Munc18a, a mammalian neuronal homologue of<i>Saccharomyces cerevisiae</i> Sec1p protein, is essential for secretion, likely as result of its high affinity interaction with the target SNARE protein syntaxin 1a (where derived from SNAP receptor (the soluble <i>N</i>-ethylmaleimide-sensitive fusion protein)). However, this inhibits vesicle interactions that are required secretory vesicles to achieve competency membrane fusion. As such, regulation between Munc18a and may provide an important...

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding molecular mechanism interference in autophagy, development therapeutic strategies for correcting such defects obese individuals is challenging. Here we show that a chronic increase cytosolic calcium concentration hepatocytes and lipotoxicity attenuates autophagic flux by preventing fusion between autophagosomes lysosomes. As pharmacological approach restore...

Dynamin is a master regulator of membrane fission in endocytosis. However, function for dynamin immediately upon fusion has also been suspected from variety experiments that measured release granule contents. The role guanosine triphosphate hydrolase (GTPase) activity controlling pore expansion and postfusion topology was investigated using polarization optics total internal reflection fluorescence microscopy (pTIRFM) amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase resulted...

The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks interaction of syntaxin with vesicle SNARE proteins. However, this is simultaneously essential for final stages secretion evidenced by necessity Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) proper these organisms. As such, any event that regulates two proteins important secretion....

Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome energy barrier for fusion, have been extensively studied, regulation of itself, determined by specific membrane lipids, has largely overlooked. Our findings reveal novel function in reducing directly binding sequestering fusogenic lipids to sites fusion. We demonstrate interaction between Syntaxin1A lipid phosphatidic acid, addition multiple...

Ghrelin, a gastric peptide hormone, has been reported to regulate growth hormone secretion and energy homeostasis. Here we show that ghrelin promotes neural proliferation in vivo vitro the rat dorsal motor nucleus of vagus (DMNV). Ghrelin receptor mRNA immunoreactivity were detected tissues from DMNV. Systemic administration (130 nmol kg −1 ) significantly increased 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation DMNV adult rats with cervical vagotomy (BrdU positive cells; 27 ± 4 69 14 n = 5, P...

Cytoplasmic free calcium concentration ((Ca2+]i) was evaluated by dual-wavelength microspectrofluorometry of fura-2-loaded individual rat pancreatic acinar cells. Resting [Ca2+]i in unstimulated acini 94.1 +/- 4.1 nM. Stimulation with high concentrations cholecystokinin (CCK, 100 pM to 1 nM) led an immediate rise 400-1,000 nM followed a fall within 2-5 min plateau only slightly above the prestimulation level. Lower and more physiological CCK (1-30 pM), after latent period 60-90 s, induced...

The nerve endings of rat neurohypophyses were acutely dissociated and a combination pharmacological, biophysical biochemical techniques was used to determine which classes Ca 2+ channels on these central nervous system (CNS) terminals contribute functionally arginine vasopressin (AVP) oxytocin (OT) secretion. Purified neurohypophysial plasma membranes not only had single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also distinct another (MVIIC), affects both...

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (Cm) in nerve terminals acutely dissociated from neurohypophysis adult rats to investigate modulation Ca2+ currents and secretion by activation opioid receptors. Bath superfusion the kappa-opioid agonists U69,593 (0.3-1 microM), dynorphin A (1 or U50,488H (1-3 microM) reversibly suppressed peak amplitude 32. 7 +/- 2.7% (in 41 56 terminals), 37.4 5.3% 5 8 33.5 8.1% 10...

The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations Ca2+ signaling in sensory neurons. naturally occurring amino acid taurine functions as an osmolyte, antioxidant, modulator, inhibitory neurotransmitter, and analgesic such that its depletion diabetes predispose one pain. This study reports the effects replacement on hyperalgesia neuron homeostasis streptozotocin-diabetic (STZ-D) rats. Nondiabetic STZ-D...

Mutations that alter signaling through the mammalian target of rapamycin complex 1 (mTORC1), a well established regulator neuronal protein synthesis, have been linked to autism and cognitive dysfunction. Although previous studies role for mTORC1 as necessary enduring changes in postsynaptic function, here we demonstrate dendritic activation rat hippocampal neurons also drives retrograde mechanism promoting enhanced neurotransmitter release from apposed presynaptic terminals. This novel mode...

1. Electrophysiological measurements of Ca2+ influx using patch clamp methodology were combined with fluorescent monitoring the free intracellular calcium concentration ([Ca2+]i) to determine mechanisms regulation in isolated nerve endings from rat neurohypophysis. 2. Application step depolarizations under voltage resulted voltage‐dependent (ICa) and increase [Ca2+]i. The [Ca2+]i was proportional time‐integrated ICa for low loads but approached an asymptote at large loads. These data...

Digital-imaging microfluorimetry, together with microinjection of marker/messenger molecules, was utilized to investigate intercellular Ca2+ signaling in rat pancreatic acinar cells. Stimulation acini low concentrations secretagogues [< 100 pM cholecystokinin (CCK), < 1 microM carbachol (CCh)] resulted asynchronous but coordinated increases that appeared pass a "wavelike" fashion between In contrast, at higher supermaximal agonists (> 300 CCK, > CCh), which induce large "peak-and-plateau"...

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 -2, occur in mammals produce seven different isoforms via alternative splicing. Here, we map the structural differences between yeast homologue of m-tomosyn-1, Sro7, tomosyn genes/isoforms to identify domains critical regulation activity are outside soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling m-tomosyn-1 based on...

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using vivo fluorescence resonance energy transfer 3T3L1 adipocytes, co-immunoprecipitation, and vitro binding assays, we provide data indicate that also associates with nearly equal affinity mutant constitutively open (unfolded) state (L173A/E174A; LE). To bind conformation 4, found requires an interaction N...

Neurotransmitter release probability (P(r)) largely determines the dynamic properties of synapses. While much is known about role presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity unclear. One such protein, tomosyn, believed reduce P(r) by interfering with SNARE complex formation. Tomosyn enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low P(r), strong facilitation, and pre-synaptic...

Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct (SV) pools. However, the molecular pathways matching neural to SV dynamics release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate pool partitioning at rat hippocampal synapses. Our analysis is based monitoring changes defined pools via...

The present study expands the contemporary view of mitochondria as important participants in cellular Ca(2+) dynamics and provides evidence that regulate supply release-competent secretory granules. Using pharmacological probes to inhibit mitochondrial import, ability modulate activity single, patch-clamped bovine chromaffin cells was examined by simultaneously monitoring rapid changes membrane surface area (DeltaC(m)) cytosolic levels ([Ca(2+)](c)). Repetitive step depolarizations or action...

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent which activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, be rapidly selectively using an assay selective for GTPases. To examine effects Rac1 on secretion, constitutively active mutants (Rac1‐V12, Rac1‐L61) were transiently expressed cells....

The sustained increase in cytosolic free Ca2+ concentration ([Ca2+]i) during maximal stimulation of rat pancreatic acini with carbamylcholine (10(-5) M) was investigated individual acinar cells by microspectrofluorometric analysis fura-2. After the large initial [Ca2+]i from intracellular stores, remained significantly elevated as long stimulus applied. amplitude this plateau dependent on median ([Ca2+]o) being 45-50 nM above prestimulation medium 1 mM [Ca2+]o increasing to 90 at 10 [Ca2+]o....

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor essential to neurotransmitter release, in isolation forms closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of H3 domain cognate SNAREs. Munc18-1, member Sec1/Munc18 family, binds syntaxin1A, stabilizing this conformation. We used fluorescence resonance energy transfer (FRET) characterize Munc18-1/syntaxin1A intact cells....

Abstract Tomosyn is a cytoplasmic protein that was shown to bind Syntaxin1 and SNAP‐25 through an R‐SNARE domain, forming complex almost identical in structure the neuronal SNARE complex. inhibits exocytosis various cell types these effects were attributed direct competition between tomosyn’s domain Synaptobrevin/VAMP. In present study, we investigated contribution of different domains tomosyn its activity. We show mutant lacks entire potent inhibitor vesicle priming, similar full‐length...