Modulation of the exocytotic reaction of permeabilised rat mast cells by ATP, other nucleotides and Mg2+

In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulatory actions, although its presence (and by implication, phosphorylation) is not obligatory for secretion to occur. These effects include (1) the enhancement of the sensitivity to both of the essential effectors (Ca2+ and guanine nucleotide); (2) the maintenance of the responsiveness of permeabilised cells; (3) restoration of responsiveness to cells rendered refractory by previous permeabilisation, and (4) induction of delays in the onset of exocytosis from permeabilised cells. We define the modulatory reactions induced by ATP by characterising their specificity to other potential phosphorylating nucleotides and their requirement for Mg2+. GTP and AppNHp are without effect in any of the modulatory actions. ATP, ATP-gamma-S, ITP, XTP, CTP and UTP all appear to support an enhancement of the sensitivity to GTP-gamma-S when applied immediately at the time of permeabilisation. However, the non-adenine nucleoside triphosphates appear to mediate their effect by transphosphorylation to ADP, and therefore the active species appears to be ATP. Only ATP is capable of maintaining and restoring responsiveness (2 and 3 above). Only ATP and ATP-gamma-S induce onset delays and do so moreover in the absence (less than 10(-8) M) of Mg2+. We conclude that three of the modulatory effects (1, 2 and 3 above) which all express a requirement for Mg2+, and can be prevented by inhibitors of protein kinase C are likely to result from phosphorylation reactions. The induction of delays by ATP is unlikely to incur phosphorylation.     

Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range

Rat peritoneal mast cells have been permeabilised by treatment with streptolysin O which generates membrane lesions of macromolecular dimensions. In the presence of Ca2+ buffered at concentrations in the micromolar range, the permeabilised mast cells release histamine, beta-N-acetylglucosaminidase and lactate dehydrogenase. Release of the two secretory components (but not lactate dehydrogenase) has an obligatory requirement for a nucleoside triphosphate and micromolar concentrations of Ca2+. Inosine triphosphate (ITP) supports the release reaction better than ATP does. It is concluded that the secretory materials are released from the cells by an exocytotic mechanism, while lactate dehydrogenase leaks from the cells through the toxin-generated lesions. By initially withholding and then supplying Ca2+ to the permeabilised cells, it is shown that the exocytotic secretory reaction can persist even when the cytosol is depleted of the bulk of soluble proteins. The streptolysin O treated mast cell preparation represents a simplified system with which to study the mechanism of exocytosis.     

ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.     

Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.     

Catecholamine inhibition of Ca2+-induced insulin secretion from electrically permeabilised islets of Langerhans

Noradrenaline (1-10 microM) inhibited Ca2+-induced insulin secretion from electrically permeabilised islets of Langerhans with an efficacy similar to that for inhibition of glucose-induced insulin secretion from intact islets. The inhibition of insulin secretion from permeabilised islets was blocked by the alpha 2-adrenoreceptor antagonist, yohimbine. Adenosine 3',5'-cyclic monophosphate (cAMP) did not relieve the noradrenaline inhibition of Ca2+-induced secretion from the permeabilised islets, although noradrenaline did not affect the secretory responses to cAMP at substimulatory (50 nM) concentrations of Ca2+. These results suggest that catecholamines do not inhibit insulin secretion solely by reducing B-cell adenylate cyclase activity, and imply that one site of action of noradrenaline is at a late stage in the secretory process.     

Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.     

Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP- gamma-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-gamma-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Evidence for protein dephosphorylation as a permissive step in GTP-gamma-S-induced exocytosis from permeabilized mast cells.

Mast cells permeabilized by streptolysin O secrete histamine and lysosomal enzymes in response to provision of a dual effector system comprising Ca2+ and a guanine nucleotide (e.g., GTP-gamma-S2) at concentrations in the micromolar range. These are both necessary and together they are sufficient. There is no requirement for adenosine triphosphate (ATP) and hence no obligatory phosphorylation reaction in the terminal stages of the exocytotic pathway. When exocytosis is induced by Ca2(+)-plus-GTP-gamma-S (i.e., no ATP) added at times after permeabilization (the permeabilization interval), cellular responsiveness declines so that there is no response to provision of the two effectors (both at 10(-5)M) if they are initially withheld and then added after 5 min. Here we show that this decline in responsiveness is characterized by a time-dependent reduction in the effective affinity for Ca2+. Affinity for Ca2+ and hence secretory competence can then be restored if ATP is added alongside the stimulus. Unlike cells stimulated to secrete at the time of permeabilization, exocytosis from cells that have undergone the cycle of permeabilization-induced refractoriness followed by ATP-induced restoration can be triggered by Ca2+ alone: after such conditioning there is no requirement for guanine nucleotide. In contrast, dependence on guanine nucleotide remains mandatory in cells that have been pretreated (i.e., before permeabilization) with okadaic acid (understood to be an inhibitor of protein phosphatases 1 and 2A) or phorbol myristate acetate (an activator of protein kinase C). These results indicate that obligatory dependence on guanine nucleotide is retained when the cells are treated under conditions conducive to maintained phosphorylation. It is concluded that the exocytotic mechanism of permeabilized mast cells is enabled by a dephosphorylation reaction and that the effector of the guanosine triphosphate (GTP)-binding protein (G epsilon) that mediates exocytosis is likely to be a protein phosphate.     

Glucose-stimulated insulin secretion is not dependent on activation of protein kinase A

The involvement of cyclic AMP-dependent protein kinase A (PKA) in the exocytotic release of insulin from rat pancreatic islets was investigated using the Rp isomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS). Preincubation of electrically permeabilised islets with Rp-cAMPS (1 mM, 1 h, 4 degrees C) inhibited cAMP-induced phosphorylation of islet proteins of apparent molecular weights in the range 20-90 kDa, but did not affect basal (50 nM Ca2+) nor Ca2(+)-stimulated (10 microM) protein phosphorylation. Similarly, Rp-cAMPS (500 microM) inhibited both cAMP- (100 microM) and 8BrcAMP-induced (100 microM) insulin secretion from electrically permeabilised islets without affecting Ca2(+)-stimulated (10 microM) insulin release. In intact islets, Rp-cAMPS (500 microM) inhibited forskolin (1 microM, 10 microM) potentiation of insulin secretion, but did not significantly impair the insulin secretory response to a range of glucose concentrations (2-20 mM). These results suggest that cAMP-induced activation of PKA is not essential for either basal or glucose-stimulated insulin secretion from rat islets.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Bromoenol lactone, an inhibitor of Group V1A calcium-independent phospholipase A2 inhibits antigen-stimulated mast cell exocytosis without blocking Ca2+ influx

Calcium-independent phospholipase A2 (iPLA2beta) has recently been suggested to regulate Ca2+ entry by activating store-operated Ca2+ channels. These studies have been conducted in mast cells using thapsigargin to deplete intracellular stores. In RBL 2H3 and bone marrow-derived mast cells (BMMCs), Ca2+ entry is critical for exocytosis and therefore we have examined whether the proposed mechanism would be relevant when a physiological stimulus is applied to these cells. Using an iPLA2beta antibody, we demonstrate that the 84kDa iPLA2beta is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA2beta it was used to probe this potential mechanism. We observe inhibition of exocytosis stimulated either with antigen or with thapsigargin. However, BEL also inhibits exocytosis when stimulated using a Ca2+ ionophore A23187, which passively transports Ca2+ down a concentration gradient and also in permeabilised mast cells where Ca2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen- or thapsigargin-stimulated Ca2+ signalling, both the release from internal stores and sustained elevation due to Ca2+ influx. These results cast doubt on the proposed mechanism involving iPLA2beta required for Ca2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA2beta activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis.     

Mechanisms of granule enzyme secretion from permeabilized guinea pig eosinophils. Dependence on Ca2+ and guanine nucleotides

The mechanisms of granule protein secretion have been studied in streptolysin-O-permeabilized guinea pig eosinophils. Secretion of the granule-associated enzyme N-acetyl-beta-D-glucosaminidase was dependent on both Ca2+ and a nonhydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting roles for both calcium and GTP binding proteins. Secretion was maximal by 7 min, and varied between 35 and 60% of the total enzyme activity. Other GTP analogues also elicited secretion, with rank order GTP-gamma-S greater than guanylyl-imidophosphate greater than guanylyl (beta-gamma-methylene-diphosphate). Unrelated nucleotide triphosphates showed little or no effect confirming the specificity of the G protein. Transmission electronmicroscopy confirmed that permeabilization alone did not result in loss of granules and that exocytosis was dependent on the addition of the effectors, Ca2+ and GTP-gamma-S. ATP enhanced the magnitude of the secretory response and also enhanced the effective affinities for both Ca2+ and GTP-gamma-S. In the presence of 10(-5) M GTP-gamma-S the ED50 (Ca2+) was pCa 5.57 +/- 0.04 (2.69 microM) in the absence of ATP and declined to pCa 6.16 +/- 0.03 (0.69 microM) in the presence of ATP (p less than 0.0001). Furthermore, ATP served to restore responsiveness in cells that had been rendered refractory by delaying stimulation after permeabilization. Pretreatment with PMA (an activator of PKC) inhibited the induction of a refractory state, whereas inhibition of PKC partially countered the ability of ATP to restore responsiveness, both observations pointing to a requirement for a specific component of the secretory mechanism to be in a phosphorylated state in order to condone the secretion process. These observations show that secretory mechanisms in eosinophils are similar to those in other myeloid cells, in particular neutrophils and mast cells, although the time course of secretion is more protracted.     

The ATP4- receptor of rat mast cells.

The concentration-dependence on exogenous ATP of activation and inhibition of mast-cell histamine secretion, phosphatidylinositol labelling and leakage of metabolites shows that all these functions are regulated by the free acid ATP4-. Maximal histamine secretion and phosphatidylinositol labelling occur with ATP4- at approx. 2 microM, but higher concentrations, which cause inhibition of secretion and phosphatidylinositol labelling, are required to maximize leakage of 32P-labelled metabolites. Both enhancement and inhibition of phosphatidylinositol labelling (due to low and high concentrations of ATP4- respectively) are rapid in onset; histamine secretion is characterized by a delay, especially at low concentrations of ATP4- (approx. 1 microM). Phosphatidylinositol labelling and histamine secretion are dependent on extracellular Ca2+. Metabolite leakage due to the presence of exogenous ATP4- is slow and does not require Ca2+. Of 18 analogues of ATP that were tested, only four were agonists for secretion, and only these four permitted leakage of 32P-labelled metabolites. It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor. For mast cells stimulated with ATP4- enhancement of phosphatidylinositol metabolism is not sufficient by itself to cause Ca2+-dependent secretion.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.     

Regulation of insulin secretion by cAMP in rat islets of Langerhans permeabilised by high-voltage discharge

Adenosine 3',5-cyclic monophosphate (cAMP) was shown to stimulate insulin secretion from electrically permeabilised islets of Langerhans incubated in Ca2+/EGTA buffers. cAMP-induced insulin secretion occurred in the presence of either sub-stimulatory (50 nM) or stimulatory (greater than 100 nM) concentrations of Ca2+. Similar effects on secretion were obtained in response to 8-bromo-cAMP (8-Br-cAMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. Forskolin (0.2-20 microM) increased adenylate cyclase activity and enhanced insulin secretion from the permeabilised islets. These results suggest that, in electrically permeabilised islets, cAMP-induced insulin secretion is not dependent on changes in cytosolic Ca2+.     

Role of protein kinase C in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary cells

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.     

Chemotaxis of rat mast cells toward adenine nucleotides.

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 approximately 0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.