - tetra acetic acid (EGTA) for 30 min. before adding carbachol an initial phase of 86Rb + efflux was observed. A second phase of 86Rb + efflux was obtained upon addition of 2.0 mM Ca 2+. However when cells were incubated for 60 min. in Ca 2+ free buffer containing 1.0 mM EGTA the initial phase of release caused by carbachol was inhibited by 95 percent. If the EGTA was titrated with Ca 2+ to give 1.0 mM Ca 2+, following the 60 min. depletion regimen, the second phase was observed. Although 60 min. of Ca 2+-depletion in EGTA buffer was required for complete inhibition of the effect of carbachol on the initial phase of 86Rb + efflux, the response was fully restored within 4 min. after the readdition of Ca 2+.

Alloxan-induced alteration of insulin release, rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues

Insulin release and 86Rb efflux were studied in perifused rat islets exposed in vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased 86Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in 86Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of 86Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde, alpha-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and alpha-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce 86Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce 86Rb efflux.

Valinomycin inhibition of insulin release and alteration of the electrical properties of pancreatic B cells

The effects of valinomycin on insulin release and rubidium efflux from perifused isolated rat islets were investigated and correlated with its effects on the electrical properties of mouse B cells studied with microelectrode techniques. Valinomycin produced a (1 · 10 −9–1 · 10 −6 M) dose- and time-dependent inhibition of (10–15 mM) glucose-stimulated insulin release but did not affect basal secretion. This inhibitory effect rapidly followed addition of the ionophore and equally affected the two phases of glucose-stimulated secretion. It was not reversible by simple washing of the islets, but could be reversed transiently by tetraethylammonium or high extracellular potassium ion levels. At low or high glucose, valinomycin rapidly augmented the rate of 86Rb + efflux from preloaded islets. Amplitude and rapidity of this effect were dose-dependent and it was antagonized by tetraethylammonium. Glucose metabolism by islet cells was reduced only slightly (15%) by 1 · 10 −7 M valinomycin. During the first 6 to 8 min of valinomycin addition, the membrane potential of B cells augmented slowly but the typical bursts of spikes disappeared rapidly. Later on, B cells hyperpolarized more quickly to a stable value of approx. −70 mV. Increasing extracellular K + immediately depolarized B cells and the linear relationship found between the logarithm of K + concentration and the membrane potential was characterized by a slope of 58 mV for a ten-fold increase in extracellular K +. These results suggest that valinomycin interferes with the insulin releasing effect of glucose by increasing the potassium permeability of the B cell membrane.

Changes in fluorescence of 8-anilino-1-naphthalene sulfonate after bacteriophage T5 infection of escherichia coli. Initial fluorescence rise coincides with onset of rubidium efflux

Escherichia coli cells pre-loaded with 86Rb + begin to lose 86Rb + immediately after phage T5 addition. The loss proceeds with negative-exponential (first-order) kinetics for up to approximately 15 min after phage addition. The constant which characterizes the rate of loss increases with increasing numbers of infecting phage per cell. It is known that anaerobic, fermenting cells of E. coli show a two-step increase in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence upon infection with bacteriophage T5; the first rise begins immediately upon phage addition, the second 6 min later. The onset of 86Rb + release, therefore, is correlated with the first fluorescence rise with respect to timing and response to the multiplicity of infection.

Tetraethylammonium potentiation of insulin release and inhibition of rubidium efflux in pancreatic islets

Summary The significance, for insulin release, of changes in B cell K permeability, was investigated with perifused rat islets. Tetraethylammonium (TEA, 20 mM) had no effect on insulin secretion at 2 mM glucose, but markedly and reversibly potentiated the stimulation by 6 mM glucose. TEA effect was not due to activation of cholinergic or β-adrenergic receptors in B cells. 6 mM glucose greatly reduced 86 Rb efflux (used as tracer of K efflux) from preloaded islets. At 2 mM glucose, TEA decreased 86 Rb efflux to the same extent as did 6 mM glucose alone; at 6 mM glucose TEA reduced 86 Rb efflux still further. These findings suggest that a decrease in K efflux from B cells is not sufficient to trigger insulin release, but may potentiate the secretory response evoked by a secretagogue.

Bioassay Method for Polyene Antibiotics Based on the Measurement of Rubidium Efflux from Rubidium-Loaded Yeast Cells

A bioassay method for the polyene antibiotics nystatin and amphotericin B is proposed based on the measurement of the efflux of rubidium ions from a rubidium-loaded yeast culture challenged with the antibiotics. For this purpose a major proportion of the intracellular K(+) ions in a Saccharomyces cerevisiae culture has been substituted by Rb(+) ions. The rubidium leakage is measured by atomic absorption spectrophotometry, and a straight-line, dose-response correlation has been obtained for both antibiotics.

Effects of norepinephrine in the presence of adrenergic antagonists and different ionic media upon the mechanical and 86 rubidium efflux responses in vascular smooth muscle.

The action of norepinephrine (NE) on the isolated guinea pig mesenteric–portal vein was studied in relation to its mechanical and 86rubidium (86Rb) efflux responses using a continuous flow technique. NE (1 × 10−8 to 2 × 10−6 g/ml) produced dose-dependent increases in both contraction and 86Rb efflux. Phentolamine but not propranolol inhibited both responses to NE. In high potassium medium the 86Rb efflux response was more markedly depressed than the mechanical response. In calcium-deficient medium the NE-induced contraction was abolished but the 86Rb efflux response was potentiated and in high calcium medium there was a significant depression of the mechanical response but no significant difference in the 86Rb efflux response. There seems to be an optimal calcium ion concentration at 2.5 mM for the mechanical responses to NE. These experiments suggest that contraction correlates strongly with an increase in membrane permeability, calcium is required for vascular muscle contraction, and contraction can be initiated by a mechanism independent of changes in membrane potential.

Interactions of serotonin with neuromuscular blocking agents

Serotonin has been shown to reverse blockade induced by the nondepolarizing agents and hemicholinium and to potentiate blockade produced by the depolarizers. It is more effective at low frequencies of stimulation. The drug augments the twitch potentiations produced by small doses of decamethonium and neostigmine. It has no effect on exogenously applied acetylcholine. The agent increases the vascular resistance but this is not a prerequisite to its myoneural actions. Rubidium efflux from vascular strips, which is a measure of potassium permeability, is not affected by the chemical. At a dose of 10 μg/ml there is no anticurare action in vitro. All of the neuromuscular actions of serotonin can be prevented by administration of methysergide. It is postulated that the agent might act presynaptically to release acetylcholine by the interaction with serotonin D-receptors.

The effects of calcium and magnesium on the response of intestine smooth muscle to drugs.

1. The sensitivity of the longitudinal muscle of the guinea-pig ileum to muscarinic drugs producing contraction depends on optimum concentrations of calcium and magnesium. It can also be reduced by changes in sodium concentration and osmolarity.2. The rubidium efflux response to these same drugs is insensitive to any of these changes in the external medium.3. Raised calcium or magnesium concentration has the effect of largely annulling the differences in structure-activity relationships of the two responses as they exist in optimal media.4. The effects are explained in terms of a labile coupling process between a single receptor and the contractile process compared with a stable coupling process of the efflux process.