Search

Research using membrane capacitance (C

Aim To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved, and to establish whether activation leads to secretion of the neuropeptide calcitonin‐gene related peptide (CGRP) and/or the translocation of NF‐κB. Methodology Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2 days. The effect of P. gingivalis LPS (20 μg/mL) on calcium signalling was assessed (by calcium imaging using Cal‐520 AM) in comparison to the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100 μM), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1 μM), and high potassium (60 mM KCl). TG cultures were pre‐treated with either 1 μM CLI‐095 to block Toll‐like receptor 4 (TLR4) signalling or with 3 μM HC‐030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF‐κB was investigated using immunocytochemistry. Data were analysed by one‐way analysis of variance, followed by Bonferroni’s post‐hoc test as appropriate. Results P. gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non‐neuronal cells (p

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.

With a prevalence of 15%, migraine is the most common neurological disorder and among the most disabling diseases, taking into account years lived with disability. Current oral medications for migraine show variable effects and are frequently associated with intolerable side effects, leading to the dissatisfaction of both patients and doctors. Injectable therapeutics, which include calcitonin gene–related peptide–targeting monoclonal antibodies and botulinum neurotoxin A (BoNT/A), provide a new paradigm for treatment of chronic migraine but are effective only in approximately 50% of subjects. Here, we investigated a novel engineered botulinum molecule with markedly reduced muscle paralyzing properties which could be beneficial for the treatment of migraine. This stapled botulinum molecule with duplicated binding domain—binary toxin-AA (BiTox/AA)—cleaves synaptosomal-associated protein 25 with a similar efficacy to BoNT/A in neurons; however, the paralyzing effect of BiTox/AA was 100 times less when compared to native BoNT/A following muscle injection. The performance of BiTox/AA was evaluated in cellular and animal models of migraine. BiTox/AA inhibited electrical nerve fiber activity in rat meningeal preparations while, in the trigeminovascular model, BiTox/AA raised electrical and mechanical stimulation thresholds in Aδ- and C-fiber nociceptors. In the rat glyceryl trinitrate (GTN) model, BiTox/AA proved effective in inhibiting GTN-induced hyperalgesia in the orofacial formalin test. We conclude that the engineered botulinum molecule provides a useful prototype for designing advanced future therapeutics for an improved efficacy in the treatment of migraine.

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated. Electronic supplementary material The online version of this article (doi:10.1007/s11302-016-9497-4) contains supplementary material, which is available to authorized users.

Inappropriate activation of mast cells via the Fcε RI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to Fcε RI‐initiated mediator release. However, until now the role of TRPC calcium channels in Fcε RI‐mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to Fcε RI‐mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to Fcε RI‐mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK‐3503A and GSK‐2934A, respectively) did not reveal evidence for TRPC6 contribution to Fcε RI‐mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1‐regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1‐KK 684‐685 EE ‐ TRPC1 gating mutant) failed to alter Fcε RI‐mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that Fcε RI‐mediated calcium influx through Orai is necessary for histamine and TNF α release but is differentially involved in the generation of cytokines and eicosanoids.

Background and purpose: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. Key results: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1–1000 µM) to cells clamped at −60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by αβmethylene ATP (10 µM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 µM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1–10 µM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1–5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 µM). P2X7-like responses were also evoked in LAD2 cells by 2′(3′)-0-(4-benzoylbenzoyl)ATP (300 µM). A P2X4-like current was evoked by 100 µM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 µM). Conclusion and implications: Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.

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.

Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.

Inappropriate activation of mast cells via the FceRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FceRI-initiated mediator release. However, until now the role of TRPC calcium channels in FceRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FceRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FceRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FceRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FceRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FceRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue. It is for this reason that, in this chapter, we provide detailed methods for the isolation and purification of human lung mast cells. However, it should be noted that the methods that are described in this chapter are generally applicable to the isolation of mast cells from different tissues, and this will also be discussed.

Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG-encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4±0.4 μM (mean±s.e.mean) and a Hill coefficient of 1.17±0.03 (n=5). 3 μM amitriptyline inhibited IHERG by 34±6% (n=3). The inhibition showed only weak voltage dependence. Using an ‘envelope of tails' comprised of pulses to +20 mV of varying durations, the τ of activation was found to be 155±30 ms for control and 132±26 ms for 3 μM imipramine (n=5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine-mediated HERG channel inhibition. Taking current measurements every second during a 10 s depolarizing pulse from −80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at −80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The τ of inactivation was 8±2 ms and 5.6±0.4 ms (n=5) in the absence and presence of 3 μM imipramine, respectively, and currents inactivated to a similar extent. Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose. Keywords: HERG, imipramine, amitriptyline, tricyclic antidepressant, ‘rapid' delayed rectifier, IKr, cardiac arrhythmia, long QT syndrome, potassium channel Introduction Imipramine and amitriptyline are agents belonging to the tricyclic class of antidepressants (TCAs), and are used for treatment of depression and other conditions including panic disorder, obsessive compulsive disorder, bulimia, and nocturnal enuresis (Gorman, 1996; Daly & Wilens, 1998). TCAs have a wide spectrum of actions including inhibition of noradrenaline and 5-hydroxytryptamine reuptake at nerve endings, as well as anti-histaminergic, anticholinergic activities, and block of sodium channels (Coupland et al., 1997; Stahl, 1998). Tricyclic antidepressants such as imipramine and amitriptyline have cardiovascular side effects including orthostatic hypotension, atrioventricular conduction delay, reduced heart rate variability in response to exercise, tachycardia, syncope, and lengthening of the QT interval (which may be associated with arrhythmias), particularly in cases with high dosages and in patients with concurrent cardiovascular disease (Coupland et al., 1997). Overdose of antidepressant agents (and particularly TCAs) is epidemiologically important from the standpoint of mortality (Henry, 1997). The cardiac actions of TCAs have been suggested to result from ‘quinidine-like' activity (Pentel & Benowitz, 1986). Quinidine, a type I antiarrhythmic, is known to cause slowing of phase 0 depolarization of the action potential resulting in slowing of conduction through the His-Purkinje system and myocardium. It is well-established that, in addition to its antiarrhythmic effects, quinidine can have pro-arrhythmic activity (Grace & Camm, 1998). Recent data suggest that quinidine and mexilitene (another type I antiarrhythmic), can also inhibit delayed rectifier potassium currents (IK; Balser et al., 1991; Mitcheson & Hancox, 1997; Po et al., 1999). The rapid component of the delayed rectifier potassium current (IKr) is widely accepted as being mediated by the HERG gene-product, and block of this current can lead to QT prolongation and its concomitant risk of the polymorphic ventricular tachyarrhythmia, torsade de pointes (Sanguinetti et al., 1995; Sanguinetti & Keating, 1997). Furthermore, a variety of pharmacological agents known to cause QT elongation with its associated risk of arrhythmia have been shown to block IHERG, including type III antiarrhythmics (Trudeau et al., 1995; Spector et al., 1996a; Busch et al., 1998), non-sedating antihistamines (Roy et al., 1996; Suessbrich et al., 1996), and antipsychotics (Suessbrich et al., 1997a). Imipramine has been shown to inhibit IKr in guinea-pig ventricular myocytes (Valenzuela et al., 1994), but the characteristics and concentration-dependence of the inhibition were not studied. In this study, therefore, we investigated the ability of the TCAs imipramine and amitriptyline to block heterologously expressed IHERG. The concentration-dependence and mechanism underlying inhibition were also studied.

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.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X 1 (or P2X 3 ) receptors had strong desensitization, whereas currents in cells expressing P2X 2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X 1 receptor of either one of two 34-residue segments from the P2X 2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X 2 receptor only by providing both these segments of the P2X 1 (or P2X 3 ) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

SummaryBackground Prostaglandin E2 (PGE2) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2-selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. Objective The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. Methods The effects of non-selective (PGE2, misoprostol), EP2-selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4-selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2-selective (PF-04418948, PF-04852946) and EP4-selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. Results Prostaglandin E2, EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4-selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2-selective antagonists caused rightward shifts in the PGE2 concentration–response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. Conclusions and Clinical Relevance Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.

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.

The best characterised calcium channel in immune cells is the store-operated calcium release activated calcium channel (CRAC); studies in heterologous expression systems and knockout mice have identified Orai and its regulator STIM1, an ER-resident calcium sensor, as the molecular constituents of CRAC. Members of the TRPC family are also likely to contribute to store-operated calcium entry (SOCE) in immune cells. The expression of Orai and TRPC channels in primary human mast cells has not yet been investigated, but is of key importance given the critical role these cells play in the pathophysiology of type-1 hypersensitivity reactions. Allergic activation occurs following cross-linking of the FceRI receptor by antigen-IgE complexes that, via PLC-γ signalling, leads to robust activation of SOCE. The resulting calcium signal stimulates secretion and production of pro-inflammatory mediators. The aim of this study was to characterize the biophysical and pharmacological properties of CRAC channels in human lung mast cells (HLMC) following SOCE.RT-PCR revealed mRNA expression for Orai1, Orai2, STIM1 and STIM2 but not Orai3 in HLMCs. Whole-cell patch clamp recordings of isolated, cultured HLMCs were carried out using a Cs-based internal solution, with added IP3 and BAPTA to activate SOCE. These conditions activated an inwardly rectifying current with Erev= 18±4mV (n=6), a value less positive than expected for a pure Orai/CRAC current. However, the current was potentiated in divalent-free solution 563.7±6% (n=6), as expected for Orai/CRAC currents, and was significantly attenuated 51.4±8% (n=6), by the Orai-specific inhibitor Synta66 (10μM) (Ng et al. 2008., Di Sabatino et al. 2009). Calcium imaging in HLMCs loaded with fura-2 AM showed that Synta66 also attenuated anti-IgE induced calcium signaling by 62.2%(n=14). Taken together the data suggest that both Orai and TRPC channels contribute to SOCE in HLMCs.

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.

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg2+. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg2+ (6 mM). Ion substitution experiments revealed cation selectivity and Ca2+ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg2+. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.

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.

Mast cells are important targets for the treatment of allergic diseases and anaphylaxis. Inappropriate and chronic activation of mast cells via the IgE receptor leads to the release of a range of pre-stored and newly synthesized inflammatory mediators and the symptoms of disease. Calcium influx is a critical regulator of mast cell signaling, with influx through ion channels absolutely required not only for the exocytosis of preformed mediators but also to direct the synthesis of eicosanoids, cytokines and chemokines.Studies in rodent and human mast cells have identified STIM-regulated Orai channels to be key players in initiating calcium influx and degranulation in antigen-stimulated mast cells; in rodent mast cells, a role for TRPC channels is also emerging. Here we report evidence for a role of TRPC channels in human mast cell signaling. Using gene microarray analysis, we find evidence for expression of multiple TRP family members in primary human lung mast cells and LAD2 cells; the expression of TRPC1 and TRPC6 was further confirmed by immunocytochemistry. Single cell fura-2 imaging experiments, showed that barium could partially substitute for calcium to support influx following antigen- and thapsigargin stimulation, consistent with a contribution of non-selective TRPC-like channels to store operated calcium entry in human mast cells. To investigate the potential involvement of TRPC1 in STIM-regulated store operated calcium signaling in human mast cells, LAD2 cells were transfected with the STIM1 (684KK685) TRPC1 gating mutant. Expression of the mutant, however failed to alter calcium signaling in either thapsigargin or antigen-stimulated cells. Further pharmacological and molecular experiments are currently being performed to further evaluate the putative role of other TRPC channels in human mast cell signaling.

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.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (-30 to -20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from -90 to +10 mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10-300 microM) and gadolinium (10-30 microM) as well as by high concentrations of nickel and cobalt. omega Conus toxin (1 microM) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3-10 microM) and antagonists, nifedipine (3-10 microM) and nimodipine (10 microM) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.

In the hippocampal slice preparation, perfusion with benzyl penicillin evokes multiple population spikes and spontaneous discharges. Doses of 0.25 to 2 mM of the drug produced this effect within 20–40 min. Cleavage products and analogues of benzyl penicillin, penicilloic acid, 6-aminopenicillanic add, cephalexin, thioproline and penicillamine, were devoid of such action. It is concluded that the structural requirements for epileptogenic action of penicillin include not only the β-lactam ring and side chain substitution on C-6, but also the thiazolidine ring.

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.