86Rb Efflux Research Articles

Effects of NaCl removal on osmolyte fluxes and regulatory volume decrease in cultured astrocytes.

Cultured cerebellar astrocytes exhibit regulatory volume decrease (RVD) after anisosmotic swelling by extrusion of potassium (K), chloride (Cl), and organic osmolytes. The volume decay curve after swelling in 50% hyposmotic solutions has a first-order rate constant k min(-1) of 0.18 and 70% cell volume recovery is attained after 15 min. Replacing NaCl in the hyposmotic medium by sugars and polyalcohols markedly accelerated RVD increasing k to 0.37-0.39 min(-1) and full recovery in 3-5 min. NaCl removal increased (80%) K efflux (measured as 86Rb), delayed the inactivation of Cl efflux (measured as 125I) and enhanced taurine and D-aspartate release by 25%. These effects appear due to NaCl removal rather than to specific actions of the organic molecules. Replacing Na by other cations or Cl by gluconate increased k to 0.22 min(-1) and 0.26 min(-1), respectively, and 86Rb efflux by 4-23% and 39, respectively. It is concluded that the electrochemical gradient after NaCl removal increasing the K driving force, delaying the equilibrium of Cl and increasing amino acid efflux, accounts for the observed RVD acceleration. Other consequences of NaCl removal possibly modulating the K efflux pathway include changes in the surface charge screening, decreased ion strength, a rise in [Ca]i and a decrease in intracellular Na.

Changes in resting membrane potential induced by active sensitization in the guinea-pig vas deferens

Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35°C, Er was a mean of -54.1 ± 0.3 mV (mean ± SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+-K+ pump sites.Key words: hyperreactivity, sensitization, Na+-K+ ATPase, guinea-pig, vas deferens, smooth muscle.

Kinetics of ATP-sensitive K+ channels in isolated rat hearts assessed by87Rb NMR spectroscopy

An experimental model was developed to evaluate the effects of activators and inhibitors of K(ATP) channels on unidirectional K+ fluxes in the whole heart. Isolated rat hearts perfused in the Langendorff mode were equilibrated with Pi-free Krebs-Henseleit buffer (KH buffer) containing 0.94-2.14 mM RbCl and 3.76 mM KCl (20-36% of K+ substituted by Rb+). Rb+ efflux was initiated by removing Rb+ from the perfusate and 87Rb spectra were acquired continuously with a 1-2 min time resolution. In hearts with normal energetics, the efflux of Rb+ fit a monoexponential function, and the rate constant did not depend on intracellular [Rb+]. Agents depressing excitability and heart rate (HR), such as 0.6 mM lidocaine (Lido), 10 microM carbachol (carb) and 20 mM MgSO4, inhibited Rb+ efflux such that the rate constant, k (10(3)/min), decreased from 50+/-1.2 in the beating heart to 26+/-1, 40+/-1.1 and 19+/-1.2, respectively. In contrast, high [K+] (21 mM) did not affect the k value (50+/-4.5), independently of the presence or absence of bumetanide (Bum, 30 microM) and glibenclamide (Glib, 5 microM). Dinitrophenol (DNP, 0.2 mM) added in the presence of high [K+] + Bum increased k three-fold, to 160+/-5. This effect was associated with a significant decrease in phosphocreatine (PCr, <10% of initial) and ATP ( 15%) levels, and a 7-fold increase in the Pi level, assessed by 31P-NMR spectroscopy. Glib completely reversed the effect of DNP. Pinacidil (Pin, 20-80 microM) did not affect the k value either in beating control hearts or in the presence of Carb or KCl + Bum. Moreover, under conditions of moderate metabolic stress induced by 0.05 mM DNP (PCr, 35%; ATP, 65%), where half-maximal activation of K(ATP) channels occurred, Pin did not further activate Rb+ efflux. We conclude that:(1) heart rate-independent Rb+ efflux accounts for 40-80% of the total Rb+ efflux in beating (300 bpm) rat hearts;(2) DNP-activated Rb+ efflux is a good model for testing inhibitors of KATP channels in whole hearts; and (3) Pin is not an effective K(ATP) channel opener in the rat heart model.

Changes in resting membrane potential induced by active sensitization in the guinea-pig vas deferens.

Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35 degrees C, Er was a mean of -54.1+/-0.3 mV (mean +/- SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+ -K+ pump sites.

Insulin secretion from isolated rat islets induced by the novel hypoglycemic agent A-4166, a derivative of D-phenylalanine.

A derivative of D-phenylalanine, A-4166, reportedly evokes a more rapid and short-lived hypoglycemic action in vivo than any of the currently available sulfonylureas. This novel oral hypoglycemic agent is structurally different from sulfonylureas. Therefore, studies were designed to elucidate the mechanisms by which A-4166 stimulates insulin secretion. Insulin release from incubated or perifused rat islets was dose-dependently stimulated by 10 to 200 mumol/l A-4166, in the presence of 2.8 mmol/l glucose. Both A-4166 and tolbutamide evoke a prompt rise in insulin secretion followed by a sustained gradually decreasing release from perfused islets in the presence of low glucose, although A-4166 appeared to be more sensitive than tolbutamide to subthreshold glucose concentration. Diazoxide abolished the initial release and blunted sustained release. Removing calcium from the perifusate abolished insulin release within 15 minutes. A-4166 inhibited [3H]-glibenclamide binding to HIT cell membranes and 86Rb efflux from ATP-depleted or diazoxide-treated cells. These results suggest that the insulin release induced by A-4166 is relevant to this agent occupying the tolbutamide binding sites. Therefore, one possible mechanism accounting for the more rapid and short-lived hypoglycemic action of A-4166 in vivo, as compared with tolbutamide, may involve the reported differences in the bioavailability of A-4166.

Insulinotropic action of alpha-D-glucose pentaacetate: functional aspects.

The functional determinants of the insulinotropic action of alpha-D-glucose pentaacetate were investigated in rat pancreatic islets. The ester mimicked the effect of nutrient secretagogues by recruiting individual B cells into an active secretory state, stimulating proinsulin biosynthesis, inhibiting 86Rb outflow, and augmenting 45Ca efflux from prelabeled islets. The secretory response to the ester was suppressed in the absence of Ca2+ and potentiated by theophylline or cytochalasin B. The generation of acetate from the ester apparently played a small role in its insulinotropic action. Thus acetate, methyl acetate, ethyl acetate, alpha-D-galactose pentaacetate, and beta-D-galactose pentaacetate all failed to stimulate insulin release. The secretory response to alpha-D-glucose pentaacetate was reproduced by beta-D-glucose pentaacetate and, to a lesser extent, by beta-L-glucose pentaacetate. It differed from that evoked by unesterified D-glucose by its resistance to 3-O-methyl-D-glucose, D-mannoheptulose, and 2-deoxy-D-glucose. It is concluded that the insulinotropic action of alpha-D-glucose pentaacetate, although linked to the generation of the hexose from its ester, entails a coupling mechanism that is not identical to that currently implied in the process of glucose-induced insulin release.

Verapamil, a phenylalkylamine Ca2+ channel blocker, inhibits ATP-sensitive K+ channels in insulin-secreting cells from rats.

Radioisotopic and electrophysiological techniques were used to assess the effects of verapamil, a phenylalkylamine Ca2+ channel blocker, on K+ permeability of insulin-secreting cells. Verapamil provoked a concentration-dependent inhibition of 86Rb (42K substitute) outflow from prelabelled and perifused rat pancreatic islets. This property appears to be inherent to the phenylalkylamine Ca2+ channel blockers since gallopamil, a methoxyderivative of verapamil, but not nifedipine, a 1,4-dihydropyridine Ca2+ channel blocker, inhibited 86Rb outflow. The experimental data further revealed that verapamil interacted with a Ca2+-independent, glucose- and glibenclamide-sensitive modality of 86Rb extrusion. Moreover, verapamil prevented the increase in 86Rb outflow brought about by BPDZ 44; a potent activator of the ATP-sensitive K+ channel. Single-channel current recordings by the patch clamp technique confirmed that verapamil elicited a dose-dependent inhibition of the ATP-dependent K+ channel. Lastly, under experimental conditions in which verapamil clearly inhibited the ATP-sensitive K+ channels, the drug did not affect 45Ca outflow, the cytosolic free Ca2+ concentration or insulin release. It is concluded that the Ca2+ entry blocker verapamil inhibits ATP-sensitive K+ channels in pancreatic beta cells. This effect was not associated with stimulation of insulin release.

Potassium transport in opossum kidney cells: Effects of Na-selective and K-selective ionizable cryptands, and of valinomycin, FCCP and nystatin

The effects of two ionizable cryptands, the Na-selective (221)C 10 and the K-selective (222)C 10, and of valinomycin, FCCP and nystatin on K + fluxes in opossum kidney (OK) cells have been quantified. The Na,K-ATPase (ouabain-sensitive 86Rb influx) was stimulated by nystatin (≥20%), and inhibited by the other ionophores (50–80%), by barium (K-channel blocker) (61%) and by amiloride (Na entry blocker) (34%). The V max of the Na,K-ATPase phosphatase activity was unmodified by the ionophores, indicating the absence of direct interaction with the enzyme. The ATPi content was unmodified by the inhibitors and nystatin, but was lowered by (221)C 10 (47%), (222)C 10 (75%), valinomycin (72%) and FCCP (88%). Amiloride was found to partially remove the inhibition caused by (222)C 10 (51%) and valinomycin (49%). Rb efflux was stimulated by nystatin (32%), unmodified by valinomycin, and was inhibited by (221)C 10 (19%), (222)C 10 (19%) and FCCP (10%). Barium (39%) and amiloride (32%) inhibited this efflux and, in their presence, the nystatin effect persisted, whereas that of the other ionophores vanished. At pH 6.4, the Rb efflux decreased by 14% of its value at pH 7.4, with no additional inhibition by cryptands. Cryptands are shown to inhibit the pH-sensitive K +-conductance, probably by inducing a K +–H + exchange at the plasma membrane, and by uncoupling oxidative phosphorylation by inducing the entry of K + and H + (and possibly Ca 2+) ions into the mitochondria.

Modulation of Ca2+ and K+ permeabilities by oxotremorine-m (Oxo-m) in rodent pancreatic B-cells.

The effects of the muscarinic agonist oxotremorine-m (Oxo-m) on 45Ca and 86Rb fluxes, insulin secretion, cytoplasmic Ca2+ concentration ([Ca2+]i) and membrane potential in pancreatic B-cells were studied. Oxo-m (40-200 microM) increased the [Ca2+]i by about 250 nM, irrespective of the glucose concentration present in the medium (2.8-22 mM). This effect was reduced by 50% upon the addition of EGTA. Oxo-m (50 microM) increased the 45Ca efflux from islets perifused in the absence or presence of [Ca2+]o, although under the former condition this efflux was transient. The difference between effluxes measured in the absence and presence of [Ca2+]o represents the sustained second component, which presumably reflects Ca2+ influx. In both the absence and presence of 11.2 mM glucose. Oxo-m (50 microM) transiently increased 86Rb efflux. In the presence of glucose, Oxo-m provoked a transient polarization of the B-cell membrane associated with an increase in the K+ permeability values. K+ permeability returned to basal values (no Oxo-m) after 1-2 min. These results indicate that the initial phase of Oxo-m-induced insulin secretion depends partially on intracellular Ca2+ release, and that the sustained enhancement of release depends on Ca2+ influx. The participation of a calcium release-activated current (ICRAC) is proposed to explain the sustained small changes in membrane potential.

Cationic and secretory effects of 6-O-acetyl-D-glucose in rat pancreatic islets.

Selected esters of D-glucose were recently proposed as tools to supply the sugar to cells affected by a defect in the carrier-mediated process of hexose transport across the plasma membrane. The present study extends this knowledge to 6-O-acetyl-D-glucose. At a 2.0 mM concentration, the ester was indeed found to stimulate insulin release from perifused rat pancreatic islets. This coincided with stimulation of calcium influx into the islet cells, as judged from comparison of the ester effects on 45Ca outflow from prelabelled islets perifused in either the absence or presence of extracellular Ca2+. The facilitated inflow of Ca2+ did not appear attributable to a decrease in K+ conductance, 6-O-acetyl-D-glucose augmenting 86Rb efflux from islets prelabelled with this radioactive cation. Both the latter phenomenon and the insulinotropic action of 6-O-acetyl-D-glucose failed to be antagonized by D-mannoheptulose. These findings indicate that the cationic events coupling the recognition of D-glucose to the stimulation of insulin release are not identical in islets exposed to the hexose or its ester.

Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S-21663: evidence for the existence of a novel imidazoline site in beta cells.

1. The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic beta cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas. 2. S-21663, at concentrations ranging from 10(-5) M to 10(-3) M was able to release insulin from MIN6 cells; its activity peaked at 10(-4) M, a drop in the stimulant factor being noted between 10(-4) and 10(-3) M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1. 3. In contrast to tGLP-1, S-21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil- and nifedipine-sensitive voltage-dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S-21663-induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP-dependent K+ (K-ATP) channels as determined by measurement of 86Rb efflux and use of a K-ATP channel opener. 4. Comparison of the effects of S-21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose-dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S-21663 acts through a novel site which displays a remarkably stable expression along the cell culture. 5. It is concluded that S-21663 is a very efficient, glucose-independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and 'efaroxan' binding sites. In vitro and in vivo features of S-21663 indicate that this compound, or new drugs derived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin-dependent) diabetes.

Evidence for an R(+)-[(dihydroindenyl)oxy]alkanoic acid-sensitive K+/Cl- co-transporter in human platelets and its interaction with the Na+/K+/2Cl- co-transporter.

1. The K+/Cl- co-transport system is activated by a number of interventions, such as cell swelling and stimulation with N-ethylmaleimide. It is specifically inhibited by R(+)-[(dihydroindenyl)oxy]alkanoic acid and requires the presence of K+ and Cl- on the same side of the cell membrane. This co-transporter has been studied extensively, mainly in erythrocytes of many species, in which it plays a key role in cell volume regulation. Here we present evidence that human platelets contain K+/Cl- co-transporters. 2. We have studied the efflux of 86Rb+ (a marker for K+) from 86Rb(+)-loaded human platelets, and have defined their response to stimulation by N-ethylmaleimide. 3. N-Ethylmaleimide (0.5 and 1 mmol/l) stimulated an increase in cumulative 86Rb+ efflux in a concentration-dependent manner. This efflux was inhibited by R(+)-[(dihydroindenyl)oxy]alkanoic acid (10 mumol/l) but was insensitive to bumetanide. It also required the presence of external Cl-. 4. These observations suggest that 86Rb+ efflux from the platelets stimulated by N-ethylmaleimide occurs via K+/Cl- co-transport. 5. When the K+/Cl- co-transporter was stimulated by N-ethylmaleimide we were unable to stimulate the Na+/K+/2Cl- co-transporter with a high external concentration of KCl or inhibit 86Rb+ efflux with bumetanide. Together with other evidence, this suggests that when the K+/Cl- co-transporter is stimulated with N-ethylmaleimide, the Na+/K+/2Cl- co-transporter is inhibited.

Pharmacological evidence of calcium-activated and voltage-gated potassium channels in human platelets.

1. Previous electrophysiological studies have suggested the presence of KCa and Kv channels in human platelets. However, the pharmacology of these channels has not been defined. 2. We have studied potassium channels in human platelets by measuring the efflux of 86Rb+ (a marker for K+) from 86Rb(+)-loaded cells, and have defined their responses to stimulation by the platelet agonist thrombin and the calcium ionophore ionomycin. 3. Thrombin (0.1-0.6 i.u./ml) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l), charybdotoxin (300 nmol/l) and alpha-dendrotoxin (100-200 nmol/l), blockers of SKCa channels, KCh channels and Kv channels respectively. Iberiotoxin (300 nmol/l), a specific inhibitor of BKCa channels, had no effect on the thrombin-stimulated 86Rb+ efflux. Although glibenclamide, an inhibitor of KATP channels, inhibited the thrombin-stimulated efflux, it did so only in a high concentration (20 mumol/l). 4. Ionomycin (1-5 mumol/l) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l) and charybdotoxin (300 nmol/l). However, iberiotoxin (300 nmol/l) had no effect on the ionomycin-stimulated 86Rb+ efflux. 5. These findings suggest that 86Rb+ efflux from platelets stimulated by thrombin and ionomycin occurs via two types of KCa channel: SKCa and KCh channels. Thrombin also stimulated efflux via Kv channels.

Isovolumetric regulation in a distal nephron cell line (A6).

The effects of gradually reducing the osmolality of the basolateral solution (pi b) were examined in a renal epithelial cell line (A6). pi b was linearly decreased with time from 260 to 140 mosmol/ kgH2O. Cell volume did not change when pi b was gradually decreased at dilution rates (D(r)) of 1-1.5 mosmol. kgH2O-1. min-1. Increasing D(r) to 3 or 6 mosmol. kgH2O-1. min-1 abolished this isovolumetric regulation (IVR). Replacing Cl- by NO3- or SCN- inhibited IVR markedly, whereas Br- substituted perfectly for Cl-. On the other hand, with all these anions, the regulatory volume decrease (RVD) was completely developed. Ba2+ (30 mM) markedly slowed down RVD but improved IVR. The discrepancies between RVD and IVR suggest that different mechanisms are used to control cell volume during gradual and shockwise hyposmotic perturbations. During gradual and shockwise reductions of pi b, cellular K+ content was reduced to the same extent; 86Rb efflux was only partially inhibited by Ba2+. The amount of intracellular K+ depletion could account for 70% of the cationic osmolyte loss, which suggests that K+ is the major cation excreted during both types of perturbations.

Cationic Events Stimulated by D-Glucose in Depolarized Islet Cells

Pancreatic islets prelabelled with either 86Rb or 45Ca were exposed to a rise in D-glucose concentration from 2.8 to 16.7 mM whilst perifused in the presence of 2 μM glibenclamide, 30 mM extracellular K + and both 30 mM K + and 250 μM diazoxide. In all three situations, the rise in glucose concentration provoked a dramatic increase in insulin output, despite unchanged or even increased efflux of 86Rb from the prelabelled islets. Also in all three situations, glucose sharply decreased effluent radioactivity from islets prelabelled with 45Ca but perifused in the absence of extracellular Ca 2+, whilst augmenting 45Ca efflux, to a variable extent, from islets perifused at normal extracellular Ca 2+ concentration (1.0 mM). It is proposed, therefore, that the insulinotropic action of D-glucose in depolarized islets, and presumably also under normal conditions, may involve the gating of voltage-insensitive Ca 2+ channels.

The Role of Potassium Channels in the Inhibitory Effects ofβ2-adrenoceptor Agonists on DNA Synthesis in Human Cultured Airway Smooth Muscle

Opening of calcium-dependent K+channels byβ2-adrenoceptor agonists has been shown to contribute to their smooth muscle relaxant activity. In this study, the influence of K+channel activity on human cultured airway smooth muscle proliferation and on its inhibition by salbutamol have been examined. Studies of86Rb+efflux from the airway smooth muscle cell cultures confirmed that both cromakalim, an activator of ATP-dependent K+channels and salbutamol increased K+channel activity in cultured airway smooth muscle. The efflux of86Rb+was significantly attenuated by non-specific K+channel blockade. Potassium channel blockers had only small and variable effects on [3H]-thymidine incorporation in unstimulated cells and those stimulated with fetal calf serum (FCS) and no effect on FCS-induced increases in cell number. Potassium channel openers also had no substantial inhibitory effects on DNA synthesis or proliferation in cells stimulated with other mitogens including thrombin. Blockade of K+channels had no consistent effects on the inhibition of DNA synthesis and cell proliferation caused by salbutamol (0.3 nm–10μm). The inhibition of DNA synthesis caused by 8 bromoadenosine 3′5′ cyclic monophosphate (1μm–1 mm) or 8 bromoguanosine 3′5′ cyclic monophosphate (1μm–1 mm) was also unaffected by K+channel blockade. These results indicate that changes in K+channel activity have no detectable influence on DNA synthesis and proliferation of human cultured airway smooth muscle cells or on the antiproliferative effects ofβ2-agonists.

The SAR of UK-78,282: A novel blocker of human T cell Kv1.3 potassium channels

UK-78,282 was identified in a human T cell 86Rb efflux high-throughput screen of our compound libraries. This compound was found to be a potent and selective blocker of human T cell voltage-gated K + channels and to inhibit T cell activation. The SAR around UK-78,282 and a general pharmacophore hypothesis are presented in this communication.

3-Morpholinosydnonimine as instigator of a glibenclamide-sensitive reduction in the insulin secretory rate

The nitric oxide (NO) donor SIN-1 (3-morpholinosydnonimine) induced a concentration-dependent inhibition of the secretory response to glucose. The negative insulinotropic action of SIN-1 was attenuated by the hypoglycemic sulfonylurea glibenclamide. Moreover, the NO donor enhanced 86Rb outflow from perfused islets and reduced the glucose-induced increase in 45Ca outflow. The present data provide further evidence that NO donors impair the secretory response to glucose, at least in part, by activating the ATP-sensitive K + channels.

A mechanism for regulatory volume decrease in cultured lens epithelial cells

PURPOSE. To identify mechanisms contributing to regulatory volume decrease in lens epithelial cells. METHODS. Cells of the lens epithelial cell line aTN4 were cultured in four-well culture dishes in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. After confluence cell water space was determined by measuring the equilibrium distribution of 3-O-methylglucose. Potassium influx and efflux in isotonic and hypotonic solutions were measured using 86 rubidium (86 Rb) as tracer. Total cell potassium and sodium content were determined with atomic absorption spectroscopy. Protein content per well was assayed with a modified Lowry assay and flux data and ion concentrations were normalized per mg of protein. RESULTS. Lens epithelial cells responded to hypotonic solutions with rapid swelling followed by regulatory volume decrease (RVD). During swelling and subsequent volume decrease the unidirectional Rb efflux was increased proportionaly to the osmotic challenge. Rubidium efflux was highly sensitive to changes in extracellular osmolarity and responded with a measurable activation to changes of 12.5 mOsm. No changes in 86 Rb influx were observed with small changes (<20%) in osmolarity and only relatively small changes occurred with larger changes in osmolarity. The resulting net loss of 86 Rb and potassium (K +) was demonstrated by measuring the change of intracellular [K + ] in hypotonic solutions using atomic absorption spectroscopy. The K + -channel blockers quinine-HCl and BaCl 2 and the Cl - -channel blockers diphenyl-2-carboxylate (DPC) and 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB) did not significantly affect the 86 Rb efflux induced by hypotonic solutions. However, [(dihydroindenyl)oxy]alkanoic acid (DIOA), reported to be a specific inhibitor of the K-Cl cotransporter, inhibited the activation of 86 Rb efflux. 86 Rb efflux could be activated in isosmotic solutions by the addition of 1 mM N-ethylmaleimide (NEM). This activation of Rb efflux could be prevented by the addition of 1 mM dithiothreitol and could be 90% blocked by DIOA. The activation of rubidium efflux by NEM led to a significant decrease of the intracellular water content. The volume regulatory changes in NEM and in hypotonic solutions could be inhibited in DIOA. CONCLUSIONS. The observations are consistent with the presence in lens epithelial cells of a K-Cl cotransporter serving as a mechanism for regulatory volume decrease.

Effects of High External Concentrations of K+ on 86Rb+ Efflux in Human Platelets: Evidence for Na+/K+/2Cl− Co-Transport

1. Na+/K+/2Cl- co-transport mediates a bidirectional symport of Na+, K+ and Cl-. The important properties of the co-transport system are its requirement for Na+, K+ and Cl- and its inhibition by loop diuretics such as bumetanide. This co-transporter has been described in a number of animal and human tissues. However, its presence in human platelets, although inferred, has not been demonstrated directly. 2. We have studied the efflux of 86Rb+ (a marker for K+) from Rb(+)-loaded platelets, and have defined their response to stimulation by high concentrations of external K+. 3. KCl (30-120 mmol/l) stimulated a concentration-dependent increase in 86Rb+ efflux from the platelets. This efflux was completely inhibited by bumetanide (10 mumol/l) but was insensitive to ouabain and R(+)-[(dihydroindenyl)oxy]alkanoic acid. It also required Cl- in the external medium, but did not depend on the presence of extracellular Na+. 4. These observations suggest that 86Rb+ efflux from platelets stimulated by external K+ occurs via Na+/K+/2Cl- co-transport acting in a K+/K+ (K+/Rb+) exchange mode. 5. Non-stimulated efflux of 86Rb+ from the platelets (i.e. in the presence of 5 mmol/l K+) had the characteristics of Na+/K+/2Cl- co-transport acting in normal mode.