Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+](i) to several microM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an approximately 1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein-protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.     

Protein kinase activation increases insulin secretion by sensitizing the secretory machinery to Ca2+

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).     

Measurement of exocytosis by amperometry in adrenal chromaffin cells: effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics

We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca(2+) to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery.     

Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.     

Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis.

The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Calcium dependencies of regulated exocytosis in different endocrine cells

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca(2+) concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca(2+) sensitivity in endocrine cells, accurate and fast determination of Ca(2+) dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that leads to a robust exocytotic activity. Slow increases in the [Ca(2+)](i) revealed exocytotic phases with different Ca(2+) sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca(2+) sensitivity 'classes' of secretory vesicles have been observed: a highly Ca(2+)-sensitive, a medium Ca(2+)-sensitive and a low Ca(2+)-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca(2+) sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca(2+) sensor protein.     

cAMP modulation of Ca(2+)-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

Effects of cAMP accumulation on ATP-dependent priming and Ca(2+)-dependent fusion in Ca(2+)-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca(2+)-regulated exocytosis activated by 1 microM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 microM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca(2+)-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca(2+)-regulated exocytosis. This suggests that the initial transient phase of Ca(2+)-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca(2+) dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca(2+) sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca(2+) sensitivity of the fusion, which potentiates Ca(2+)-regulated exocytosis in antral mucous cells.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.     

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used G?6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.     

Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.     

Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells

The second messenger cAMP exerts powerful stimulatory effects on Ca(2+) signaling and insulin secretion in pancreatic beta-cells. Previous studies of beta-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic beta-cells and INS-1 insulin-secreting cells to mobilize Ca(2+) from intracellular Ca(2+) stores via Epac-mediated Ca(2+)-induced Ca(2+) release (CICR). The cAMP-dependent increase of [Ca(2+)](i) that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus.     

Distinct potentiation of L-type currents and secretion by cAMP in rat chromaffin cells

We have investigated the potentiating action of cAMP on L-currents of rat chromaffin cells and the corresponding increase of Ca(2+)-evoked secretory responses with the aim of separating the action of cAMP on Ca(2+) entry through L-channels and the downstream effects of cAMP/protein kinase A (PKA) on exocytosis. In omega-toxin-treated rat chromaffin cells, exposure to the permeable cAMP analog 8-(4-chlorophenylthio)-adenosine 3',5'-monophosphate (pCPT-cAMP; 1 mM, 30 min) caused a moderate increase of Ca(2+) charge carried through L-channels (19% in 10 mM Ca(2+) at +10 mV) and a drastic potentiation of secretion ( approximately 100%), measured as membrane capacitance increments (deltaC). The apparent Ca(2+) dependency of exocytosis increased with pCPT-cAMP and was accompanied by 83% enhancement of the readily releasable pool of vesicles with no significant change of the probability of release, as evaluated with paired-pulse stimulation protocols. pCPT-cAMP effects could be mimicked by stimulation of beta(1)-adrenoreceptors and reversed by the PKA inhibitor H89, suggesting strict PKA dependence. For short pulses to +10 mV (100 ms), potentiation of exocytosis by pCPT-cAMP was proportional to the quantity of charge entering the cell and occurred independently of whether L, N, or P/Q channels were blocked, suggesting that cAMP acts as a constant amplification factor for secretion regardless of the channel type carrying Ca(2+). Analysis of statistical variations among depolarization-induced capacitance increments indicates that pCPT-cAMP acts downstream of Ca(2+) entry by almost doubling the mean size of unitary exocytic events, most likely as a consequence of an increased granule-to-granule rather than a granule-to-membrane fusion.     

Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase

Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-independent but stimulated by cAMP. Recombinant protein kinase A (PKA) could phosphorylate phogrin directly. High performance liquid chromatography analysis of tryptic phosphopeptides, combined with site-directed mutagenesis of candidate sites, revealed the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680 and Thr-699 had been replaced either by alanines or by aspartic acid residues, were targeted to secretory granules in transfected AtT20 neuroendocrine cells. Stimulation of these cells with a range of secretagogues, including K(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.     

Mechanisms of signal transduction in photo-stimulated secretion in Phaeocystis globosa

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.     

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.     

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.     

Alpha-latrotoxin Triggers Extracellular Ca^2＋-dependent Exocytosis and Sensitizes Fusion Machinery in Endocrine Cells

α-Latrotoxin from the venom of black widow spider induces and augments neurotransmitterand hormone release by way of extracellular Ca~(2 ) influx and cellular signal transduction pathways.By usingwhole cell current and capacitance recording,the photolysis of card Ca~(2 ),and Ca~(2 ) microfluorometry andamperometry,we investigated the stimulating effect and mechá(?)ism of α-latrotoxin on exocytosis in ratpancreatic β cells,LβT2 cells and latrophilin plasmid-transfected INS-1 cells.Our data indicated that:(1)α-latrotoxin increased cytosolic Ca~(2 ) concentration through the formation of cation-permitting pores and sub-sequent Ca~(2 ) influx with the presence of extracellular Ca~(2 );(2)α-latrotoxin stimulated exocytosis in normalbath solution and its stimulating effect on secretion was eradicated in Ca~(2 )-free bath solution; and (3)α-latrotoxin sensitized the molecular machinery of fusion through activation of protein kinase C and increasedthe response of cells to Ca~(2 ) photolysed by a flash of ultraviolet light.In summary,α-latrotoxin inducedexocytosis by way of Ca~(2 ) influx and accelerated vesicle fusion by the sensitization of fusion machinery.     

Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools

Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.