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51.
Jacques J. H. Hens Marina De Wit Wim E. J. M. Ghijsen A. G. Miriam Leenders †Hendrik W. G. M. Boddeke ‡Roland Kissmehl Victor M. Wiegant §Ulrich Weller Willem-Hendrik Gispen Pierre N. E. De Graan 《Journal of neurochemistry》1998,71(5):1978-1986
Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+ -regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+ /calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+ -induced release of [3 H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca2+ /calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles. 相似文献
52.
Lopez I Giner D Ruiz-Nuño A Fuentealba J Viniegra S Garcia AG Davletov B Gutiérrez LM 《Cell calcium》2007,41(6):547-558
Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis. 相似文献
53.
de la Sancha CU Martínez-Cadena G López-Godínez J Castellano LE Nishigaki T Nishisaki T Darszon A García-Soto J 《Biochemical and biophysical research communications》2007,364(3):470-475
Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (∼135 kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na+-H+ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR. 相似文献
54.
Leonardo E. Pelletán Laila Suhaiman Cintia C. Vaquer Matías A. Bustos Gerardo A. De Blas Nicolas Vitale Luis S. Mayorga Silvia A. Belmonte 《The Journal of biological chemistry》2015,290(15):9823-9841
Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization. 相似文献
55.
Lee M Chung S Uhm DY Park MK 《Biochemical and biophysical research communications》2005,334(4):1241-1247
The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+. 相似文献
56.
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis. 相似文献
57.
Chen X Li C Izumi T Ernst SA Andrews PC Williams JA 《Biochemical and biophysical research communications》2004,323(4):1157-1162
To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptide mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis. 相似文献
58.
Karim S Ramakrishnan VG Tucker JS Essenberg RC Sauer JR 《Biochemical and biophysical research communications》2004,324(4):1256-1263
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands. 相似文献
59.
The sodium bicarbonate cotransporter (NBC1) mediates bicarbonate reabsorption in the renal proximal tubule. NBC1 activity is stimulated by 10% CO2, however, the mechanism is poorly understood. Here, we examined the mechanism of NBC1 regulation by 10% CO2 using an immortalized human proximal tubule cell line (HK2). In cells exposed to 10% CO2, the cotransporter activity (measured as ΔpH/min) increased within minutes and this increase was maintained for 6 to 24 h. Early NBC1 stimulation was accompanied by increased NBC1 phosphorylation. Basolateral membrane NBC1 protein increased by 30 min and reached a maximum at 6 h. Increased NBC activity at 6 h was accounted for by increased NBC exocytosis to the basolateral membrane and not by decreased endocytosis. Latruncullin B (an actin cytoskeleton inhibitor) did not prevent CO2-induced stimulation, while nocodazole (a microtubule-disrupting agent) abrogated the stimulatory effect of 10% CO2. A significant increase in NBC1 mRNA expression level was observed at 6 h and maintained for 24 h. Total NBC1 protein increased at 12 to 24 h with 10% CO2 incubation and this effect was blocked by cycloheximide. In summary, the present study demonstrates that early activation of NBC1 activity by 10% CO2 was mediated by NBC1 phosphorylation. The stimulation of cotransporter activity observed at 6 h was due to exocytosis, while the late effect starting from 12 h was accounted for by increased protein synthesis.This revised version was published online in August 2005 with a corrected cover date. 相似文献
60.
Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases. 相似文献