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The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o-proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 ( ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/ PKC-, PLDs-, and epidermal growth factor receptor ( EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions ( OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. [ABSTRACT FROM AUTHOR]

A low-affinity Ca2+/H+-antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca2+-binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca2+/H+-antiport. We measured Ca2+/H+-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca2+-induced dissipation of the vesicular H+-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca2+/H+-antiport activity is detectable only in acidic organelles expressing functional synaptotagmin-1. In contrast, synaptotagmin-1-deficient cells - and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated - were devoid of any Ca2+/H+-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca2+ sequestration through a Ca2+/H+ antiport. [ABSTRACT FROM AUTHOR]

In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca2+ increase that develops after the activation of voltage- dependent Ca2+ channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source. The immediately releasable pool is a small group of ready-to-fuse vesicles, whose fusion is tightly coupled to Ca2+ entry through channels. Recent work indicates that such coupling is not produced by a random distribution between vesicles and channels, but would be the result of a specific interaction of immediately releasable vesicles with particular Ca2+ channel subtypes. The immediately releasable pool is able to sustain, with high efficiency, the secretion triggered by the small and localized Ca2+ gradients produced by brief depolarizations at low frequencies, like action potentials at basal conditions in adrenal chromaffin cells. [ABSTRACT FROM AUTHOR]

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC. [ABSTRACT FROM AUTHOR]

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense-core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full-collapse fusion and clathrin-mediated endocytosis as essential modes of exo-endocytosis. Kiss-and-run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo-endocytosis across a wide range of neurosecretory systems. Full-collapse fusion and kiss-and-run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss-and-run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high-frequency inputs. Full-collapse fusion and kiss-and-run will each contribute to maintaining cellular communication over a wide range of frequencies. [ABSTRACT FROM AUTHOR]

Abstract The specificity of Ca[sup 2+] signals is conferred in part by limiting changes in cytosolic Ca[sup 2+] to subcellular domains. Mitochondria play a major role in regulating Ca[sup 2+] in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca[sup 2+] increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 µm, 1 min) evoked a greater increase in cytosolic Ca[sup 2+] in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca[sup 2+] efflux, enhanced Ca[sup 2+] influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca[sup 2+] stores. Ca[sup 2+] uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca[sup 2+] influx. A subset of mitochondria close to a Ca[sup 2+] source may preferentially regulate Ca[sup 2+] microdomains, set the threshold for Ca[sup 2+]-induced toxicity and participate in local ATP production. [ABSTRACT FROM AUTHOR]

Investigates the neurotransmitter release through the proteolipid membrane sector of the V-adenosine triphosphatase. Presence of a proteolipid oligomer in synaptosomal membranes; Pre-concentration of the neurotransmitter in synaptic vesicles; Collapse of synaptic vesicles in the plasma membrane.

The secretion of neurotransmitters is a rapid Ca[sup 2+]-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 µs) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca[sup 2+] channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca[sup 2+] influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca[sup 2+] channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca[sup 2+] channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca[sup 2+] channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization. [ABSTRACT FROM AUTHOR]

Abstract: The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca2+, ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 μM), or tyramine (10 μM), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca2+ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer with out external Ca2+ ; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca2+. Amphetamine-induced spikes in Ca2+-free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and mitochondrial Ca2+ stores. Atomic absorption measurements of amphetamine- and methylamine-treated vesicles reveal that intravesicular Ca2+ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca2+ can escape and trigger exocytosis. [ABSTRACT FROM AUTHOR]