Paradoxical inhibitory effect of cromakalim on 86Rb outflow from pancreatic islet cells

Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the "K+ channel openers." The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ channels.     

Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.     

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets

Summary— Na/Ca exchange was recently shown to regulate cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated ⁴⁵Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase ⁴⁵Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased ⁴⁵Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca²⁺ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.     

Does calcium mediate the increase in potassium permeability due to phenylephrine or angiotensin II in the liver?

Two agonists, phenylephrine and angiotensin II, which have been shown to alter K+ permeability in the liver were investigated as to the possible role of Ca++ in the K+ release response (measured as 86Rb efflux) in liver slices. Both phenylephrine and angiotensin II caused transient increases in 86Rb efflux from liver slices. For both agonists, the first in a series of responses was independent of extracellular Ca++, but Ca++ was required to obtain a subsequent response. This dependence on extracellular Ca++ for a second response was not receptor-specific suggesting that activation of either receptor elicited the release of the same cellular pool of Ca++. The cationophore, A-23187, only slightly increased 45Ca++ efflux and was without effect on 86Rb efflux. In contrast to the ionophore, phenylephrine stimulated a precipitous rise in 45Ca++ efflux. It is proposed that the liver may be similar to a number of other tissues in that Ca++ mediates changes in K+ permeability, but that the source is a bound Ca++ store, rather than the extracellular space.     

Similarities between the effects of pinacidil and diazoxide on ionic and secretory events in rat pancreatic islets

The present study aimed at comparing the effects of pinacidil, a putative K+ channel opener, and diazoxide on ionic and secretory events in rat pancreatic islets. Pinacidil and diazoxide provoked a dose-dependent increase in 86Rb outflow from pancreatic islets perifused in the presence of glucose. Both drugs inhibited the glucose- and tolbutamide-induced increase in 45Ca outflow and insulin release whereas failing to affect the ionic and secretory responses to K+ depolarization. Pinacidil and diazoxide, in contrast to quinine, failed to affect 86Rb outflow from pancreatic islets stimulated by the Ca++-ionophore A23187. Pinacidil as well as diazoxide abolished the glucose-induced increase in [Ca++]i but did not modify the rise in [Ca++]i provoked by KCl. Lastly, both drugs were shown to stimulate an ouabain-resistant modality of 86Rb inflow into the islet cells. The close similarities between the ionic and secretory events mediated by pinacidil and diazoxide suggest that pinacidil could interfere with the same target site as diazoxide; namely the beta-cell ATP-sensitive K+ channel.     

Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the "K+ channel openers." The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.     

Insulinotropic effect of new glibenclamide isosteres

The aim of the present study was to characterize the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano- N"- cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene), two newly synthesized isosteres of glibenclamide, on ionic and secretory events in rat pancreatic islet cells. Both compounds inhibited 86Rb (42K substitute) outflow from rat pancreatic islets perifused throughout at low (2.8 mM) D-glucose concentration. In excised inside-out membrane patches, BM 208 and BM 225 reduced the frequency of KATP+ channel openings. The inhibition of 86Rb outflow induced by BM 208 and BM 225 coincided with an increase in 45Ca outflow. The latter phenomenon was abolished in islets exposed to Ca2+-free media. Both isosteres of glibenclamide increased the [Ca2+]i in single pancreatic islet cells. This effect was counteracted by verapamil, a Ca2+ entry blocker. In islets exposed to 2.8 mM glucose and extracellular Ca2+, BM 208 and BM 225 stimulated insulin output. The secretory capacity of BM 225 was more marked than that of BM 208, but the time courses of the cationic and secretory responses exhibited obvious dissociations. These data suggest that the secretory capacity of BM 208 and BM 225 results, at least in part, from the inhibition of ATP-sensitive K+ channels with subsequent increase in Ca2+ inflow. The dissociation between cationic and secretory variables further suggests that the modifications in Ca2+ handling are not solely attributable to a primary inhibition of the ATP-sensitive K+ channels.     

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.     

Glucose-Stimulated 45Calcium Efflux from Isolated Rat Pancreatic Islets

Kinetics of (45)Ca efflux and insulin release were studied in collagenase-isolated rat islets during 2-h perifusions with calcium-depleted (0.05 mM) bicarbonate-phosphate buffer containing 2.2 mM glucose. Addition of glucose (16.7 mM) suppressed (45)Ca efflux by 30%. Removal of glucose caused an "off response" of insulin release. The perifusion of a normal concentration of Ca (2.3 mM) greatly stimulated (45)Ca efflux, indicating Ca <--> (45)Ca exchange. When Ca and glucose were superimposed, the effects on (45)Ca efflux and insulin release depended upon the order of presentation of the stimuli: when Ca was added to an ongoing 16.7-mM glucose perifusion, biphasic patterns of (45)Ca and insulin release were seen; when glucose was superimposed on a Ca perifusion, an inhibition of the Ca-stimulated (45)Ca efflux occurred, and a reduced but clearly biphasic insulin response was seen. The subsequent insulin off response after with-drawal of the glucose was also reduced.Mathematical "peeling" of (45)Ca efflux curves from unstimulated islets suggests that there are at least two, and probably three, different intracellular Ca compartments (not including the extracellular sucrose space). At the beginning of perifusion, these three compartments (I, II, III) contain 25, 56, and 19% of the intracellular (45)Ca, and their rates of efflux are 6.7, 1.2, and 0.1%/min, respectively. Glucose appears to suppress efflux from the largest compartment (II); Ca appears to exchange with (45)Ca from a more inert compartment (III). The relationship between insulin and (45)Ca release is not stoichiometric.     

Somatostatin- and Epinephrine-Induced Modifications of 45Ca++ Fluxes and Insulin Release in Rat Pancreatic Islets Maintained in Tissue Culture

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets.Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-adrenergic mechanism, since is was abolished in the presence of phentolamine.Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca(++) uptake.     

Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.     

Rapid Transient Efflux of Phosphate Ions from Pancreatic Islets as an Early Action of Insulin Secretagogues

Anionic fluxes during the membrane realignments of stimulated insulin release have not been characterized previously although cations have been implicated in stimulus-secretion coupling. We have shown that a limited packet pulse of phosphate release ("phosphate flush") begins at the same time that the first phase of insulin secretion may occur. To demonstrate this phenomenon, we have prelabeled islets, obtained from rat pancreas by collagenase digestions, by incubation with [(32)P]orthophosphate. When such prelabeled islets are perifused with Krebs-Ringer bicarbonate containing 0.5 mg/ml D-glucose, a basal rate of efflux of radioactivity is established; transfer to perifusates containing 3.0 mg/ml D-glucose elicits an increased (32)P efflux within 1-2 min to peak values which are 7- to 21-fold greater than basal. The total duration of this "phosphate flush" approximates 10 min and exceeds the duration of the first phase of stimulated insulin secretion. With lesser concentrations of glucose, the flush exhibits dose-response relationships, and with 3 mg/ml glucose, a second flush can be elicited by restoring basal conditions and stimulating anew with 3 mg/ml glucose. The phenomenon is highly specific and can be reduplicated by other secretagogues (L-leucine) or sugars (D-mannose) which are also known to elicit insulin release but not by sugars which fail to affect insulin secretion (D-galactose, D-fructose, i-inositol, L-glucose). The efflux of radioactivity consists entirely of [(32)P]orthophosphate. Phosphate flush persists in phosphate-free media, Ca(++)-free media, and when insulin release is obtunded by adding Ni(++) (2 mM) to the perifusates. Thus, efflux of [(32)P]orthophosphate can be dissociated from insulin extrusion, and from net influx of ionic phosphate or calcium. Membrane stabilization with D(2)O or 1.0 mM tetracaine reversibly inhibits phosphate flush. Although the mechanism by which this effect occurs has not yet been established, the phosphate flush appears to constitute one of the earliest and hitherto unknown indices of the excitatory state in pancreatic islets.     

The Roles of Intracellular and Extracellular Ca++ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response.The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.     

Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets.

The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process.     

Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3-pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance.

This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.     

Muscarine-stimulated neurotransmitter release from PC12 cells

The effect of muscarine on neurosecretion was studied in the rat pheochromocytoma cell line, PC12. When PC12 cells were exposed to muscarine the cells responded rapidly with elevation of cellular inositol trisphosphate levels, elevation of intracellular free Ca++ and release of stored transmitter. These three phenomena were totally inhibited by the muscarinic antagonist, atropine, but were unaffected by the nicotinic antagonist, d-tubocurarine. Muscarine did not stimulate the production of cyclic GMP in these cells. The muscarine-stimulated increases in inositol trisphosphate, intracellular free Ca++ and neurotransmitter release displayed similar time courses and concentration dependencies suggesting that the secretion observed may be associated with the formation of inositol trisphosphate and elevation of intracellular free Ca++. The increase in intracellular free Ca++ appeared to be due to a mobilization of Ca++ from intracellular stores inasmuch as the increase in intracellular free Ca++ was not inhibited by the voltage-dependent Ca++ channel antagonist, nifedipine, at concentrations demonstrated to block K+-induced Ca++ influx into the cells, and little or no uptake of 45Ca++ was noted when cells were stimulated with muscarine. Elevation of inositol trisphosphate, intercellular free Ca++ and stimulation of transmitter release were, however, inhibited by the absence of extracellular Ca++. The results suggest that muscarine-stimulated release of neurotransmitter may be associated with an inositol trisphosphate-induced mobilization of intracellular Ca++.     

Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+.

Pancreatic islets are known to respond to a raise of the glucose concentration with Ca2+ -induced 2-3-min pulses of insulin release. The reports of cyclic variations of circulating insulin in the fasting state made it important to explore whether insulin release is also pulsatile in the absence of stimulated entry of Ca2+. Individual pancreatic islets were isolated from a local colony of ob/ob mice and perifused under conditions allowing dual wavelength recordings of the cytoplasmic Ca2+ concentration ([Ca2+]i) with fura-2 and measurements of insulin with ELISA technique. At 3 mM of glucose, [Ca2+]i remained at a stable low level, but insulin was released in pulses with a frequency of 0.41+/-0.02 min-1, determined by Fourier transformation of original and autocorrelated data. Pulses of basal insulin release were also seen when glucose was omitted and 1 microM clonidine or 400 microM diazoxide was added to a glucose-free medium. The results indicate that pulsatile insulin release can be generated in the absence of stimulated entry of Ca2+. A tentative explanation for this phenomenon is inherent fluctuations in the ATP production of the beta cells.     

Possible involvement of ATP-sensitive K+ channels in the relaxant response of dog middle cerebral artery to cromakalim

To determine the functions of ATP-sensitive K+ (KATP) channels in cerebral arterial smooth muscle, the effects of cromakalim, an opener of these channels, on tension and 86Rb efflux were investigated in endothelium-removed strips of dog middle cerebral arteries (MCAs). Cromakalim relaxed the strips that were precontracted with 20.9 mM K+ with a small maximum response. The relaxant responses to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. In strips precontracted with 65.9 mM K+, cromakalim failed to relax the strips. The addition of cromakalim to a resting strip caused a dose-dependent relaxation. In the resting strips of MCAs preloaded with 86Rb, cromakalim did not increase the 86Rb efflux. With 42K as the tracer ion, cromakalim still had no effect on the efflux from the resting strips. On the other hand, cromakalim increased the 86Rb and 42K efflux from the strips of dog coronary arteries (CAs). In 20.9 mM K(+)-contracted strips of MCAs, cromakalim significantly decreased the 86Rb efflux. However, after the inactivation of Ca(++)-activated K+ channels by the addition of 1 x 10(-7) M nifedipine to the 20.9 mM K(+)-contracted strips of MCAs, cromakalim produced a small but significant increase in the 86Rb efflux. Similarly, when the resting strips of MCAs were placed in the Ca(++)-free 12 mM-Mg(+)+ solution, cromakalim increased the 86Rb efflux. In 65.9 mM K(+)-contracted strips, cromakalim increased the 86Rb efflux from both arteries. However, the extent of the increase in 86Rb efflux was significantly smaller in the MCA than in the CA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca++ utilization in the constriction of rat aorta to stimulation of protein kinase C by phorbol dibutyrate

To determine the role of activated protein kinase C in vascular smooth muscle contraction, phorbol dibutyrate was used to stimulate this enzyme in order to evaluate the source(s) of Ca++ (10(-8) to 3 X 10(-6) M) elicited a concentration-dependent sustained contraction which was slow in onset but progressive in developed tension. The maximal contractile response induced by phorbol dibutyrate was only partly dependent on influx of extracellular Ca++ as shown by similar reductions (40%) produced by Ca++-free buffer, LaCl3 (1 mM) or nifedipine (10(-6) M). These data suggest that phorbol dibutyrate is able to open Ca++ channels which are sensitive to nifedipine blockade. However, unlike norepinephrine or K+-depolarization, phorbol dibutyrate evoked a slow 45Ca++ influx which occurred only after extended contact time. Pretreatment with nifedipine again abolished this response. In contrast to norepinephrine, phorbol dibutyrate did not cause 45Ca++ efflux indicating that intracellular Ca++ was not mobilized. It is concluded that the residual 60% contraction to phorbol dibutyrate most likely occurs via a mechanism independent of the Ca-calmodulin pathway.