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We report a novel excitatory effect of cannabinoid agonists on action potential-independent GABAergic transmission in the rat dentate gyrus. Specifically, we find that both WIN55,212-2 and anandamide increase the frequency of miniature IPSCs (mIPSCs)recorded from hilar mossy cells without altering event amplitude, area, rise time, or decay. The effect of WIN55,212-2 on mIPSCs is insensitive to AM251 and preserved in CB1 −/− animals,indicating that it does not depend on activation of CB1 receptors. It is also insensitive to AM630 and unaffected by capsazepine suggesting that neither CB2 nor TRPV1 receptors are involved. Further, it is blocked by pre-incubation in suramin and by a selective protein kinase A inhibitor (H-89), and is mimicked (and occluded) by bath application of forskolin. Similar CB1 receptor-independent facilitation of exocytosis is not apparent when recording evoked IPSCs in the presence of AM251, suggesting that the exocytotic mechanism that produces WIN55,212-2 sensitive mIPSCs is distinct from that which produces CB1 sensitive and action potential-dependent release. Despite clear independence from action potentials, WIN55,212-2 mediated facilitation of mIPSCs requires calcium, and yet is insensitive to chelation of calcium in the postsynaptic cell. Finally, we demonstrate that both bath application of 2-arachidonoylglycerol(2-AG) and depolarization-induced release of endogenous cannabinoids have minimal effect on mIPSC frequency. Cumulatively, our results indicate that cannabinoid ligands can selectively facilitate action potential-independent exocytosis of GABA in the rat dentate gyrus, and further emphasize that this new cannabinoid sensitive signalling system is distinct from previously described CB1 receptor-dependent systems in numerous respects.

Patterns of action potentials (APs), often in the form of bursts, are critical for coding and processing information in the brain. However, how AP bursts modulate secretion at synapses remains elusive. Here, using the calyx of Held synapse as a model we compared synaptic release evoked by AP patterns with a different number of bursts while the total number of APs and frequency were fixed. The ratio of total release produced by multiple bursts to that by a single burst was defined as 'burst-effect'.We found that four bursts of 25 stimuli at 100 Hz increased the totalcharge of EPSCs to 1.47 ± 0.04 times that by a single burst of 100 stimuli at the same frequency.Blocking AMPA receptor desensitization and saturation did not alter the burst-effect, indicating that it was mainly determined by presynaptic mechanisms. Simultaneous dual recordings of presynaptic membrane capacitance (Cm) and EPSCs revealed a similar burst-effect, being 1.58±0.13by Cm and 1.49±0.05 by EPSCs. Reducing presynapticCa2+ influx by lowering extracellular Ca2+concentration or buffering residual intracellular Ca2+ with EGTA inhibited the burst-effect. We further developed a computational model largely recapitulating the burst-effect and demonstrated that this effect is highly sensitive to dynamic change in availability of the releasable pool of synaptic vesicles during various patterns of activities. Taken together, we conclude that AP bursts modulate synaptic output mainly through intricate interaction between depletion and replenishment of the large releasable pool. This burst-effect differs from the somatic burst-effect previously described from adrenal chromaffin cells, which substantially depends on activity-induced accumulation of Ca2+ to facilitate release of a limited number of vesicles in the releasable pool. Hence, AP bursts may play an important role in dynamically regulating synaptic strength and fidelity during intense neuronal activity at central synapses.

Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca(2+) and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A(2b) receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins.

Direct recordings from the large axon terminals of goldfish retinal bipolar cells (BCs) have revealed detailed information about the properties and regulation of exocytosis at this ribbon-type synapse. However, the relationship between BC exocytosis and evoked postsynaptic responses in amacrine and ganglion cells is not known. To address this, I have made paired recordings from BC terminals (BCTs) and neurons in the ganglion cell layer (GCL) in goldfish retinal slices. BCT depolarisation evoked short-latency, AMPA/kainate receptor-mediated EPSCs in connected GCL neurons. NMDA receptors contributed to the response at +40 mV but not at 60 mV. Evoked EPSCs contained multiple temporal components that differed in their relative amplitudes between pairs. Changing the duration or amplitude of the presynaptic stimulus affected the size and kinetics of the EPSC, with weaker stimuli slowing the EPSC activation rate. Paired-pulse stimulation caused greater depression of fast than slow EPSC components. A linear relationship was found between the amount of BCT exocytosis, measured via changes in membrane capacitance, and the charge of evoked EPSCs, whether they were mediated by AMPA/kainate receptors alone or in combination with NMDA receptors. In addition, analysis of miniature EPSCs in GCL neurons provided estimates of the quantal content of evoked EPSCs. The results demonstrate the feasibility of using this paired recording system to study synaptic transfer at ribbon synapses, and indicate that both the rapid and sustained phases of BC exocytosis are encoded in the postsynaptic response.

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

The small GTPase Rab27a, along with the isoforms of Rab3, is present on insulin secretory granules and has been implicated in regulation of Ca(2+)-triggered exocytosis. We have used membrane capacitance measurements to define the role of Rab27a in regulating the size and refilling of distinct pools of insulin granules by comparison of evoked secretory responses from Rab27a-null ashen and strain-matched wild-type control pancreatic beta-cells. We find that ashen beta-cells display a kinetic defect in refilling of readily releasable and immediately releasable vesicle pools (RRP and IRP, respectively) in response to depolarization-evoked Ca(2+) influx. The deficit in IRP refilling was not observed in the presence of stimulatory glucose concentrations (16.7 mm), though incomplete refilling of the RRP persisted. Comparatively, beta-cells from Rab3a(-/-) mice exhibited complete refilling of the IRP and RRP, demonstrating that Rab27a and Rab3a exert distinct roles in the insulin granule secretory pathway. Further, depletion of the RRP in ashen beta-cells was twofold faster than that of control beta-cells. These deficits in refilling and exocytotic rate in ashen beta-cells were absent when cAMP-regulatory pathways were activated. Elevated cAMP increased the RRP pool size, and complete refilling of the RRP occurred in ashen beta-cells; responses were comparable to wild-type controls. These effects of cAMP were largely eliminated by Rp-cAMP inhibition of PKA, indicating that PKA acts on vesicle priming downstream or via pathways independent of Rab27a. In summary, Rab27a exerts dual roles in glucose-mediated insulin granule exocytosis, facilitating refilling of releasable granule pools while also limiting the rate of release from these pools.

Synaptotagmin I (Syt I), the putative Ca(2+) sensor in regulated exocytosis, has two Ca(2+)-binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca(2+)-dependent fashion. The role of Ca(2+) binding to these domains remains unclear, as efforts to address questions about Ca(2+)-triggered effector interactions have led to conflicting results. We have studied the effects of Ca(2+) on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyse the kinetics of fusion pore transitions. Elevating [Ca(2+)] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca(2+) during the actual fusion process. Analysing the Ca(2+) dependence of the fusion pore lifetime, together with the frequency of pore openings and the proportion of openings that close without dilating (kiss-and-run events) enabled us to resolve exocytosis into a sequence of kinetic steps representing functional transitions in the fusion pore. Fusion pore opening and dilation were both accelerated by Ca(2+), indicating separate Ca(2+) control over each of these steps. Ca(2+) ligand mutations in either the C2A or C2B domains of Syt I reduced fusion pore opening, but had opposite actions on the rate of fusion pore closure. These studies resolve two separate and distinct Ca(2+)-triggered steps during regulated exocytosis. The C2A and C2B domains of Syt I have different actions during these steps, and these actions may be linked to their distinctive effector interactions.

The role of protein kinase A (PKA) in insulin exocytosis was investigated with the use of two-photon excitation imaging of mouse islets of Langerhans. Inhibitors of PKA selectively reduced the number of exocytic events during the initial period (< 250 s) of the first phase of glucose-induced exocytosis (GIE), without affecting the second phase, in intact islets or small clusters of islet cells. The PKA inhibitors did not reduce the extent of the glucose-induced increase in [Ca(2+)](i). The actions of glucose and PKA in Ca(2+)-induced insulin exocytosis (CIE) triggered by photolysis of a caged-Ca(2+) compound, which resulted in large increases in [Ca(2+)](i) and thereby bypassed the ATP-sensitive K(+) channel-dependent mechanism of glucose sensing, were therefore studied. A high concentration (20 mM) of glucose potentiated CIE within 1 min, and this effect was blocked by inhibitors of PKA. This PKA-dependent action of glucose required glucose metabolism, given that increasing the intracellular concentration of cAMP by treatment with forskolin potentiated CIE only at the high glucose concentration. Finally, PKA appeared to reduce the frequency of 'kiss-and-run' exocytic events and to promote full-fusion events during GIE. These data indicate that a PKA-dependent mechanism of glucose sensing, which is operative even at the basal level of PKA activity, plays an important role specifically in the first phase of GIE, and they suggest that the action of PKA is mediated at the level of the fusion reaction.

Cyclic AMP regulates Ca(2+)-dependent exocytosis through a classical protein kinase A (PKA)-dependent and an alternative cAMP-guanine nucleotide exchange factor (GEF)/Epac-dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high-time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 microM cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole-cell patch-clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and approximately 34% increase of high-voltage activated Ca(2+) channel amplitude. cAMP-dependent mechanisms did not affect [Ca(2+)](i), since the application of forskolin failed to change [Ca(2+)](i) in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization-induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP-independent) probably represented the exocytosis of the release-ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP-dependent reaction(s) and > 800 nM [Ca(2+)](i). The linear component was modulated by 8-pCPT-2Me-cAMP (Epac agonist), while either H-89 (PKA inhibitor) or Rp-cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6-Phe-cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP-GEFII/Epac2-dependent mechanisms.

alpha-Latrotoxin (alpha-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of alpha-LT on single human, mouse and canine beta-cells. In isolated and combined measurements, alpha-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca(2+), into the micromolar range, that are dependent on extracellular Ca(2+); (ii) large conductance non-selective cation channels; and (iii) Ca(2+)-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, alpha-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca(2+). These results strongly support the hypothesis that alpha-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) alpha-LT inserts into the plasma membrane to form Ca(2+) permeable channels and promote Ca(2+) entry thereby triggering Ca(2+)-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, alpha-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca(2+)-sensitive pool of insulin granules activated by global cytosolic Ca(2+) concentrations in the range of approximately 1 microm.

Developmental changes in the coupling between Ca2+ entry and exocytosis were studied in mouse inner hair cells (IHCs) which, together with the afferent endings, form the primary synapse of the mammalian auditory system. Ca2+ currents (ICa) and changes in membrane capacitance (DeltaCm) were recorded using whole-cell voltage clamp from cells maintained at body temperature, using physiological (1.3 mM) extracellular Ca2+. The magnitudes of both ICa and DeltaCm increased with maturation from embryonic stages until postnatal day 6 (P6). Subsequently, ICa gradually declined to a steady level of about -100 pA from P13 while the Ca2+-induced DeltaCm remained relatively constant, indicating a developmental increase in the Ca2+ efficiency of exocytosis. Although the size of ICa changed during development, its activation properties did not, suggesting the presence of a homogeneous population of Ca2+ channels in IHCs throughout development. The Ca2+ dependence of exocytosis changed with maturation from a fourth power relation in immature cells to an approximately linear relation in mature cells. This change applies to the release of both a readily releasable pool (RRP) and a slower secondary pool of vesicles, implying a common release mechanism for these two kinetically distinct pools that becomes modified during development. The increased Ca2+ efficiency and linear Ca2+ dependence of mature IHC exocytosis, especially over the physiological range of intracellular Ca2+, could improve the high-fidelity transmission of both brief and long-lasting stimulation. These properties make the mature cell ideally suited for fine intensity discrimination over a wide dynamic range.

SPOC1 airway goblet cells secrete mucin in response to P2Y2 receptor agonists and to secretagogues, phorbol 12-myristate 13-acetate (PMA) and ionomycin, which mobilize elements of the phospholipase C pathway, PKC and Ca2+, respectively. Previous studies demonstrated that mucin secretion from SLO-permeabilized, EGTA-buffered SPOC1 cells was stimulated by PMA at low Ca2+ levels (< 0.1 microm), consistent with the notion that regulated exocytosis may occur by Ca2+-independent pathways. We tested the alternative hypothesis that PMA-induced mucin secretion is, in fact, a Ca2+-dependent process under the conditions of low bulk Ca2+, one that is permitted in the typical SLO-permeabilized cell model by the slow binding kinetics of EGTA. Both IP3 and elevated bulk Ca2+ activated mucin secretion in SPOC1 cells buffered by EGTA, suggesting that IP3 generates a local Ca2+ gradient in the vicinity of the secretory granules to the degree necessary to trigger exocytosis. BAPTA, which binds Ca2+ approximately 100-fold faster than EGTA, diminished IP3-induced mucin release over a range of concentrations by > or = 69%, yet maintained an essentially normal mucin secretory response to elevated bulk Ca2+ in permeabilized SPOC1 cells. BAPTA also diminished the mucin secretory response of permeabilized cells to PMA, relative to the EGTA-buffered control: at PMA below 30 nm, BAPTA abolished the secretory response, and at higher concentrations it was reduced significantly relative to the EGTA-buffered controls. PMA-induced secretion in EGTA was insensitive to heparin. These results suggest that Ca2+ is released locally during PMA-induced exocytosis, by an IP3-independent mechanism.

We have investigated the regulation of hormone secretion from rat pancreatic islets by the GABAB receptors (GABABRs). Inclusion of the specific GABABR antagonist CGP 55845 in the extracellular medium increased glucose-stimulated insulin secretion 1.6-fold but did not affect the release of glucagon and somatostatin. Conversely, addition of the GABABR agonist baclofen inhibited glucose-stimulated insulin secretion by approximately 60%. Using RT-PCR, transcription of GABABR1a-c,f and GABABR2 subunits was detected in beta-cells. Measurements of membrane currents and cell capacitance were applied to single beta-cells to investigate the mechanisms by which GABABR activation inhibits insulin secretion. In perforated-patch measurements, baclofen inhibited exocytosis elicited by 500-ms voltage-clamp depolarizations to 0 mV by < or = 80% and voltage-gated Ca2+ entry by only approximately 30%. Both effects were concentration-dependent with IC50 values of approximately 2 microm. The inhibitory action of baclofen was abolished in the presence of CGP 55845. The ability of baclofen to suppress exocytosis was prevented by pre-treatment with pertussis toxin and by inclusion of GDPbetaS in the intracellular medium, and became irreversible in the presence of GTPgammaS as expected for a process involving inhibitory G-proteins (Gi/o-proteins). The inhibitory effect of baclofen resulted from activation of the serine/threonine protein phosphatase calcineurin and pre-treatment with cyclosporin A or intracellular application of calcineurin autoinhibitory peptide abolished the effect. Addition of baclofen had no effect on [Ca2+]i and electrical activity in glucose-stimulated beta-cells. These data indicate that GABA released from beta-cells functions as an autocrine inhibitor of insulin secretion in pancreatic islets and that the effect is principally due to direct suppression of exocytosis.

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic beta-cells by a combination of electrophysiological recordings, intracellular Ca(2+) ([Ca(2+)](i)) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid-free albumin) to intact islets exposed to 15 mm glucose increased the [Ca(2+)](i) by approximately 30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca(2+)](i) and insulin release in the presence of tolbutamide and in islets depolarized by high K(+) in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K(+) channels (K(ATP) channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single beta-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca(2+) currents rose by approximately 25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 microm isradipine indicating that the free fatty acid specifically acts on L-type Ca(2+) channels. The actions of palmitate on exocytosis and Ca(2+) currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K(ATP) channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca(2+) currents and an increased size of the RRP.

Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for beta-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.

Capacitance measurements of exocytosis were applied to functionally identified alpha-, beta- and delta-cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in beta-cells during a depolarization to 0 mV was equivalent to 14 granules s(-1), <5% of that observed in isolated beta-cells. Beta-cell secretion exhibited bell-shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to approximately -20 mV) the maximum rate of release was approximately 4 granules s(-1). Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L-type Ca(2+) channel blocker nifedipine (25 microm) but only marginally (<20%) affected by the R-type Ca(2+) channel blocker SNX482 (100 nm). Exocytosis in the glucagon-producing alpha-cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s(-1)) and a sustained late component (30 granules s(-1)). Whereas addition of the N-type Ca(2+) channel blocker omega-conotoxin GVIA (0.1 microm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L-type Ca(2+) channel blocker nifedipine (25 microm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca(2+)-influx through N-type rather than L-type Ca(2+) channels. Exocytosis in the somatostatin-secreting delta-cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s(-1)) component followed by a sustained slower (60 granules s(-1)) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in beta-cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Cerebellar granule cells are inhibited phasically by GABA released synaptically from Golgi cells, but are inhibited more powerfully by tonic activity of high affinity alpha 6 subunit-containing GABAA receptors. During development the tonic activity is generated by the accumulation of GABA released by action potentials, but in the adult the tonic activity is independent of action potentials. Here we show that in adult rats the tonic activation of GABAA receptors is produced by non-vesicular transmitter release and is reduced by the activity of GAT-1 and GAT-3 GABA transporters, demonstrating that alterations of GABA uptake will modulate information flow through granule cells. Acetylcholine (ACh) evokes a large Ca2+-dependent but action potential-independent release of GABA, which activates alpha 6 subunit-containing GABAA receptors. These data show that three separate modes of transmitter release can activate GABAA receptors in adult cerebellar granule cells: action potential-evoked exocytotic GABA release, non-vesicular release, and ACh-evoked Ca2+-dependent release independent of action potentials. The relative magnitudes of the inhibitory charge transfers generated by action potential-evoked release (during high frequency stimulation of the mossy fibres), tonic inhibition and superfused ACh are 1:3:12, indicating that tonic and ACh-mediated inhibition may play a major role in regulating granule cell firing.

Functional coupling between Ca(2+)-induced Ca(2+) release (CICR) and quantal exocytosis in 5-hydroxytryptamine-loaded INS-1 beta cells was assessed through the use of carbon fibre amperometry in combination with Fura-2. CICR was evoked by the glucagon-like-peptide-1 (GLP-1) receptor agonist exendin-4 (Ex-4) and was accompanied by quantal secretory events appearing as amperometric current spikes time-locked to the increase of [Ca(2+)](i). The action of Ex-4 was reproduced by treatment with caffeine, and the source of Ca(2+) serving as a stimulus for exocytosis originated from ryanodine and thapsigargin-sensitive Ca(2+) stores. Two distinct patterns of exocytosis occurred within 5 s following the initiation of CICR. Non-summating exocytosis (NS-type) was defined as multiple asynchronous current spikes, and the half-height duration of each spike was 12-48 ms. Summating exocytosis (S-type) was defined as a cluster of spikes. It generated a macroscopic current, the half-height duration of which was 243-682 ms. The release charge of S-type exocytosis was 3.2-fold greater than that of NS-type when measured 2 s following the initiation of secretion. NS-type exocytosis was observed frequently under conditions in which the basal Ca(2+) concentration ([Ca(2+)](B)) was low (75-150 nM), whereas S-type exocytosis predominated under conditions in which the [Ca(2+)](B) was elevated (200-275 nM). Depolarization-induced Ca(2+) influx triggered NS-type exocytosis in most cells tested, irrespective of [Ca(2+)](B). It is concluded that CICR is a highly effective stimulus for exocytosis in INS-1 cells. The increase of [Ca(2+)](i) that accompanies CICR stimulates the asynchronous release of a small number of secretory granules under conditions of low [Ca(2+)](B). When [Ca(2+)](B) is slightly elevated, CICR targets a much larger pool of secretory granules that undergo summating exocytosis. The transition from NS-type to S-type exocytosis may represent an amplification mechanism for Ca(2+)-dependent exocytosis.

Phorbol esters are hypothesised to produce a protein kinase C (PKC)-dependent increase in the probability of transmitter release via two mechanisms: facilitation of vesicle fusion or increases in synaptic vesicle number and replenishment. We used a combination of electrophysiology and computer simulation to distinguish these possibilities. We constructed a stochastic model of the presynaptic contacts between a pair of hippocampal pyramidal cells that used biologically realistic processes and was constrained by electrophysiological data. The model reproduced faithfully several forms of short-term synaptic plasticity, including short-term synaptic depression (STD), and allowed us to manipulate several experimentally inaccessible processes. Simulation of an increase in the size of the readily releasable vesicle pool and the time of vesicle replenishment decreased STD, whereas simulation of a facilitation of vesicle fusion downstream of Ca(2+) influx enhanced STD. Because activation of protein kinase C with phorbol ester enhanced STD of EPSCs in rat hippocampal slice cultures, we conclude that an increase in the sensitivity of the release process for Ca(2+) underlies the potentiation of neurotransmitter release by PKC.

Increasingly the role of rapid mechanisms of steroid action in physiological regulation are being recognised. We have investigated rapid effects of 17beta-oestradiol (E) on prolactin (PRL) release in vitro. Pituitary segments from male rats were incubated for 5, 10 or 20 min in Earle's balanced salt solution containing 1.2 mM tannic acid (to enable visualisation of exocytosed secretory granules by electron microscopy) either alone (control) or containing 10(-10)-10(-8) M E conjugated to bovine serum albumin (E-BSA). PRL and leuteinising hormone (LH) release from pituitary segments were also determined in response to E and E-BSA by radioimmunoassay. Within 10 min E-BSA and E (10(-12)-10(-6) M) stimulated a significant (P < 0.05) concentration-dependent release of PRL but not LH. After exposure to experimental media for 5 min, only occasional exocytosis from type I lactotrophs (characterised by large polymorphic secretory granules) was observed in either control or E-BSA treated tissue. In contrast, E-BSA (10(-10)-10(-8) M) induced a significant (P < 0.05) increase in the number of exocytotic profiles from type II lactotrophs (characterized by smaller, spherical granules). This effect was not inhibited by removal of extracellular calcium, or by pre-treatment of cells with the RNA synthesis inhibitor actinomycin-D (0.5 microg ml(-1)), the protein synthesis inhibitor cycloheximide (1 microg ml(-1)) or the anti-oestrogen ICI 182,780 (1 microM). FACS analysis demonstrated binding of E-BSA-fluorescein isothiocyanate (FITC) (10(-10)-10(-7) M) to a subpopulation of anterior pituitary cells. The E-BSA-FITC binding sites assumed a patchy distribution across the cell surface. In conclusion, we report for the first time a rapid, non-genomic effect of E on PRL secretion in normal pituitary tissue.

1. Simultaneous pre- and postsynaptic patch recordings were obtained from the varicosity synapses formed by Xenopus motoneurons on muscle cells in embryonic cultures, in order to elucidate the contribution of N- and L-type Ca(2+) channels to the varicosity Ca(2+) current (I(Ca)) and evoked transmitter release. 2. Although N-type channels are predominant in the varicosities and generally thought to be responsible for all evoked release, in most synapses a fraction of I(Ca) and release could be reversibly blocked by the L-type channel antagonist nifedipine, and enhanced by the agonist Bay K8644. Up to 50 % (mean, 21 %) of the I(Ca) evoked by a voltage clamp waveform mimicking a normal presynaptic action potential (APWF) is composed of L-type current. 3. Surprisingly, the nifedipine-sensitive (L) channels activated more rapidly (time-constant, 0.46 ms at +30 mV) than the nifedipine-insensitive (N) channels (time constant, 1.42 ms). Thus the L-type current would play a disproportionate role in the I(Ca) linked to a normal action potential. 4. The relationship between I(Ca) and release was the same for nifedipine-sensitive and -resistant components. The N- and L-components of I(Ca) are thus equally potent in evoking release. This may represent an immature stage before N-type channels become predominant. 5. Replacing Ca(2+) in the medium with Ba(2+) strongly enhanced the L-type component, suggesting that L-type channels may be inactivated at Ca(2+) levels close to those at rest. 6. We speculate that populations of L-type channels in different parts of the neuron may be recruited or inactivated by fluctuations of the cytosolic Ca(2+) concentration within the physiological range.

1. Gastrin and PACAP stimulate secretion of histamine and pancreastatin from isolated rat stomach ECL cells. We have examined whether or not secretion depends on the free cytosolic Ca2+ concentration ([Ca2+]i) and the pathways by which gastrin and PACAP elevate [Ca2+]i. Secretion was monitored by radioimmunoassay of pancreastatin and changes in [Ca2+]i by video imaging. The patch clamp technique was used to record whole-cell currents and membrane capacitance (reflecting exocytosis). 2. In the presence of 2 mM extracellular Ca2+, gastrin and PACAP induced secretion and raised [Ca2+]i. Without extracellular Ca2+ (or in the presence of La3+) no secretion occurred. The extracellular Ca2+ concentration required to stimulate secretion was 10 times higher for gastrin than for PACAP. Depletion of intracellular Ca2+ pools by thapsigargin had no effect on the capacity of gastrin and PACAP to stimulate secretion. 3. Gastrin-evoked secretion was inhibited 60-80 % by L-type channel blockers and 40 % by the N-type channel blocker omega-conotoxin GVIA. Combining L-type and N-type channel blockers did not result in greater inhibition than L-type channel blockers alone. Whole-cell patch clamp measurements confirmed that the ECL cells are equipped with voltage-dependent inward Ca2+ currents. A 500 ms depolarising pulse from -60 mV to +10 mV which maximally opened these channels resulted in an increase in membrane capacitance of 100 fF reflecting exocytosis of secretory vesicles. 4. PACAP-evoked secretion was reduced 40 % by L-type channel blockers but was not influenced by inhibition of N-type channels. SKF 96365, a blocker of both L-type and receptor-operated Ca2+ channels, inhibited PACAP-evoked secretion by 85 %. Combining L-type channel blockade with SKF 96365 abolished PACAP-evoked secretion. 5. The results indicate that gastrin- and PACAP-evoked secretion depends on Ca2+ entry and not on mobilisation of intracellular Ca2+. While gastrin stimulates secretion via voltage-dependent L-type and N-type Ca2+ channels, PACAP acts via L-type and receptor-operated Ca2+ channels.

1. A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells). 2. Tall hair cell depolarization resulted in changes in cell membrane capacitance (DeltaC(m)) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (I(Ca)) and DeltaC(m) were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around -10 mV. 3. Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed I(Ca) and blocked DeltaC(m). Conversely, the DHP agonist Bay K 8644 increased both I(Ca) and DeltaC(m) amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels.

1. We investigated the properties of Ca2+-sensitive steps in the cycling of synaptic vesicles by comparing the actions of Ca2+, Ba2+ and Sr2+ in the synaptic terminal of depolarizing bipolar cells isolated from the retina of goldfish. FM1-43 fluorescence and capacitance measurements demonstrated that exocytosis, endocytosis and vesicle mobilization were maintained when external Ca2+ was replaced by either Ba2+ or Sr2+. 2. The rapidly releasable pool of vesicles (RRP) was equivalent to 1.5 % of the membrane surface area when measured in the presence of 2.5 mM Ca2+, but only 0.4 % in 2.5 mM Sr2+. The relative sizes of the RRP in Ca2+, Sr2+ and Ba2+ were 1.0, 0.28 and 0.1, respectively. We conclude that a smaller proportion of docked vesicles are available for fast exocytosis triggered by the influx of Sr2+ or Ba2+ compared to Ca2+. 3. The slow phase of exocytosis was not altered when Ca2+ was replaced by Ba2+, but it was accelerated 1.6-fold in Sr2+. The peak concentrations of Ca2+, Sr2+ and Ba2+ (measured using Mag-fura-5) were approximately 4, approximately 14 and approximately 60 microM, respectively. The order of efficiency for the stimulation of slow exocytosis was Ca2+ approximately Sr2+ > Ba2+. 4. Exocytosis was prolonged after the influx of Sr2+ and Ba2+. Sr2+ was cleared from the synaptic terminal with the same time constant as Ca2+ (1.3 s), but Ba2+ was cleared 10-100 times more slowly. Although Ba(2+) stimulates the slow release of a large number of vesicles, it did so less efficiently than Ca2+ or Sr2+. 5. The recovery of the membrane capacitance was equally rapid in Sr2+ and Ca2+, demonstrating that the fast mode of endocytosis could be triggered by either cation.

1. Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca(2+)-induced exocytosis in single rat glucagon-secreting pancreatic alpha-cells. 2. Somatostatin decreased the exocytotic responses elicited by voltage-clamp depolarisations by 80 % in the presence of cyclic AMP-elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half-maximal within 22 s. 3. The inhibitory action of somatostatin was concentration dependent with an IC(50) of 68 nM and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to G(i1/2) and by antisense oligonucleotides against G proteins of the subtype G(i2). 4. Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L-type Ca(2+) channel blocker nifedipine. The size of the omega-conotoxin-sensitive and forskolin-independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L-type Ca(2+) channel-dependent exocytosis in alpha-cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5. The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP-PCP indicate that somatostatin acts by depriming of granules. 6. We propose that somatostatin receptors associate with L-type Ca(2+) channels and couple to G(i2) proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L-type Ca(2+) channels.

1. The ion selectivities of the Ca(2+) sensors for the two components of exocytosis in rat phaeochromocytoma (PC12) cells were examined by measurement of membrane capacitance and amperometry. The cytosolic concentrations of metal ions were increased by photolysis of caged-Ca(2+) compounds and measured with low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothiazole coumarin (BTC-5N). 2. The Ca(2+)-induced increases in membrane capacitance comprised two phases with time constants of 30--100 ms and 5 s. Amperometric events reflecting the exocytosis of large dense-core vesicles occurred selectively in the slow phase, even with increases in the cytosolic Ca(2+) concentration of > 0.1 mM. 3. The slow component of exocytosis was activated by all metal ions investigated, including Cd(2+) (median effective concentration, 18 pM), Mn(2+) (500 nM), Co(2+) (900 nM), Ca(2+) (8 microM), Sr(2+) (180 microM), Ba(2+) (280 microM) and Mg(2+) (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd(2+) (26 pM), Mn(2+) (620 nM), Ca(2+) (24 microM) and Sr(2+) (320 microM), but was only slightly increased by Ba(2+) (> 2 mM) and Co(2+) and not at all by Mg(2+). 4. The fast component, but not the slow component, was competitively blocked by Na(+) (median effective concentration, 44 mM) but not by Li(+), K(+) or Cs(+). Thus, the Ca(2+) sensor for the fast component of exocytosis is more selective than is that for the slow component; moreover, this selectivity appears to be based on ionic radius, with cations with radii of 0.84 to 1.13 A (1 A = 0.1 nm) being effective. 5. These data support a role for synaptotagmin--phospholipid as the Ca(2+) sensor for the exocytosis of large dense-core vesicles and they suggest that an additional Ca(2+)-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.

1. We used the patch-clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca(2+)](i)), and flash photolysis of caged Ca(2+) to study exo- and endocytosis in identified rat corticotrophs. 2. Exocytosis stimulated by depolarization pulses was typically followed by a 'slow' endocytosis that retrieved the membrane with a time constant of approximately 6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of 'slow' endocytosis was approximately 1.2 at [Ca(2+)](i) < 3 microM and increased to approximately 1.6 at [Ca(2+)](i) > 3 microM. 3. Whole-cell dialysis through a patch pipette did not affect the kinetics and the efficiency of 'slow' endocytosis, but the amplitude of exocytosis was reduced. 4. 'Slow' endocytosis did not require sustained [Ca(2+)](i) elevation and its kinetics was only weakly [Ca(2+)](i) dependent. Our results suggest that 'slow' endocytosis involves a Ca(2+) sensor with a high Ca(2+) affinity (approximately 500 nM). 5. At high [Ca(2+)](i) (> 10 microM), the 'slow' endocytosis was frequently preceded by a 'fast' endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. 6. Neither calmodulin nor calcineurin appeared to be the Ca(2+) sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 microM) or the calcineurin inhibitors cyclosporin A (1 microM) and calcineurin autoinhibitory peptide (1 mg ml(-1)). Ba(2+), a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of 'slow' endocytosis approximately 2-fold. 7. Non-hydrolysable analogues of GTP (GDP-beta-S) and ATP (ATP-gamma-S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis. 8. We suggest that the high Ca(2+) affinity of 'slow' endocytosis may be important for maintaining continuous cycles of exocytosis-endocytosis during sustained adrenocorticotropin secretion in corticotrophs.

We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 +/- 0.9 % and the fluorescence by 6.6 +/- 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner. Intracellular dialysis (0.5-4.5 min) with elevated Ca2+ (1.5-100 microM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 +/- 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 +/- 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. Sustained endocytosis was not blocked when the intracellular GTP (300 microM) was replaced with GTP[gamma]S. Replacing intracellular Ca2+ (100 microM) with Ba2+ (300 microM) or Sr2+ (200 microM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

Glucose-induced insulin secretion is determined by signals generated in the mitochondria. The elevation of ATP is necessary for the membrane-dependent increase in cytosolic Ca2+, the main trigger of insulin exocytosis. Beta cells depleted of mitochondrial DNA fail to respond to glucose while still secreting insulin in response to membrane depolarisation. This cell model resembles the situation of defective insulin secretion in patients with mitochondrial diabetes. On the other hand, infants with activating mutations in the mitochondrial enzyme glutamate dehydrogenase are characterised by hyperinsulinism and hypoglycaemia. We have recently proposed that glutamate, generated by this enzyme, participates in insulin secretion as a glucose-derived metabolic messenger. In this model, glutamate acts downstream of the mitochondria by sensitising the exocytotic process to Ca2+. The evidence in favour of such a role for glutamate is discussed in the present review.

An adequate understanding of secretion requires the measurement of exocytosis on the same time scale as that used for second messenger dynamics. To investigate the kinetics of ACh-evoked secretion in pancreatic acinar cells, exocytosis of zymogen granules was quantified by continuous, time-differential analysis of digital images. The validity of this method was confirmed by simultaneous fluorescence imaging of quinacrine-loaded zymogen granules. Basal rates of exocytosis were low (0.2 events min(-1)). ACh stimulated a biphasic increase in secretory activity, maximal rates exceeding 20 events min(-1) after 10 s of ACh application (10 microM). Over the next 15 s the rate of exocytosis fell to less than 4 events min(-1); then began a second phase of secretion that peaked 15 s later at approximately 11 events min(-1), but subsequently declined in the continued presence of agonist. Measurements of fura-2 fluorescence demonstrated a biphasic increase in intracellular [Ca2+] ([Ca2+]i). Comparison of the [Ca2+]i records and time-differential analysis revealed that the fall in exocytotic rate following the initial burst occurred despite the fact that [Ca2+]i remained high. The second phase of secretion depended on both [Ca2+]i and [ACh]. At 10 microM ACh there was a decrease in the steepness of the relationship between [Ca2+]i and exocytosis that led to an enhancement of the slow secretory phase. We propose that acinar cells contain two pools of secretory vesicles: a small pool of granules that is exocytosed rapidly, but is quickly depleted; and a reserve pool of granules that can be recruited by ACh in a process that is modulated by second messengers other than calcium.

1. The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo-1, or indo-1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and exocytosis (changes in membrane capacitance) in single, identified rat corticotrophs. 2. Exocytosis could be stimulated by extracellular Ca2+ entry (via voltage-gated Ca2+ channels). A train of depolarizations could exhaust the pool of readily releasable granules and the pool replenished with a time constant of 42 s (at 22-25 C). 3. Recordings from cells with 0.5 mM intracellular cAMP showed that the amplitude of the depolarization-triggered exocytosis, the Ca2+ sensitivity of exocytosis, as well as the rate of replenishment of the readily releasable pool, were similar to the controls. 4. Exocytosis could also be stimulated by intracellular Ca2+ release from the inositol 1,4, 5-trisphosphate (IP3)-sensitive store (via flash photolysis of caged IP3). At comparable [Ca2+]i, extracellular Ca2+ entry and intracellular Ca2+ release had similar efficacy in triggering exocytosis. 5. The rate of exocytosis triggered via depolarization or intracellular Ca2+ release was much faster than that triggered via uniform elevation of [Ca2+]i (Ca2+ dialysis or flash photolysis of caged Ca2+). 6. The above findings suggest that both intracellular Ca2+ release and voltage-gated extracellular Ca2+ entry generate a spatial Ca2+ gradient, such that the local [Ca2+] near the exocytic sites was approximately 3-fold higher than the mean cytosolic [Ca2+]. However, neither cAMP nor the spatial Ca2+ gradient generated during depolarization could account for the high efficacy of corticotropin-releasing hormone (CRH) in stimulating adrenocorticotropic hormone (ACTH) secretion from corticotrophs.

1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 microM and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

We employed the whole-cell recording technique in conjunction with fluorometry to measure cytosolic Ca(2+) concentration ([Ca(2+)](i)) and exocytosis (capacitance measurement) in single, identified rat gonadotrophs. Direct activation of G-protein (via intracellular dialysis of non-hydrolysable analogues of GTP, but not of GDP) triggered a slow rise in capacitance even in the presence of a fast intracellular Ca(2+) chelator. The broad-spectrum kinase inhibitors H7 and staurosporine did not prevent this Ca(2+)-independent exocytosis, ruling out the involvement of the cAMP and PKC pathways. AlF(4)(-), a potent stimulator of heterotrimeric G-proteins, failed to stimulate any exocytosis when the intracellular Ca(2+) store was depleted, implicating the involvement of AlF(4)(-)-insensitive G-protein(s). Maximal stimulation of Ca(2+)-independent exocytosis by GTP analogues did not reduce the number of readily releasable granules that were available subsequently for Ca(2+)-dependent release. The last finding raises the possibility that the G-protein-stimulated Ca(2+)-independent exocytosis may regulate a pool of granules that is distinct from the Ca(2+)-dependent pool.

1. A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)-induced amylase secretion. 2. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose-response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). 3. The CCh-induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3-sensitive stores was relatively ATP independent. 4. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca(2+)-free medium evoked a second rapid and large peak of amylase secretion. 5. Phorbol 12,13-dibutyrate (PDBu) potentiated the CCh-induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh-induced [Ca2+]i changes. 6. PKC inhibitors such as Ro 31-8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. 7. These results suggest that a CCh-induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP-dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh-induced sustained plateau of amylase secretion. PDBu-induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

1. Tight-seal whole-cell patch clamp experiments were performed to examine the ability of different intracellular Ca2+ mobilising agents to activate the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL-1) cells under conditions of weak cytoplasmic Ca2+ buffering. 2. Dialysis with a maximal concentration of inositol 1,4,5-trisphosphate (IP3) routinely failed to activate macroscopic ICRAC in low buffer (0.mM EGTA, BAPTA or dimethyl BAPTA), whereas it activated the current to its maximal extent in high buffer (10 mM EGTA). Dialysis with a poorly metabolisable analogue of IP3, with ionomycin, or with IP3 and ionomycin all failed to generate macroscopic ICRAC in low Ca2+ buffering conditions. 3. Dialysis with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin was able to activate ICRAC even in the presence of low cytoplasmic Ca2+ buffering, albeit at a slow rate. Exposure to IP3 together with the SERCA blockers thapsigargin, thapsigargicin or cyclopiazonic acid rapidly activated ICRAC in low buffer. 4. Following activation of ICRAC by intracellular dialysis with IP3 and thapsigargin in low buffer, the current was very selective for Ca2+ (apparent KD of 1 mM) Sr2+ and Ba2+ were less effective charge carriers and Na+ was not conducted to any appreciable extent. The ionic selectivity of ICRAC was very similar in low or high intracellular Ca2+ buffer. 5. Fast Ca2+-dependent inactivation of ICRAC occurred at a similar rate and to a similar extent in low or high Ca2+ buffer. Ca2+-dependent inactivation is not the reason why macroscopic ICRAC cannot be seen under conditions of low cytoplasmic Ca2+ buffering. 6. ICRAC could be activated by combining IP3 with thapsigargin, even in the presence of 100 microM Ca2+ and the absence of any exogenous Ca2+ chelator, where ATP and glutamate represented the only Ca2+ buffers in the pipette solution. 7. Our results suggest that a threshold exists within the IP3-sensitive Ca2+ store, below which intraluminal Ca2+ needs to fall before ICRAC activates. Possible models to explain the results are discussed.

1. We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerizes cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 microM Ca2+i increased Cm by 23 +/- 4 % (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 +/- 5 % (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 +/- 0.85 fF s-1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s-1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 microM Ca2+i decreased Cm by 9 +/- 3 % (n = 7), in CST-treated cells Cm increased by 11 +/- 3 % (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s-1; CST treatment: 1-2 fF s-1). 4. Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis.

1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.

1. The giant presynaptic terminal of chick ciliary ganglion was used to examine how protein kinase C (PKC) modulates neurotransmitter release. Cholinergic excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp. 2. Although the EPSC was potentiated by phorbol ester (phorbol 12-myristate 13-acetate, PMA; 0.1 microM) in a sustained manner, the nicotine-induced current was unaffected. PMA increased the quantal content to 2.4 +/- 0.4 (n = 9) of control without changing the quantal size. 3. The inactive isoform of PMA, 4alpha-PMA, showed no significant effect on EPSCs. The PMA-induced potentiation was antagonized by two PKC inhibitors with different modes of action, sphingosine (20 microM) and bisindolylmaleimide I (10 microM). 4. When stimulated by twin pulses of short interval, the second EPSC was on average larger than the first EPSC (paired-pulse facilitation; PPF). PMA significantly decreased the PPF ratio with a time course similar to that of the potentiation of the first EPSC. 5. PMA did not affect resting [Ca2+]i or the action potential-induced [Ca2+]i increment in the giant presynaptic terminals. 6. The effect of PMA was less at 10 mM [Ca2+]o than at 1 mM [Ca2+]o. 7. When a train of action potentials was generated with a short interval, the EPSC was eventually depressed and reached a steady-state level. The recovery process followed a simple exponential relation with a rate constant of 0.132 +/- 0.029 s-1. PMA did not affect the recovery rate constant of EPSCs from tetanic depression. In addition, PMA did not affect the steady-state EPSC which should be proportional to the refilling rate of the readily releasable pool of vesicles. 8. These results conflict with the hypothesis that PKC upregulates the size of the readily releasable pool or the number of release sites. PKC appears to upregulate the Ca2+ sensitivity of the process that controls the exocytotic fusion probability.

1. We have reported previously that in mouse pancreatic beta-cells H2O2 hyperpolarizes the membrane and increases the ATP-sensitive K+ current recorded in the perforated patch configuration of the patch-clamp technique. The present study was undertaken to elucidate the underlying mechanisms. 2. The intracellular ATP concentration measured by chemoluminescence was reduced by H2O2. The ADP concentration increased in parallel during the first 10 min, resulting in a pronounced decrease in the ATP/ADP ratio. 3. Consistent with these results, glucose-stimulated insulin secretion from isolated islets was inhibited by H2O2. 4. Membrane hyperpolarization measured with intracellular microelectrodes in intact islets and inhibition of insulin secretion were counteracted by tolbutamide, indicating that the channels are still responsive to inhibitors and that the ATP concentration is not too low to trigger exocytosis. However, the sensitivity of the beta-cells to tolbutamide was reduced after treatment with H2O2. 5. H2O2 increased the intracellular Ca2+ activity ([Ca2+]i) in a biphasic manner. A first transient rise in [Ca2+]i due to mobilization of Ca2+ from intracellular stores was followed by a sustained increase, which was at least partly dependent on Ca2+ influx. The first phase seems to reflect Ca2+ mobilization from mitochondria. 6. Our results demonstrate that H2O2 interferes with glucose metabolism, which influences the membrane potential and ATP-sensitive K+ current via the intracellular concentration of ATP. These events finally lead to an inhibition of insulin secretion despite an increase in [Ca2+]i.

1. Vesicular secretion from single human umbilical vein endothelial cells (HUVECs) was monitored by changes in membrane capacitance (Cm). Secretion was evoked by dialysis with strongly buffered intracellular free Ca2+ concentrations ([Ca2+]i), flash photolysis of Ca2+-loaded DM-nitrophen or caged InsP3, or by thrombin. [Ca2+]i was monitored spectrofluorimetrically with furaptra. The results show that a large, slowly rising component of vesicular secretion requires prolonged exposure to high [Ca2+]i. 2. Cm increased during intracellular perfusion with [Ca2+] buffered in the range 1.0-20 microM. Changes in Cm comprised an initial slowly rising small component of 0.1-0.5 pF followed by a faster rising larger component of up to approximately 7 pF, seen when [Ca2+]i > 2 microM and which was maximal at 10-20 microM Ca2+. 3. Thrombin evoked rapid initial elevations of [Ca2+]i to a peak of 7.1 +/- 1.5 microM (mean +/- s.e. m., n = 5) that declined within approximately 20-30 s with thrombin present either to resting levels or to a maintained elevated level of 2.0 +/- 0.7 microM (mean +/- s.e.m., range 1.0-3.6 microM, n = 3). Transient [Ca2+]i rises were associated with small, slowly rising increases in Cm of 0.1-0.2 pF, that recovered to pre-application levels over 2-3 min. Maintained elevations of [Ca2+]i caused larger, faster-rising sustained increases in Cm to 1.14 +/- 0.12 pF (mean +/- s.e.m., n = 3). Separate specific enzyme-linked immunosorbent assay (ELISA) showed that 1.0 U ml-1 thrombin produced secretion of von Willebrand factor in HUVEC cultures. 4. Short-lived [Ca2+]i elevations with a peak of 3-25 microM and a duration of approximately 20 s generated by flash photolysis of caged InsP3 or DM-nitrophen produced either no net change in Cm, or small slow increases of approximately 0.1-0.6 pF at up to 5 fF s-1 that recovered to pre-flash levels over 2-3 min. 5. Maintained elevations of [Ca2+]i in the range 1-28 microM produced by flash photolysis of DM-nitrophen caused large increases in Cm, up to approximately 4 pF, corresponding to approximately 25-30 % of the initial cell Cm. The maximum rate of change of Cm was up to 50 fF s-1 at steady [Ca2+] up to 20 microM; Cm recovered towards pre-flash levels only when [Ca2+] had declined.

1. The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo-1, or indo-1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2. Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via alpha-adrenergic receptors and the guanosine trisphosphate (GTP) binding protein-coupled phosphoinositide pathway. 3. Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA-induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 +/- 0.3 % (n = 6). 4. When Ca2+ was directly released from the inositol 1,4, 5 trisphosphate (IP3)-sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 +/- 0.5 % (n = 6). Thus, NA-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release. 5. Large and rapid elevation of [Ca2+]i (>15 microM) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in approximately 100 ms and a slow burst that continued for many seconds. 6. The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 +/- 0.1 % (n = 14). 7. We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.

1. Changes in cell capacitance were monitored in whole-cell patch-clamp recordings from calf adrenal chromaffin cells using a software-based phase-tracking technique. Rapid endocytosis and exocytosis were observed in extracellular solutions containing either Ca2+ or Ba2+. 2. There was no significant difference in the magnitude or the time course of rapid endocytosis of cells stimulated in Ca2+ as compared to Ba2+. When cells were pretreated with caffeine and thapsigargin in order to deplete intracellular Ca2+ stores, rapid endocytosis in Ba2+ was not affected. This indicates that Ba2+ itself is capable of supporting rapid endocytosis. 3. The application of the calmodulin inhibitor calmidazolium via the intracellular pipette solution did not inhibit rapid endocytosis. Although our findings are inconsistent with an immediate requirement for calmodulin in rapid endocytosis, they do not rule out an involvement on a longer time scale. 4. While rapid endocytosis was not affected by the substitution of Ca2+ with Ba2+, the maximum rate of exocytosis was higher in cells stimulated in Ca2+ than in Ba2+. Since Ba2+ currents were much larger than Ca2+ currents during depolarizations to +10 mV (the test potential used in these experiments), Ba2+ appears to be less efficient at promoting exocytosis than Ca2+.

1. Using whole-cell patch-clamp recordings of NMDA EPSCs from co-cultured rat hippocampal (CA region) neurones, developmental changes in the kinetic and pharmacological properties of synaptic NMDA receptors were investigated. During in vitro differentiation a fast decaying component increasingly contributed to NMDA EPSCs. 2. Extracellular Mg2+ (1 mM) strongly blocked NMDA EPSCs at all stages in culture. Using the NR2B subunit-specific NMDA receptor antagonist ifenprodil (3 microM), we observed a developmental decrease in ifenprodil sensitivity of NMDA EPSCs. This suggests developmental changes in the expression of NMDA receptor subtypes. 3. To transiently block presynaptic exocytosis, we incubated presynaptic explants with tetanus toxin (TeTx) prior to cultivation. In TeTx-pretreated cultures the occurrence of fast decaying components of NMDA EPSCs and the developmental decrease in ifenprodil sensitivity was inhibited. Our results indicate a regulatory role of presynaptic exocytosis in the expression of NMDA receptor subtypes.

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.

1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.

1. The mechanisms by which cAMP stimulates Ca(2+)-dependent insulin secretion were investigated by combining measurements of whole-cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B-cells maintained in tissue culture. 2. Cyclic AMP stimulated exocytosis > 4-fold in whole-cell experiments in which secretion was evoked by intracellular dialysis with a Ca(2+)-EGTA buffer with a [Ca2+]i of 1.5 microM. This effect was antagonized by inhibitors of protein kinase A (PKA). 3. Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (> or = 60 nM), but was without effect in the complete absence of Ca2+. 4. Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6-fold PKA-dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated-patch whole-cell recordings. 5. Exocytosis triggered by single depolarizations in standard whole-cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition. 6. When exocytosis was triggered by Ca2+ released from Ca(2+)-NP-EGTA ('caged Ca2+'), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA-independent phase and a late PKA-dependent component. 7. We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.

1. The giant presynaptic terminal of chick ciliary ganglion was used to examine how noradrenaline (NA) modulates neurotransmitter release. The cholinergic excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp of the postsynaptic neuron. 2. Although the EPSC was potentiated by NA, the current directly activated by acetylcholine (IACh) was unaffected. NA also increased the quantal contents without changing the quantal size. 3. The NA-dependent potentiation was antagonized by neither phentolamine nor propranolol. The EPSC was also potentiated by adrenaline and dopamine but not by normetanephrine, phenylephrine or isoprenaline. The EPSC was attenuated by clonidine. Therefore, NA potentiated the transmitter release through a receptor pharmacologically different from both alpha- and beta-adrenergic receptors. 4. The Ca2+ increment produced by an action potential (delta[Ca2+]pre) was reduced by NA through an alpha 2-adrenergic receptor. However, when alpha 2-adrenergic receptors were blocked, neither delta[Ca2+]pre nor resting Ca2+ were changed by NA. 5. The [Ca2+]o-EPSC relation was shifted by NA, decreasing the half-saturating [Ca2+]o, without changing the maximum. 6. It is concluded that NA-dependent potentiation of transmitter release was due to an increase in the Ca2+ sensitivity of the exocytotic process. The enhancement of the fusion probability is suggested.

1. We have monitored changes in membrane capacitance (delta C) and conductance (delta G) induced by muscarinic acetylcholine stimulation in single rat pancreatic acinar cells. 2. Acetylcholine (ACh, 500 nM) induced simultaneous increases of delta C and delta G. In contrast, a low concentration (50 nM) of ACh exclusively induced delta C increases without delta G. These responses were abolished by the internal perfusion of heparin. This indicates that inositol 1,4,5-trisphosphate-mediated internal Ca2+ mobilization either simultaneously activates exocytosis and ion channels or exclusively initiates exocytosis. In comparison, a low concentration of A23187 selectively activated ion channels but a high concentration activated exocytosis and ion channels simultaneously. 3. These selective response patterns of delta C and delta G depend on the choice of agonist and the internal EGTA concentration. From this, we postulated two explanations for the selective action of muscarinic ACh stimulation on exocytosis. First, an area of high [Ca2+]i, spatially close to secretory granules, activates exocytosis. Second, an as yet unknown signalling factor sensitizes the Ca2+ affinity of the exocytotic apparatus.

1. Whole-cell patch recordings were used to examine the EPSC generated by the calyx of Held in neurones of the medial nucleus of the trapezoid body (MNTB). Each neurone receives a somatic input from a single calyx (giant synapse). 2. A slow NMDA receptor-mediated EPSC peaked in 10 ms and decayed as a double exponential with time constants of 44 and 147 ms. A fast EPSC had a mean rise time of 356 microseconds (at 25 degrees C), while the decay was described by a double exponential with time constants of 0.70 and 3.43 ms. 3. Cyclothiazide slowed the decay of the fast EPSC, indicating that it is mediated by AMPA receptors. The slower time constant was slowed to a greater extent than the faster time constant. Cyclothiazide potentiated EPSC amplitude, partly by a presynaptic mechanism. 4. The metabotropic glutamate receptor (mGluR) agonists, 1S,3S-ACPD, 1S,3R-ACPD and L-2-amino-4-phosphonobutyrate (L-AP4) reversibly depressed EPSC amplitude. A dose-response curve for 1S,3S-ACPD gave an EC50 of 7 microM and a Hill coefficient of 1.2. 5. Analysis of the coefficient of variation ratio showed that the above mGluR agonists acted presynaptically to reduce the probability of transmitter release. Adenosine and baclofen also depressed transmission by a presynaptic mechanism. 6. alpha-Methyl-4-carboxyphenylglycine (MCPG; 0.5-1 mM) did not antagonize the effects of 1S,3S-ACPD, while high concentrations of L-2-amino-3-phosphonopropionic acid (L-AP3; 1 mM) and 4-carboxy-3-hydroxyphenyglycine (4C3HPG; 500 microM) depressed transmission. 7. There was a power relationship between [Ca2+]o and EPSC amplitude with co-operativity values ranging from 1.5 to 3.4. 8. The mechanism by which mGluRs modulate transmitter release appeared to be independent of presynaptic Ca2+ or K+ currents, since ACPD caused no change in the level of paired-pulse facilitation or the duration of the presynaptic action potential (observed by direct recording from the terminal), indicating that the presynaptic mGluR transduction mechanism may be coupled to part of the exocytotic machinery. 9. Our data are not consistent with the presence at the calyx of Held of any one known mGluR subtype. Comparison of the time course and pharmacology of the fast EPSC with data from cloned AMPA receptors is consistent with the idea that GluR-Do subunits dominate the postsynaptic channels.