Humanin derivative, HNG, enhances neurotransmitter release.

BACKGROUND: Humanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated. METHODS: The extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells. RESULTS: The hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells. CONCLUSIONS: These findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions. GENERAL SIGNIFICANCE: The regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.

8-Nitro-cGMP modulates exocytosis in adrenal chromaffin cells.

Nitric oxide (NO)-mediated production of cyclic guanosine 3',5'-monophosphate (cGMP) is a crucial signaling pathway that controls a wide array of neuronal functions, including exocytotic neurotransmitter release. A novel nitrated derivative of cGMP, 8-nitro-cGMP, not only activates cGMP-dependent protein kinase (PKG), but also has membrane permeability and redox activity to produce superoxide and S-guanylated protein. To date, no studies have addressed the effects of 8-nitro-cGMP on exocytotic kinetics. Here, we aimed to assess the 8-nitro-cGMP-mediated modulation of the depolarization-evoked catecholamine release from bovine chromaffin cells. 8-Nitro-cGMP was produced in bovine chromaffin cells dependent on NO donor. Amperometric analysis revealed that 8-nitro-cGMP modulated the kinetic parameters of secretory spikes from chromaffin cells, particularly decreased the speed of individual spikes, resulting in a reduced amperometric spike height, slope ß, and absolute value of slope γ. The modulatory effects were independent of the PKG signal and superoxide production. This is the first study to demonstrate that 8-nitro-cGMP modulates exocytosis and provide insights into a novel regulatory mechanism of exocytosis.

Involvement of the α(1D)-adrenergic receptor in methamphetamine-induced hyperthermia and neurotoxicity in rats.

Methamphetamine (METH) is a psychostimulant that damages nigrostriatal dopaminergic terminals, primarily by enhancing dopamine and glutamate release. α1-adrenergic receptor (AR) subtype involved in METH-induced neurotoxicity in rats was investigated using selective α1-AR antagonists. METH neurotoxicity was evaluated by (1) measuring body temperature; (2) determining tyrosine hydroxylase (TH) immunoreactivity levels; (3) examining levels of dopamine and its metabolites; and (4) assessing glial fibrillary acidic protein (GFAP) and microglial immunoreactivity in the striatum. METH caused a decrease in dopamine and TH levels and induced hyperthermia which is an exacerbating factor of METH neurotoxicity. Concurrently, METH increased GFAP expression and the number of activated microglia. Pretreatment with prazosin, a nonselective α1-AR antagonist, completely abolished METH-induced decrease in both dopamine and TH and caused a partial reduction in hyperthermia. Prazosin also prevented METH-induced increase in both GFAP expression and the number of activated microglia. In vivo microdialysis analysis revealed that prazosin, however, does not alter the METH-induced dopamine release in the striatum. The neuroprotective effects of prazosin could be mimicked by a selective α(1D) antagonist, BMY 7378, but not by selective α(1A) or α(1B) antagonists. These results suggest that the α(1D)-AR is involved in METH-induced hyperthermia and neurotoxicity in rats.

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species.

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.

Dissociation of inositol polyphosphates from the C2B domain of synaptotagmin facilitates spontaneous release of catecholamines in adrenal chromaffin cells. A suggestive evidence of a fusion clamp by synaptotagmin.

Synaptotagmins (Syts) serve as a Ca²+ sensor in the release of neurotransmitters and hormones. Inositol polyphosphates (InsPPs) such as Inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) bind to Ca²+-binding C2B domain of Syt I and II, and inhibit transmitter release. We have shown that the inhibition by InsPPs is reversed by Ca²+ in adrenal chromaffin cells, while a rapid accumulation of endogenous InsP5 and InsP6 upon depolarizing stimuli have been reported in these and some other cells. Such a rapid accumulation of InsPPs, if not all, might reflect their dissociation from C2B domain of Syt. To elucidate the functional relevance, we studied the effects of antibodies against C2A and C2B domains (anti-C2A Ab, anti-C2B Ab) on the accumulation of InsPPs induced by Ca²+ in digitonin-permeabilized adrenal chromaffin cells. Anti-C2B Ab by itself caused an accumulation of InsPPs in the permeabilizing medium, and increased spontaneous release of catecholamines (CA). Anti-C2A Ab abolished Ca²+-induced increase of InsPPs in cytosolic component, and inhibited Ca²+-evoked release of CA with little effect on the spontaneous release. Microinjection of InsP6 but not inositol hexakissulfate into intact chromaffin cells inhibited both spontaneous and nicotine-evoked exocytotic events. These results suggest that endogenous InsPPs bound to the C2B domain clamp spontaneous fusion of the docked or primed vesicles at resting level of intracellular Ca²+, and binding of Ca²+ to the C2A or/and C2B domain facilitate fusion dissociating InsPPs from Syt in adrenal chromaffin cells. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Characterization of exocytotic events from single PC12 cells: amperometric studies in native PC12h, DA-loaded PC12h and bovine adrenal chromaffin cells.

Exocytotic events from rat pheochromocytoma (PC12) cells were characterized by amperometric analysis. For single-cell amperometric recordings, PC12h cells cultured onto poly-L-lysine corted glass-base dish were incubated with 1 mM dopamine (DA) for 60 min. Amperometric recordings, with a carbon fiber microelectrode (5 mum diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 degrees C. To characterize a single exocytotic event that is detected as a single spike current, the spike number, spike parameters (rise time, middle width and area) and spike shape were analyzed. Exposure of DA-loaded PC12h cells to 60 mM KCl (1000 hps) for 5 min and for 4 s evoked a train of events with the event number of 114+/-19 (spikes/response for 5 min) and 12+/-3 (spikes/response for 15 s), respectively. We observed distinctive kinetics in the events (rise time=0.83+/-0.19 ms, middle width=2.89+/-0.62 ms, area=62+/-7.6 fC and the spikes with a "foot"=15.4+/-2.7% of total spikes). The number and mean height of the events were 3- to 4-fold higher than that in DA-unloaded cells, and the values of rise time and middle width in DA-loaded PC12h cells were approx. 5- and 10-fold less than those observed in cultured adrenal chromaffin cells. The successful application of amperometry to monitor DA released from secretory vesicles in DA-loaded PC12h cell suggest that this technique is applicable to characterize exocytotic events in neurons.

Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A.

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.

Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis.

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.