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Glucagon-like peptide-1 receptor (GLP-1R) agonists’ ability to increase insulin secretion and promote satiety have led to their development for the treatment of type 2 diabetes and obesity; while these beneficial effects have been attributed to receptors in the pancreas and hypothalamus, the function and importance of extra-pancreatic GLP-1Rs in other organ systems is not well understood despite the possibilities for targeting these receptors for other indications. We have analysed the contribution of GLP-1R to the secretory responses of adrenal medulla, a key player in stress responses. Exendin-4 (Ex-4), a synthetic analogue of GLP-1, increased the synthesis and the release of catecholamines (CA) by increasing cAMP production, without the participation of EPAC (Exchange Proteins Activated by cAMP). Ex-4 increased CA synthesis by promoting the activation of tyrosine hydroxylase (TH), and moreover increased the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of glycaemia and blood pressure by sympathetic tissues.

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1Gq-protein-coupled receptors (GqPCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1H567Kcompletely abolished it.This is the first endogenous mammalian GqPCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. GqPCRs are therefore able to control exocytosis at the level of SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.

Nucleotides are released from bovine chromaffin cells and take part in a feedback loop to inhibit further exocytosis. To identify the nucleotide receptors involved, we measured the effects of a range of exogenous nucleotides and related antagonists on voltage-operated calcium currents (I(Ca)), intracellular calcium concentration ([Ca(2+)](i)), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y(12) receptor from chromaffin cells and determined its properties by coexpression in Xenopus laevis oocytes with inward-rectifier potassium channels made up of Kir3.1 and Kir3.4. In both systems, the agonist order of potency was essentially identical (2-methylthio-ATP approximately 2-methylthio-ADPATP approximately ADPUDP). alphabeta-Methylene-ATP and adenosine were inactive. UTP inhibited I(Ca) in chromaffin cells (pEC(50) = 4.89 +/- 0.11) but was essentially inactive at the cloned P2Y(12) receptor. The relatively nonselective P2 antagonist pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid blocked nucleotide responses in both chromaffin cells and X. laevis oocytes, whereas the P2Y(12)- and P2Y(13)-selective antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP (ARC69931MX) blocked responses to ATP in both chromaffin cells and X. laevis oocytes but not to UTP in chromaffin cells. These results identify the P2Y(12) purine receptor as a key component of the nucleotide inhibitory pathway and also demonstrate the involvement of a UTP-sensitive G(i/o) -coupled pyrimidine receptor.

1. Oxytocin and vasopressin secretion from the neurohypophysis (NHP) is evoked by strongly patterned bursts of action potentials. We studied excitation-secretion coupling in single isolated terminals of rat NHP using patch clamp and capacitance detection techniques. 2. The secretory response evoked by trains of depolarizing pulses consisted of two discrete phases. Ca2+ entry during pulses early in the train did not elicit secretion. Exocytotic responses began only after a characteristic amount of total Ca2+ entry called "threshold". 3. In the postthreshold secretory phase, exocytotic events occurred during or immediately after depolarizing pulses, indicating that the final Ca(2+)-dependent step is triggered by high Ca2+ concentrations near the plasma membrane that dissipate rapidly after channel closure. Secretion was sensitive to both the concentration and species of Ca2+ chelator. BAPTA, a Ca2+ chelator with rapid Ca2+ binding kinetics, was more effective than EGTA in diminishing secretion. 4. The "threshold" amount of Ca2+ was determined by the concentration, but not species, of Ca2+ chelator. The threshold value was constant even when Ca2+ entry parameters were varied over a broad range of current amplitudes, pulse durations, and number of pulses, indicating that it did not require high Ca2+ concentrations near the plasma membrane. 5. These results suggest that the secretory response to a train of pulses consists of a Ca(2+)-dependent preparatory step that must be completed before subsequent Ca2+ entry can elicit exocytosis. 6. Exocytotic responses during single trains showed strong depression at a step subsequent to Ca2+ entry. Recovery from depression required 30-60 sec. 7. The properties of threshold secretion observed in NHP terminals are discussed in terms of current models of secretion.

The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a “shell” rather than “microdomain” model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two- step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.

Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exoctyosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin networkthat excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0–50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca 2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0–50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.

P/Q-type (CaV2.1) and N-type (CaV2.2) Ca2+channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the CaV2.1 α1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native CaV2 channels and suggest that the ability of individual Ca2+channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.

Angiotensin II (AngII) receptors couple to a multitude of different types of G-proteins resulting in activation of numerous signaling pathways. In this study we examined the consequences of this promiscuous G-protein coupling on secretion. Chromaffin cells were voltage-clamped at −80 mV in perforated-patch configuration, and Ca2+-dependent exocytosis was evoked with brief voltage steps to +20 mV. Vesicle fusion was monitored by changes in membrane capacitance (ΔCm), and released catecholamine was detected with single-cell amperometry. Ca2+signaling was studied by recording voltage-dependent Ca2+currents (ICa) and by measuring intracellular Ca2+([Ca2+]i) with fura-2 AM.AngII inhibitedICa(IC50= 0.3 nm) in a voltage-dependent, pertussis toxin (PTX)-sensitive manner consistent with Gi/o-protein coupling to Ca2+channels. ΔCmwas modulated bi-directionally; subnanomolar AngII inhibited depolarization-evoked exocytosis, whereas higher concentrations, in spite ofICainhibition, potentiated ΔCmfivefold (EC50= 3.4 nm). Potentiation of exocytosis by AngII involved activation of phospholipase C (PLC) and Ca2+mobilization from internal stores. PTX treatment did not affect AngII-dependent Ca2+mobilization or facilitation of exocytosis. However, protein kinase C (PKC) inhibitors decreased the facilitatory effects but not the inhibitory effects of AngII on stimulus-secretion coupling. The AngII type 1 receptor (AT1R) antagonist losartan blocked both inhibition and facilitation of secretion by AngII. The results of this study show that activation of multiple types of G-proteins and transduction pathways by a single neuromodulator acting through one receptor type can produce concentration-dependent, bi-directional regulation of exocytosis.

We have used combined membrane capacitance measurements (Cm) and voltage-clamp recordings to examine the mechanisms underlying modulation of stimulus–secretion coupling by a Gi/o-coupled purinoceptor (P2Y) in adrenal chromaffin cells. P2Y purinoceptors respond to extracellular ATP and are thought to provide an important inhibitory feedback regulation of catecholamine release from central and sympathetic neurons. Inhibition of neurosecretion by other Gi/o-protein-coupled receptors may occur by either inhibition of voltage-operated Ca2+channels or modulation of the exocytotic machinery itself. In this study, we show that the P2Y purinoceptor agonist 2-methylthio ATP (2-MeSATP) significantly inhibits Ca2+entry and changes inCmevoked by single 200 msec depolarizations or a train of 20 msec depolarizations (2.5 Hz). We found that P2Y modulation of secretion declines during a train such that only ∼50% of the modulatory effect remains at the end of a train. The inhibition of both Ca2+entry and ΔCmare also attenuated by large depolarizing prepulses and treatment with pertussis toxin. Inhibition of N-type, and to lesser extent P/Q-type, Ca2+channels contribute to the modulation of exocytosis by 2-MeSATP. The Ca2+-dependence of exocytosis triggered by either single pulses or trains of depolarizations was unaffected by 2-MeSATP. When Ca2+channels were bypassed and exocytosis was evoked by flash photolysis of caged Ca2+, the inhibitory effect of 2-MeSATP was not observed. Collectively, these data suggest that inhibition of exocytosis by Gi/o-coupled P2Y purinoceptors results from inhibition of Ca2+channels and the Ca2+signal controlling exocytosis rather than a direct effect on the secretory machinery.

Regulated exocytosis involves the Ca 2+ -triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca 2+ -binding proteins [1]. The Ca 2+ -binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca 2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca 2+ called a yellow ‘cameleon' (Ycam2) – comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP – which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin–Ycam2) to monitor changes in Ca 2+ concentration ([Ca 2+ ]) at the secretory vesicle surface ([Ca 2+ ] gd ) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic β cells, apparent resting values of cytosolic [Ca 2+ ] and [Ca 2+ ] gd were similar throughout the cell. In MIN6 cells following the activation of Ca 2+ influx, the minority of vesicles that were within ∼1 μm of the plasma membrane underwent increases in [Ca 2+ ] gd that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca 2+ ] change. The ability to image both global and compartmentalised [Ca 2+ ] changes with recombinant targeted cameleons should extend the usefulness of these new Ca 2+ probes.

The coupling between divalent cations and exocytosis of large dense- cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are “stimulus- coupled” to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus- coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20–50 sec. “Stimulus- decoupled” exocytosis is slow (approximately 25–40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5–12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.

Stimulus-secretion coupling in bovine chromaffin cells was investigated with whole-cell patch-clamp recordings and capacitance detection techniques to monitor exocytosis in response to trains of depolarizing pulses. Two kinetically discrete modes of exocytotic responses were observed. In one mode, the first depolarization of a train elicited a large increase in membrane capacitance (Cm; mean approximately 70 fF). This secretory mode was characterized by small Ca2+ requirements, relative insensitivity to the pipette Ca2+ chelator concentration, and rapid depletion of the secretory response. This mode of stimulus-secretion coupling was labile and was seen only in response to the first and, occasionally, the second stimulus train of whole-cell recordings. The second type of exocytotic response persisted for the remainder of the whole-cell recordings and consisted of two distinct phases. During the earliest pulses of a stimulus train, Ca2+ entry did not evoke Cm increases. Instead, Cm responses were elicited by later pulses, despite diminished Ca2+ entry per pulse caused by Ca2+ channel inactivation. The secretory phase was initiated after a specific "threshold" amount of Ca2+ had entered the cell, which was determined by the concentration, but not the binding kinetics, of the Ca2+ chelator in the pipette. In both the early and the secretory phases, the response of the cell was proportional to cumulative Ca2+ entry, regardless of current amplitude, pulse duration, or number of pulses. Threshold-type secretory kinetics has been described previously in peptide-secreting neurohypophysial (NHP) nerve terminals (Seward et al., 1995). Secretory kinetics with minimal Ca2+ requirements has not been observed in that preparation. Chromaffin cells appear to possess a broader repertoire of stimulus-secretion coupling modes than NHP terminals.

Mast cells form an integral part of both innate and adaptive immunity, playing an important role in the inflammatory response. Adverse reaction to allergens can lead to activation of mast cells, causing degranulation and release of a range of pro-inflammatory mediators contributing to the onset of allergy. Mast cells have been further implicated in many other diseases associated with aberrant mediator release, including irritable bowel disease. Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins have been identified as essential proteins for the exocytosis of vesicles containing these mediators. Inhibiting secretion though disruption of SNARE-mediated vesicle fusion could provide a novel therapeutic strategy for a variety of inflammatory diseases.The aim of this study was to identify and characterise SNARE proteins involved in the release of inflammatory mediators in human mast cells. using LAD 2 human mast cells, PCR identified expression of a variety of Syntaxins and VAMPS, as well as the ubiquitously expressed SNAP 23.To study the roles of individual VAMPS in exocytosis a novel technique utilising pH sensitive pHluorins was developed. using VAMPS tagged with pHluorins, we have studied the cellular distribution of VAMP 3 and VAMP 8 containing vesicles, and their behaviour upon IgE stimulation in live cells. In unstimulated cells, VAMPS 3 and 8 were found to have distinct cellular distributions. Upon IgE stimulation both VAMP 3 and VAMP 8 containing vesicles translocate to the membrane and undergo membrane fusion, consistent with roles in exocytosis. However, their responses show distinct time courses and calcium dependences. The data presented here supports the notion that distinct vesicle pools, defined in part by expression of VAMP 3 and VAMP 8, regulate the release of inflammatory mediators from mast cells.

The traditional function of neurotransmitter-gated ion channels is to induce rapid changes in electrical activity. Channels that are Ca(2+)-permeable, such as N-methyl-D-aspartate receptors at depolarized membrane potentials, can have a broader repertoire of consequences, including changes in synaptic efficacy, developmental plasticity, and excitotoxicity. Neuronal nicotinic receptors for acetylcholine (nAChRs) are usually less Ca(2+)-permeable than N-methyl-D-aspartate receptors but have a significant Ca2+ permeability, which is greater at negative potentials. Here we report that in neuroendocrine cells, activation of nAChRs can trigger exocytosis at hyperpolarized potentials. We used whole-cell patch-clamp recordings to record currents and the capacitance detection technique to monitor exocytosis in isolated bovine chromaffin cells. Stimulation of nAChRs at hyperpolarized potentials (-60 or -90 mV) evokes a large current and a maximal capacitance increase corresponding to the fusion of approximately 200 large dense-core vesicles. The amount of exocytosis is controlled both by the Ca2+ influx through nAChRs and by a contribution from thapsigargin-sensitive Ca2+ sequestering stores. This is a form of neurotransmitter action in which activation of nAChRs triggers secretion through an additional coupling pathway that coexists with classical voltage-dependent Ca2+ entry.

Previously we demonstrated that activation of Gq protein-coupled H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine chromaffin cells, despite inhibiting Ca2+ influx through voltage-gated calcium channels (VGCCs). This potentiation is restricted to the immediately releasable pool (IRP) of vesicles coupled to VGCCs, and requires the priming protein Munc13-1. Doc2B is a calcium-binding protein, known to interact with Munc13 to promote exocytosis; whether it plays a role in agonist-regulated exocytosis is unknown. To address this question, we isolated chromaffin cells from Doc2B (+/+), (+/-) and (-/-) mice and examined the effects of PLC-coupled GPCR activation on depolarization-evoked exocytosis.We found that under control stimulus conditions, depolarization-evoked exocytosis, measured as changes in membrane capacitance, was not significantly different between Doc2B (+/+), (+/-) and (-/-) cells; similarly no differences were found between the sizes of the releasable vesicle pools (IRP, RRP, SRP). Equimolar replacement of extracellular Ca2+ by Ba2+ also revealed no significant differences in the synchronous and asynchronous phases of Ba2+-evoked exocytosis in Doc2B (+/+), (+/-) or (-/-) cells. Finally, although more than 80% of cells, independent of their genotype, responded to histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and, even in Doc2 (+/+) cells rarely observed (5/17 cells, (+/-): 5/24 cells, (-/-): 2/18). This is in stark contrast to bovine cells, where 80% of the cells show a 5-fold potentiation of exocytosis. Importantly, Doc2B (+/+), (+/-) and (-/-) cells responded to the DAG analog PMA with potentiation. Taken together, the results suggest that there are profound species differences in the coupling between GqPCRs and the exocytotic machinery in chromaffin cells and that Doc2B is not required for agonist-dependent potentiation of exocytosis in mice.