Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

Decreased insulin secretory response of pancreatic islets during culture in the presence of low glucose is associated with diminished 45Ca2+ net uptake, NADPH/NADP+ and GSH/GSSG ratios

In isolated rat pancreatic islets maintained at a physiologic glucose concentration (5.6 mM) the effect of glucose on parameters which are known to be involved in the insulin secretion coupling such as NADPH, reduced glutathione (GSH), 86Rb+ efflux, and 45Ca++ net uptake were investigated. The insulinotropic effect of 16.7 mM glucose was decreased with the period of culturing during the first 14 days being significant after 2 days though in control experiments both protein content and ATP levels per islet were not affected and insulin content was only slightly decreased. Both NADPH and GSH decreased with time of culture. 86Rb+ efflux which is decreased by enhancing the glucose concentration from 3 to 5.6 mM in freshly isolated islets was not affected by culturing whatsoever, even not after 14 days of culture when there was no longer any insulin responsiveness to glucose. The 45Ca++ net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2) that more likely it is the result of a diminished 45Ca++ net uptake as a consequence of the inability of islet cells to maintain proper NADPH and GSH levels, and (3) that potassium (86Rb+) efflux may not be related to changes of NADPH and GSH.     

Distinct mechanisms for two amplification systems of insulin release.

The mechanisms whereby activation of the cyclic AMP-dependent protein kinase A or the Ca2+-phospholipid-dependent protein kinase C amplifies insulin release were studied with mouse islets. Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) were used to stimulate adenylate cyclase and protein kinase C respectively. The sulphonylurea tolbutamide was used to initiate insulin release in the presence of 3 mM-glucose. Tolbutamide alone inhibited 86Rb+ efflux, depolarized beta-cell membrane, triggered electrical activity, accelerated 45Ca2+ influx and efflux and stimulated insulin release. Forskolin alone only slightly inhibited 86Rb+ efflux, but markedly increased the effects of tolbutamide on electrical activity, 45Ca2+ influx and efflux, and insulin release. In the absence of Ca2+, only the inhibition of 86Rb+ efflux persisted. TPA (100 nM) alone slightly accelerated 45Ca2+ efflux and insulin release without affecting 45Ca2+ influx or beta-cell membrane potential. It increased the effects of tolbutamide on 45Ca2+ efflux and insulin release without changing 86Rb+ efflux, 45Ca2+ influx or electrical activity. Omission of extracellular Ca2+ suppressed all effects due to the combination of TPA and tolbutamide, but not those of TPA alone. Though ineffective alone, 10 nM-TPA amplified the releasing action of tolbutamide without affecting its ionic and electrical effects. In conclusion, the two amplification systems of insulin release involve at least partially distinct mechanisms. The cyclic AMP but not the protein kinase C system initiating signal (Ca2+ influx) triggered by the primary secretagogue.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Comparison of the inhibition of insulin release by activation of adenosine and alpha 2-adrenergic receptors in rat beta-cells.

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Mechanisms of the stimulation of insulin release by arginine-vasopressin in normal mouse islets

The mechanisms by which arginine-vasopressin (AVP) affects pancreatic B-cell function were studied in normal mouse islets. AVP produced a dose-dependent (0.1-1000 nM; EC50 approximately 1-2 nM) amplification of glucose-induced insulin release. This amplification was of slow onset and reversibility. AVP was ineffective when the concentration of glucose was less than 7 mM, but was still very effective in 30 mM glucose. The increase in insulin release produced by AVP was accompanied by small accelerations of 86Rb and 45Ca efflux from islet cells. Omission of extracellular Ca2+ accentuated the effect of AVP on 86Rb efflux, attenuated that on 45Ca efflux, and abolished that on release. Under no condition did AVP inhibit 86Rb efflux. AVP did not significantly affect cAMP levels, but increased inositol phosphate levels in islet cells, even in the absence of extracellular Ca2+. AVP did not affect the membrane potential in unstimulated B-cells and augmented glucose-induced electrical activity only slightly. This was not due to a direct action on ATP-sensitive K+ channels as revealed by patch-clamp recordings (whole cell and outside-out patches). In conclusion, AVP is not an initiator of insulin release, but it potently amplifies glucose-induced insulin release in normal mouse B-cells. This effect involves a stimulation of phosphoinositide metabolism, and presumably an activation of protein kinase C, rather than a change in cAMP levels or a direct control of the membrane potential.     

Furosemide and Ca2+ affect 86Rb+ efflux from pancreatic beta-cells by different mechanisms

The interaction between furosemide, calcium and D-glucose on the 86Rb+ efflux from beta-cell-rich mouse pancreatic islets was investigated in a perifusion system with high temporal resolution. Raising the glucose concentration from 4 to 20 mM induced an initial decrease in 86Rb+ efflux, which was followed by a steep increase and then a secondary decrease. Removal of extracellular calcium increased the 86Rb+ efflux at 4 mM D-glucose but reduced it at 20 mM. The initial biphasic changes in 86Rb+ efflux induced by 20 mM D-glucose were inhibited by calcium deficiency. Furosemide (100 microM) reduced the 86Rb+ efflux rate both at 4 and 20 mM D-glucose and the magnitudes appeared to be similar at either glucose concentration. Furosemide (100 microM) reduced the glucose-induced (10 mM) 45Ca+ uptake but did not affect the basal (3 mM D-glucose) 45Ca+ uptake. However, the ability of furosemide (100 microM) to reduce the 86Rb+ efflux at a high glucose concentration (20 mM) was independent of extracellular calcium. The inhibitory effects of furosemide and calcium deficiency on the 86Rb+ efflux rate appeared to be additive. It is concluded that the effect of furosemide on 86Rb+ efflux is not secondary to reduced calcium uptake and that the effects of furosemide and calcium deficiency are mediated by different mechanisms. The effect of furosemide is compatible with inhibition of loop diuretic-sensitive co-transport of Na+, K+ and Cl- and the effect of calcium deficiency with reduced activity of calcium-regulated potassium channels.     

The coupling of metabolic to secretory events in pancreatic islets. The possible role of glutathione reductase

The participation of glutathione reductase in the process of nutrient-stimulated insulin release was investigated in rat pancreatic islets exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU caused a time-and dose-related, irreversible inhibition of glutathione reductase activity. This coincided with a fall in both GSH/GSSG ratio and the thiol content of the islets. Pretreatment of the islets with BCNU inhibited the oxidation of glucose and its stimulant action upon both 45Ca net uptake and insulin release. Although BCNU (up to 0.5 mM) failed to affect the oxidation of L-leucine and L-glutamine, it also caused a dose-related inhibition of insulin release evoked by the combination of these two amino acids. The latter inhibition was apparently not fully accounted for by the modest to negligible effects of BCNU upon 45Ca uptake, 45Ca efflux, 86Rb efflux and cyclic AMP production. Since BCNU failed to inhibit insulin release evoked by the association of Ba2+ and theophylline, these results support the view that glutathione reductase participates in the coupling of metabolic to secretory events in the process of nutrient-stimulated insulin release. However, the precise modality of such a participation, for example the control of intracellular Ca2+ distribution, remains to be elucidated.     

Effects of maitotoxin on ionic and secretory events in rat pancreatic islets

Maitotoxin (MTX) provoked a dose-dependent increase in both 45Ca efflux and insulin release from rat pancreatic islets perifused in the presence or absence of glucose, provided that Ca2+ was present in the perifusate. The stimulatory effect of MTX on 45Ca outflow was enhanced by CGP 28392. The toxin did not reduce 86Rb outflow and 86Rb inflow. It is suggested that the secretory response to MTX is mediated by direct activation of voltage-dependent Ca2+ channels.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Relationships between hormone-induced calcium release and 86rubidium uptake stimulation in starfish oocytes

86Rubidium+ uptake, but not 86Rubidium efflux, is strongly stimulated after addition of the meiosis inducing hormone 1-methyladenine (1-MeAde) to prophase blocked oocytes of the starfish Marthasterias glacialis. This stimulation is a transient process which does not require the continuous presence of 1-MeAde and is elicited within 1 minute of contact. 1-MeAde and its biologically active structural analogs fully stimulate Rb+ uptake at concentrations which are about two orders of magnitude lower than those required to trigger meiosis reinitiation but which already release underthreshold levels of Ca2+ from the inner part of the plasma membrane. External Ca2+ concentrations effective in triggering meiosis reinitiation also stimulate Rb+ influx, while drugs like D600, theophyllin and caffein which suppress the hormone induced Ca2+ release, simultaneously preclude the stimulation of Rb+ uptake. Dithiothreitol (DTT) which mimicks 1-MeAde action in releasing Ca2+ and inducing meiosis acts both on the efflux and on active and passive Rb+ influxes. Ouabain, the classical inhibitor of the Na+, K+ pump does not preclude meiosis reinitiation under the influence of 1-MeAde, its agonists of mimetics. It suppresses the active component of Rb+ uptake both in control or stimulate oocytes. When applied only in preincubation before starting the hormone treatment, it cannot however inhibit the stimulation of Rb+ uptake, while basal pump inhibition is preserved. These results demonstrate that stimulation of the active Rb+ or K+ transport is not indispensable to meiosis reinitiation. They suggest moreover that the hormone induced Ca2+ release from the plasma membrane may be responsible for unmasking new ouabain sensitive transport sites.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

K+ transport in 'tight' epithelial monolayers of MDCK cells. Evidence for a calcium-activated K+ channel

Measurements of 86Rb efflux across the apical and basal-lateral aspects of intact monolayers of 'high-resistance' MDCK cells mounted in Ussing chambers have been made. A transient increase in 86Rb efflux across both epithelial borders upon stimulation with adrenalineeeeeee or ionophore A23187 is observed. The increased 86Rb across the basal cell aspects is of greatest quantitative importance. Measurements of total cellular K+ contents by flame photometry of tissue extracts indicate a net loss of K+ following adrenalin addition. The effects of adrenalin and ionophore A23187 upon 86Rb efflux are abolished in 'Ca2+ -free' media. The properties of the Ca2+ -dependent increase in 86Rb efflux show similarities to Ca2+ -activated K+ conductances in other tissues, notably human red cells, including inhibition by quinine (1 mM), tetraethylammonium (25 mM) and insensitivity to bee venom toxin (apamin) (25 nM). Adrenalin is only effective when applied to the basal bathing solution suggesting that the receptors mediating adrenalin action are located upon the basal-lateral membranes. Half maximal stimulation of 86Rb efflux by adrenalin is observed at 9.1 X 10(-7) M. The action of various adrenergic receptor agonists and antagonists are consistent with adrenalin action being mediated by an alpha-adrenergic receptor.