Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells

Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of 86Rb and 45Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated 86Rb and 45Ca efflux, and increased insulin release. This latter effect, but not the acceleration of 45Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated 86Rb and 45Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of 45Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents.     

Mechanism of the stimulation of insulin release in vitro by HB 699, a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide

Summary HB 699 is a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide. The mechanisms whereby it affects B-cell function have been studied in vitro with mouse islets. In the presence of 3 mmol/l glucose, HB 699 decreased ⁸⁶Rb⁺ efflux and accelerated ⁴⁵Ca²⁺ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in ⁴⁵Ca²⁺ efflux and insulin release, but not the inhibition of ⁸⁶Rb⁺ efflux, were abolished in the absence of Ca²⁺. In the presence of 10 mmol/l glucose, HB 699 increased ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ efflux from the islets, caused a persistent depolarization of the B-cell membrane with continuous electrical activity, and markedly potentiated insulin release. All these changes were suppressed by omission of extracellular Ca²⁺. In the presence of 15 mmol/l glucose, diazoxide increased ⁸⁶Rb⁺ efflux, hyperpolarized the B-cell membrane, suppressed electrical activity and inhibited insulin release. HB 699 reversed these effects of diazoxide. It is suggested that HB 699 decreases K⁺ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channnels and Ca²⁺ influx, and eventually increases insulin release. A sulphonylurea group is thus not a prerequisite to trigger the sequence of events that is also thought to underlie the releasing effects of tolbutamide and glibenclamide.     

Is there a role for osmotic events in the exocytotic release of insulin?

The possible role of an osmotic lysis of insulin granules during exocytosis has been studied in perifused mouse pancreatic islets. Raising the osmolarity of the extracellular medium by addition of 400 mM sucrose reversibly inhibited glucose-stimulated insulin release. This inhibition was accompanied by a decrease in the rates of 86Rb+ or 45Ca2+ efflux from the islets. Increasing the osmolarity and restoring a normotonic medium in the presence of a nonstimulatory concentration of glucose accelerated 86Rb+ and 45Ca2+ efflux and augmented basal insulin release in both the presence and absence of Ca2+. Hyperosmolarity did not prevent a rise in glucose concentration from decreasing 86Rb+ efflux from islet cells or from inhibiting 45Ca2+ efflux in Ca2+-free medium. However, the stimulation of 45Ca2+ efflux otherwise produced by glucose in the presence of Ca2+ was abolished, and the stimulation of insulin release was almost suppressed. Hyperosmolarity also strongly impaired the release of insulin during stimulation by eight experimental conditions known to act through at least partially different mechanisms. The changes in 45Ca2+ efflux brought about by these different agents were also altered by hyperosmolarity, whether they resulted from direct mobilization of intracellular Ca2+ or were secondary to increased Ca2+ influx. The blockade of insulin release by hyperosmolarity, whatever the mode of action of the stimulus, is compatible with the participation of osmotic events in exocytosis. However, the marked alterations in Ca2+ handling that occur concomitantly and might account for the inhibition of release make it impossible to demonstrate their exact role in intact islet cells.     

Effect of low temperatures on glucose-induced insulin secretion and ionic fluxes in rat pancreatic islets

The direct effect of cold on the inhibition of B cell secretion is well known in hibernating and experimentally hypothermic mammals. This temperature dependency may result from the inhibition of ion transport across the membranes. In order to verify this hypothesis, ionic effluxes and insulin secretion from rat islets loaded with 86Rb+ and 45Ca+ were measured during perifusion. At 37 degrees C, the rise in glucose concentration from zero to 16.7 mmol/l provoked a rapid decrease in 86Rb+ efflux, an early fall and subsequent rise in 45Ca2+ efflux and a typical biphasic pattern of insulin secretion. At 27 degrees C, glucose induced only a very slight increase in insulin secretion, while the fluxes of radioactive ions were not significantly modified in amplitude but were clearly delayed. At 17 degrees C, no insulin response to glucose was observed and the decrease in K+ conductance indicated by 86Rb+ flux decrease was less temperature-dependent than the movement of Ca2+. After supplementary stimulation with a high extracellular concentration of Ca2+, insulin secretion was enhanced at 27 degrees C and reached levels induced by glucose alone at 37 degrees C. An increase in hormone secretion occurred even at 17 degrees C, but only during a first phase of secretion. Regular increases in temperature potentiated insulin secretion and provoked changes in ionic fluxes which suggest that B cell depolarization (86Rb+ flux decrease) induced by glucose can occur at 15 degrees C but cannot induce the opening of voltage-dependent Ca2+ channels (increase in 45Ca2+ efflux) until temperatures higher than 27 degrees C are reached.(ABSTRACT TRUNCATED AT 250 WORDS)     

Are ionic fluxes of pancreatic beta cells a target for gastric inhibitory polypeptide?

Gastric inhibitory polypeptide (GIP), an incretin candidate, is suggested to amplify the glucose-induced insulin secretion. To evaluate its mode of action we examined whether GIP affects 86Rb+ efflux, 45Ca2+ uptake or efflux, and intracellularly recorded electrical activity of mouse pancreatic islets. GIP (5 nM) neither inhibited 86Rb+ efflux at 3 mM glucose nor modulated 86Rb+ efflux that was inhibited by 5.6 mM glucose or stimulated by the calcium ionophore A23187. 45Ca2+ uptake was increased by GIP in the presence of 16.7 mM which was not observed at 3 or 11 mM glucose. GIP elevated 45Ca2+ efflux from islets, but did not modify 45Ca2+ efflux when a virtually Ca2+ free medium was used. Electrical activity of beta cells induced by 16.7 mM glucose was significantly increased by 5 nM GIP. It is concluded that the amplification of insulin release by GIP is based on the effect of GIP on Ca2+ uptake.     

The ionic, electrical, and secretory effects of protein kinase C activation in mouse pancreatic B-cells: studies with a phorbol ester

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to study the effects of protein kinase C activation on stimulus-secretion coupling in mouse pancreatic B-cells. At a nonstimulatory concentration of glucose (3 mM), 100 nM TPA, but not 10 nM TPA, slightly and slowly increased insulin release and 45Ca2+ efflux and decreased 86Rb+ efflux, but did not affect the membrane potential of B-cells. At a threshold concentration of glucose (7 mM), 100 nM TPA markedly increased insulin release without triggering electrical activity in B-cells. At a stimulatory concentration of glucose (10 mM), TPA caused a dose-dependent irreversible increase in insulin release, 45Ca2+ efflux, and 86Rb+ efflux and slightly augmented islet cAMP levels. Omission of extracellular Ca2+ abolished the effects of 10 nM TPA and partially inhibited those of 100 nM TPA on insulin release and 45Ca2+ efflux. In contrast, their effect on 86Rb+ efflux was paradoxically augmented. Glucose-induced electrical activity in B-cells was only marginally affected by TPA; the duration of the slow waves with spikes was not modified, but a small shortening of the polarized intervals raised their frequency and slightly increased the overall activity. This increase was significant only with 10 nM TPA, whereas only 100 nM TPA brought about a minute increase in 45Ca2+ influx. These results thus show that TPA induces insulin release or potentiates glucose-induced insulin release without mimicking or amplifying the initial ionic and electrical signals triggered by glucose. They suggest that protein kinase C activation affects stimulus-secretion coupling by modulating intracellular and/or nonelectrogenic membrane events.     

Regulation of calcium fluxes in pancreatic islets: the role of membrane depolarization

The effect of K+-induced depolarization on calcium fluxes and insulin release from isolated islets were investigated in order to elucidate the mechanism by which glucose initially reduces and later increases 45Ca efflux from prelabeled and perifused rat pancreatic islets. Raising the extracellular K+ concentration from 5.0 to 20.0 mM produced a 2- to 3-fold increase in 45Ca net uptake and efflux from isolated islets. The latter effect was dependent on the presence of extracellular Ca2+, suggesting that it resulted from the entry of calcium into the islet cells. In the presence of 20 mM K+, 16.7 mM glucose failed to stimulate 45Ca efflux, while 20 mM K+ further enhanced 45Ca efflux from islets perifused in the presence of the high concentration of glucose. These findings suggest that the effect of glucose to stimulate 45Ca efflux from perifused islets depends mainly on the glucose-induced depolarization of the cell membrane. In the absence of extracellular calcium, 20 mM K+ failed to mimick the effect of glucose to reduce 45Ca efflux. Glucose (16.7 mM) decreased 45Ca efflux from islets perifused in the presence of 20 mM K+ and antagonized the effect of 20 mM K+ to stimulate 45Ca efflux from perifused islets. It is concluded that K+-induced plasma membrane depolarization reproduces the effect of glucose to stimulate but not to inhibit 45Ca efflux from perifused islets.     

Effect of extracellular phosphate on Ca2+ and K+ fluxes in pancreatic islets

The idea that a lowering in cytosolic Ca2+ concentration may cause a decrease in K+ conductance in the pancreatic B-cell was tested by investigating the effect of a high extracellular phosphate concentration on 45Ca and 86Rb efflux from prelabelled rat pancreatic islets. Whether in the absence or presence of glucose, 20 mM phosphate tended to decrease 45Ca efflux. This effect was not suppressed in the absence of extracellular Ca2+, at least in glucose-deprived islets, suggesting that it may reflect a fall in cytosolic Ca2+ concentration. The administration of phosphate failed, however, to decrease 86Rb efflux from the islets. In the presence of extracellular Ca2+, 20 mM phosphate also failed to stimulate insulin release from islets perifused at low glucose concentration and inhibited insulin release stimulated by a high glucose concentration. These data indicate that the sequestration of Ca2+ in intracellular organelles and concomitant decrease in cytosolic Ca2+ concentration, as presumably provoked by a rise in extracellular phosphate concentration, is not sufficient to simulate the effect of glucose on K+ conductance.     

Stimulus-secretion coupling of arginine-induced insulin release. Uptake of metabolized and nonmetabolized cationic amino acids by pancreatic islets

In order to assess the possible role of L-arginine accumulation in islet cells as a determinant of its insulinotropic action, the uptake of L-arginine and other cationic amino acids (L-ornithine, L-homoarginine, D,L-alpha-methylornithine, D,L-alpha-difluoromethylornithine) by rat pancreatic islets was compared to the ionic and secretory responses of the islets to the same amino acids. A tight correlation was found between the net uptake of these amino acids and their capacity to stimulate 86Rb efflux, 45Ca uptake and efflux, and insulin release. In the latter respect, there was little difference between metabolized and nonmetabolized amino acids. Thus, although L-homoarginine and 4-amino-1-guanylpiperidine-4-carboxylic acid failed to act as a substrate for either arginase or amino acid aminotransferase in islet homogenates, they both stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin secretion in intact islets. These findings are compatible with the view that the accumulation of these positively charged amino acids in islet cells represents an essential determinant of their secretory action. Hence, the release of insulin evoked by these amino acids could be due to depolarization of the plasma membrane with subsequent gating of voltage-sensitive Ca2+ channels and/or to some other biophysical effect, as suggested by the persistence of a sizeable secretory response to L-arginine or L-ornithine in islets perifused at a high concentrations of extracellular K+ (50 mM).     

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.     

Inhibition by corticosterone of calcium inflow and insulin release in rat pancreatic islets

Corticosterone (0.6 mumol/l) inhibited both 45Ca outflow and insulin release evoked by glucose, the combination of leucine and glutamine, 2-ketoisocaproate, gliclazide or the association of gliclazide and a tumour-promoting phorbol ester in rat pancreatic islets perifused at normal extracellular Ca2+ concentration (1.0 mmol/l). In all cases, the inhibitory action of corticosterone reached statistical significance within 10-22 min of exposure to this steroid and failed to be rapidly reversible. Corticosterone failed to affect basal 45Ca outflow and insulin release. The steroid also failed to affect the inhibitory action of glucose upon 45Ca outflow, as judged from either the glucose-induced early fall in effluent radioactivity from islets maintained at normal extracellular Ca2+ concentration or the steady-state values for 45Ca outflow from glucose-stimulated but Ca2+-deprived islets. Corticosterone caused a modest increase in 86Rb outflow from islets perifused in the presence of glucose (16.7 mmol/l). It is concluded that corticosterone impairs Ca2+ inflow into the islet cells and, by doing so, causes a progressive inhibition of insulin release. The pancreatic B cell might thus serve as a further model for the study of the rapid biological response to steroids, as presumably mediated by alteration in the biophysical properties of the plasma membrane.     

Gastrin releasing peptide augments glucose mediated 45Ca2+ uptake, electrical activity, and insulin secretion of mouse pancreatic islets

Gastrin releasing peptide (GRP) has recently been shown to increase glucose-induced insulin secretion in vivo. Being present in pancreatic tissue, the 27-amino acid peptide could play a role in the control of the glucose-induced insulin secretion of islets of Langerhans. In the presence of a stimulatory glucose concentration, GRP augmented insulin secretion of isolated islets in batch incubations. The peptide did not affect 56Rb+ efflux in the presence of 3 or 5.6 mM glucose but reduced the increase of 86Rb+ efflux evoked by the calcium ionophore A23187. 45Ca2+ uptake and intracellular recorded electrical activity induced by glucose were amplified by GRP. It is suggested that GRP plays a role in the regulation of glucose-induced insulin secretion by increasing the uptake of Ca2+ directly or by inhibition of the Ca(2+)-dependent K+ channel activity and reduced repolarization of the cell.     

Calcium and pancreatic beta-cell function. 12. Modification of 45Ca fluxes by excess of K+

Glucose stimulation of insulin release is supposed to result from depolarization of the pancreatic beta-cells with subsequent influx of Ca2+. Isolated islets from non-inbred ob/ob-mice were employed for elucidating whether the glucose effects on the beta-cell handling of Ca2+ could be simulated by the depolarization evoked by excess of K+. Addition of 25 mM K+ was as effective as 20 mM glucose in stimulating the intracellular uptake of 45Ca. In both instances the additional amounts of incorporated 45Ca appeared in the mitochondria and the secretory granules. When analysing the washout pattern for 45Ca it was evident that the effects of raising K+ differed from those evoked by glucose. Whereas glucose inhibited 45Ca efflux during perifusion with Ca2+-deficient medium the addition of K+ resulted in a slight stimulation. Furthermore, the 45Ca incorporated in response to K+ was more readily mobilised.     

Mobilization of Ca2+ from intracellular stores of pancreatic beta-cells by the calcium store blocker TMB-8

TMB-8 has been used experimentally in many cell types, including endocrine cells, because of its ability to block the efflux of Ca2+ from intracellular stores without affecting influx. Unexpectedly, TMB-8 potentiates stimulated insulin release from pancreatic islets, a process believed to be dependent on the level of cytosolic Ca2+. In the present study, while having no effect on basal insulin release (in the presence of 2.8 mM glucose), TMB-8 (10, 30, and 100 microM) caused a concentration-dependent increase in 45Ca2+ efflux from 45Ca2+-preloaded islets. TMB-8 (100 microM) stimulated 45Ca2+ efflux even in the absence of extracellular Ca2+. In the presence of 5.6 mM glucose, TMB-8 (30 and 100 microM) potentiated insulin release and again increased 45Ca2+ efflux in a concentration-dependent manner. Similarly, insulin release stimulated by isobutylmethylxanthine (IBMX) was potentiated significantly, and IBMX-stimulated 45Ca2+ efflux was increased by the simultaneous introduction of 30 microM TMB-8. Thus, in pancreatic islets, TMB-8 appears to mobilize Ca2+ from intracellular stores, rather than inhibit the efflux as has been commonly accepted. In further studies, using insulin-secreting beta-cells of the RINm5F cell line, TMB-8 was shown to increase the cytosolic Ca2+ concentration in the presence and absence of extracellular Ca2+. This confirmed that mobilization of intracellular Ca2+ was occurring in the pancreatic beta-cell in response to TMB-8. Furthermore, a rise in cytosolic Ca2+ of not more than 10 nM (as induced with KCl) was found to mimick the effect of TMB-8 in conjunction with IBMX. No additional effect of TMB-8 to alter Ca2+ handling at the plasma membrane was found when 45Ca2+ uptake experiments were performed. Therefore, the paradoxical mobilization of beta-cell Ca2+ by TMB-8 appears to be a sufficient explanation for its potentiating effect on the rate of insulin secretion.     

The permissive effect of glucose,tolbutamide and high K+ on arginine stimulation of insulin release in isolated mouse islets

Summary Mouse islets were used to study how glucose modulates arginine stimulation of insulin release. At 3 mmol/l glucose, arginine (20 mmol/l) decreased the resting membrane potential of B cells by about 10 mV, but did not evoke electrical activity. This depolarisation was accompanied by a slight but rapid acceleration of ⁸⁶Rb⁺ efflux and ⁴⁵Ca²⁺ influx. However, ⁴⁵Ca²⁺ efflux and insulin release increased only weakly and belatedly. When the membrane was depolarised by threshold (7 mmol/l) or stimulatory (10–15 mmol/l) concentrations of glucose, arginine rapidly induced or augmented electrical activity, markedly accelerated ⁸⁶Rb⁺ efflux, ⁴⁵Ca²⁺ influx and efflux, and triggered a strong and fast increase in insulin release. When glucose-induced depolarisation of the B-cell membrane was prevented by diazoxide, arginine lost all effects but those produced at low glucose. However, the delayed increase in release still exhibited some glucose-dependency. In contrast, depolarisation by tolbut amide, at low glucose, largely mimicked the permissive effect of high glucose. Depolarisation by high K⁺ also amplified arginine stimulation of insulin release, but did not accelerate it as did glucose or tolbutamide. Omission of extracellular Ca²⁺ abolished the releasing effect of arginine under all conditions. The results thus show that the permissive action of glucose mainly results from its ability to depolarise the B-cell membrane. It enables the small depolarisation by arginine itself to activate Ca channels more rapidly and efficiently. Changes in the metabolic state of B cells may also contribute to this permissive action by increasing the efficacy of the initiating signal triggered by arginine.     

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide

The activity of the beta-cell Na+/K+ pump was studied by using ouabain-sensitive (1mM ouabain) 86Rb+ influx in beta-cell-rich islets of Ume?-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0 mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx during the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect showed its apparent maximum at 0.5 microM. Pre-treatment (60 min) of islets with 1 microM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1 microM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.     

Cationic determinants of D-fructose insulinotropic action

In the absence of any other exogenous nutrient, D-fructose stimulates insulin release from rat pancreatic islets provided that it is tested at high concentrations in excess of a threshold value close to 80 mM, an optimal secretory response being recorded at 240 mM. In the present study, the cationic determinants of the insulinotropic action of D-fructose, used at the latter high concentration, were explored in perifused rat islets that had been prelabelled with either ⁸⁶Rb or ⁴⁵Ca. The changes in ⁸⁶Rb outflow and ⁴⁵Ca efflux evoked by 240 mM D-fructose were comparable to those caused by 11.1 mM D-glucose in that both hexoses inhibited ⁸⁶Rb and ⁴⁵Ca outflow and, at normal Ca²⁺ concentration, caused a secondary rise in ⁴⁵Ca efflux. These cationic changes coincided wit stimulation of insulin release. The major differences between the two series of experiments consisted, in the islets exposed to D-fructose, in the occurrence of an early and transient increase in ⁴⁵Ca efflux at normal extracellular Ca²⁺ concentration, a secondary reascension in ⁸⁶Rb outflow and a dramatic off-response in both ⁸⁶Rb and ⁴⁵Ca outflow as well as insulin release. These phenomena were also observed in islets exposed to 240 mM 3-O-methyl-D-glucose, suggesting that they may be linked to the massive influx (or efflux) of monosaccharides, as possibly accompanied by Na⁺ inward co-transport, mobilization of Ca²⁺ from intracellular stores and activation of voltage- and/or Ca²⁺-sensitive K⁺ channels. This interpretation was supported by the finding that, at high concentrations (80.0 mM) of D-glucose or D-mannose, the aldohexoses, also provoked a reascension in ⁸⁶Rb outflow and off-response in insulin release. The cationic determinants of the insulinotropic action of D-fructose, in high concentration (240 mM), thus appear similar, if not identical, to those currently incriminated in the stimulation of insulin release by D-glucose. Received: 31 May 1999 / Accepted in revised form: 22 December 1999     

Stimulus-secretion coupling of glucose-induced insulin release. Timing of early metabolic,ionic, and secretory events

The timing of the early metabolic, ionic, and secretory responses to glucose in rat pancreatic islets was monitored by measuring, at 12 sec intervals, the concentrations of glucose, lactic, and pyruvic acids, ³²P, ⁸⁶Rb, ⁴⁵Ca, and insulin in the effluent of perifused prelabeled islets. The increase in glucose concentration from zero to 16.7 mM was complete within 133 sec. The output of organic acids increased after 24 sec of exposure to glucose and, in the case of lactic acid, fell slightly after the initial elevation. The phosphate flush was initiated only after 96 sec of exposure to glucose, whereas the decreases in ⁸⁶Rb and ⁴⁵Ca outflow were both detectable within 72 sec of stimulation. The secondary rise in ⁴⁵Ca efflux was first seen after 157 sec of stimulation and its time course was not vastly different from that of insulin release. These data indicate that, in the secretory sequence, metabolic changes precede both the remodelling of ionic fluxes and the stimulation of insulin release. The results are compatible with the view that the secondary rise in ⁴⁵Ca outflow is attributable, in part at least, to the glucose-induced decrease in K conductance (but not to the increase in phosphate outflow), with resulting membrane depolarization and gating of voltage-dependent Ca channels.     

Proinflammatory interactions of pneumolysin with human neutrophils

The effects of pneumolysin on the proinflammatory activity of human neutrophils, as well as on cation fluxes in these cells, have been investigated. Superoxide production, release of elastase, CR3 expression, phospholipase A2 activity, and alterations in membrane potential were measured by use of lucigenin-enhanced chemiluminescence and colorimetric, flow cytometric, radiometric, and spectrofluorimetric procedures, respectively; and cation fluxes were measured by use of 45Ca2+ and 86Rb+ and by fura-2 spectrofluorometry. Pneumolysin at concentrations >1.67 ng/mL caused influx of Ca2+ and increased phospholipase A2 activity and CR3 expression, which were associated with enhanced superoxide production and release of elastase after activation of the cells with the chemotactic tripeptide FMLP. At the same concentrations, pneumolysin caused efflux of K+ and membrane depolarization. The effects of pneumolysin on cation fluxes were not attributable to inhibition of Ca2+-adenosine triphosphatase (ATPase) or Na+, K+-ATPase. Pneumolysin potentiates the proinflammatory activities of neutrophils by a pore-forming mechanism resulting in Ca2+ influx.     

Stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester

The role currently ascribed to the accumulation of L-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to L-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on D-glucose metabolism, more severe inhibition of the catabolism of endogenous L-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-mediated entry of L-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.