Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

The role of calcium in the secretion of surfactant by rat alveolar type II cells

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.     

Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas

To determine the role of free cytosolic calcium ([Ca+2]i) in stimulated enzyme secretion from exocrine pancreas, we determined the effects of various pancreatic secretagogues on [Ca+2]i and amylase release in dispersed acini from the guinea pig pancreas. Cholecystokinin-octapeptide (CCK-OP), carbachol, and bombesin, but not vasoactive intestinal peptide, stimulated rapid increases in [Ca+2]i from 100 to 600-800 nM that were independent of extracellular calcium. The increases in [Ca+2]i were transient (lasting less than 5 min) and correlated with an initial rapid phase of amylase release. After 5 min, secretagogue-stimulated amylase release occurred at basal [Ca+2]i. Carbachol pretreatment of the acini abolished the effects of CCK-OP and bombesin on [Ca+2]i and the initial rapid phase of amylase release. 4 beta-phorbol 12-myristate 13-acetate (PMA) had no effect on [Ca+2]i but stimulated an increase in amylase release. The addition of CCK-OP or A23187 to PMA-stimulated acini caused an increase in [Ca+2]i and PMA-stimulated amylase release only during the first 5 min after addition of these agents. These results indicate that CCK-OP, carbachol, and bombesin release calcium from an intracellular pool, resulting in a transient increase in [Ca+2]i and that this increase in [Ca+2]i mediates enzyme secretion during the first few minutes of incubation. The results with PMA suggest that secretagogue-stimulated secretion not mediated by increased [Ca+2]i (sustained secretion) is mediated by 1,2-diacylglycerol.     

Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C.

Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

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.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

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.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Mathematical modeling of stimulus-secretion coupling in the pancreatic beta-cell. III. Glucose-induced inhibition of calcium efflux.

The inhibitory effect of glucose upon 45Ca efflux from prelabeled pancreatic islets was simulated in a mathematical model for Ca2+-cyclic AMP interaction in the process of glucose-induced insulin release. At variance with a previous interpretation, it was postulated that glucose inhibits 45Ca efflux by facilitating the uptake of the cation by the vacuolar system. The latter facilitation did not hinder glucose from provoking a rapid accumulation of cytosolic Ca2+ and, hence, insulin release. The postulated facilitation was also suitable in simulating the effect of glucose upon 45Ca efflux, uptake, and intracellular distribution in the pancreatic islets.     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

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 bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.

delta-Haemolysin, a small surface-active polypeptide purified from the culture media of Staphylococcus aureus, was observed to stimulate the release of insulin from isolated rat islets of Langerhans. This effect was dose-dependent and saturable, with the half-maximal response elicited by a delta-haemolysin concentration of 10 micrograms/ml. Stimulation of insulin release by delta-haemolysin (10 micrograms/ml) was not dependent on the presence of glucose in the incubation medium, but was augmented by increasing concentrations of the sugar. The release of insulin in response to delta-haemolysin could be inhibited by depletion of extracellular Ca2+ or by adrenaline (epinephrine) (10 microM) and was readily reversible when delta-haemolysin was removed from the medium. In addition, the response was potentiated by incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). These observations suggest that delta-haemolysin induced a true activation of the beta-cell secretory mechanism. Stimulation of islets of Langerhans with delta-haemolysin was found to be associated with a modest increase in intracellular cyclic AMP levels, although the adenylate cyclase activity of islet homogenates was not increased by delta-haemolysin. delta-Haemolysin was observed to induce a dose-dependent net accumulation of 45Ca2+ by islet cells and to stimulate the efflux of 45Ca2+ from preloaded islets. The efflux of 45Ca2+ was modest in size and short-lived, but dramatically increased in medium depleted fo 40Ca2+. Incubation in the presence of verapamil augmented delta-haemolysin-induced 45Ca2+ efflux and insulin secretion. delta-Haemolysin was found to be a potent 45Ca2+-translocating ionophore in an artificial system. This response was dose-dependent and could be augmented by verapamil. In addition, phosphatidylcholine (25 micrograms/ml) was found to inhibit both delta-haemolysin induced 45Ca2+ translocation and insulin release in a precisely parallel manner. These studies suggest that the ability of delta-haemolysin to stimulate insulin release may be due, in part, to the facilitation of Ca2+ entry into the beta-cells of islets of Langerhans, mediated directly by an ionophoretic mechanism.