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James E. Melvin - One of the best experts on this subject based on the ideXlab platform.
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the apical anion exchanger slc26a6 promotes oxalate secretion by murine submandibular gland Acinar Cells
Journal of Biological Chemistry, 2018Co-Authors: Taro Mukaibo, Alvin T George, Manoocher Soleimani, Gary E. Shull, Takashi Munemasa, Duy T Tran, Xin Gao, Jesse L Herche, Chihiro Masaki, James E. MelvinAbstract:The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct Cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland Acinar Cells with no detectable immunostaining in the ducts. CHO-K1 Cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland Acinar Cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in Acinar Cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland Acinar Cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.
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ae4 slc4a9 anion exchanger drives cl uptake dependent fluid secretion by mouse submandibular gland Acinar Cells
Journal of Biological Chemistry, 2015Co-Authors: Gaspar Penamunzenmayer, Gary E. Shull, Marcelo A Catalan, Yusuke Kondo, Yasna Jaramillo, Frances Liu, James E. MelvinAbstract:Transcellular Cl− movement across Acinar Cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na+-K+-2Cl− cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl− above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl− uptake pathway concentrates Cl− ions in Acinar Cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl−/HCO3− exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4−/− mice. The decrease in salivation was not related to loss of Na+-K+-2Cl− cotransporter or Na+/H+ exchanger activity in Ae4−/− mice but correlated with reduced Cl− uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl−/HCO3− exchanger activity revealed that HCO3−-dependent Cl− uptake was reduced in the Acinar Cells of Ae2−/− and Ae4−/− mice. Moreover, Cl−/HCO3− exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl−/HCO3− exchanger activity in submandibular Acinar Cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular Acinar Cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.
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tmem16a encodes the ca2 activated cl channel in mouse submandibular salivary gland Acinar Cells
Journal of Biological Chemistry, 2010Co-Authors: Victor G Romanenko, Marcelo A Catalan, David A Brown, Ilva Putzier, Criss H Hartzell, Alan D Marmorstein, Mireya Gonzalezbegne, Jason R Rock, Brian D Harfe, James E. MelvinAbstract:Activation of an apical Ca2+-dependent Cl− channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland Acinar Cells to the Ca2+-gated Cl− currents generated by Tmem16A and Best2, members from two distinct families of Ca2+-activated Cl− channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 Cells produced Ca2+-activated Cl− currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca2+-activated Cl− current found in native salivary Acinar Cells. Best2−/− and Tmem16A−/− mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca2+-activated Cl− current in submandibular gland Acinar Cells from Best2-deficient mice were the same as in wild type Cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland Acinar Cells from Tmem16A−/− mice lacked a Ca2+-activated Cl− current and a Ca2+-mobilizing agonist failed to stimulate Cl− efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2−/− mice. Our results demonstrate that both Tmem16A and Best2 generate Ca2+-activated Cl− current in vitro with similar properties to those expressed in native Cells, yet only Tmem16A appears to be a critical component of the Acinar Ca2+-activated Cl− channel complex that is essential for saliva production by the submandibular gland.
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loss of hyperpolarization activated cl current in salivary Acinar Cells from clcn2 knockout mice
Journal of Biological Chemistry, 2002Co-Authors: Keith Nehrke, Gary E. Shull, Linda Richardson, Jorge Arreola, Havan Nguyen, Jodi Pilato, Gbolahan Okunade, Raymond B Baggs, James E. MelvinAbstract:ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl− current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl− current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of theClcn2 gene was generated. The resulting homozygousClcn2 −/− mice lacked detectable hyperpolarization-activated chloride currents in parotid Acinar Cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these Cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal inClcn2 −/− mice following in vivostimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid Acinar Cells from Clcn2 −/−mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl− channel in salivary Acinar Cells but is not essential for maximum chloride flux during stimulated secretion of saliva or Acinar cell volume regulation.
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salivary Acinar Cells from aquaporin 5 deficient mice have decreased membrane water permeability and altered cell volume regulation
Journal of Biological Chemistry, 2001Co-Authors: Carissa M Krane, James E. Melvin, Ha Van Nguyen, Linda Richardson, Jennifer E Towne, Thomas Doetschman, Anil G MenonAbstract:Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive Cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of Acinar Cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary Acinar Cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted byAqp5 −/− mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual Acinar Cells isolated from Aqp5 −/− mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual Acinar Cells fromAqp5 −/− mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in Acinar Cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.
Irene Schulz - One of the best experts on this subject based on the ideXlab platform.
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hormonal control of adp ribosyl cyclase activity in pancreatic Acinar Cells from rats
Journal of Biological Chemistry, 2003Co-Authors: Lutz Sternfeld, Andreas H Guse, Elmar Krause, Irene SchulzAbstract:Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different Cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic Acinar Cells by measuring the conversion of the β-NAD+ analogs 1,N 6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N 6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact Acinar Cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the Cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic Acinar Cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic Acinar Cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.
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high and small molecular weight gtp binding proteins in zymogen granule membranes of rat pancreatic Acinar Cells
Cellular Physiology and Biochemistry, 1992Co-Authors: Susanne Schnefel, Winfried Haase, Petra Zimmermann, Andre Profrock, Reinhard Jahn, Klaus Aktories, Stefan Zeuzem, Irene SchulzAbstract:We have detected high- and small-molecular-weight GTP-binding proteins (G- and smg-proteins, respectively) in zymogen granule membranes (ZGM) of rat pancreatic Acinar Cells. Gi-proteins wit
Hiromi Shimomura - One of the best experts on this subject based on the ideXlab platform.
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characterization of cysteine string protein in rat parotid Acinar Cells
Archives of Biochemistry and Biophysics, 2013Co-Authors: Hiromi Shimomura, Akane Imai, Tomoko NashidaAbstract:Abstract Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid Acinar Cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid Acinar Cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized Acinar Cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11–82, containing the J-domain only, nor GST-CSP183–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in Acinar cell exocytosis.
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exocyst subunits are involved in isoproterenol induced amylase release from rat parotid Acinar Cells
European Journal of Oral Sciences, 2012Co-Authors: Akane Imai, Sumio Yoshie, Tomoko Nashida, Maiko Hagatsujimura, Hiromi ShimomuraAbstract:Exocytosis of secretory granules in parotid Acinar Cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian Cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid Acinar Cells. The expression of mRNA for all eight exocyst subunits was detected in parotid Acinar Cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid Acinar Cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid Acinar Cells.
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relation of rab26 to the amylase release from rat parotid Acinar Cells
Archives of Oral Biology, 2006Co-Authors: Tomoko Nashida, Akane Imai, Hiromi ShimomuraAbstract:Summary Amylase secretion from rat parotid Acinar Cells is induced by the accumulation of cAMP in response to β-adrenergic agonists as well as by the elevation of intracellular Ca 2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised Acinar Cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca 2+ -induced release of amylase. Finally, in the early stage of β-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon β-adrenergic stimulation.
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the small gtpase rab27b regulates amylase release from rat parotid Acinar Cells
Journal of Cell Science, 2004Co-Authors: Akane Imai, Sumio Yoshie, Tomoko Nashida, Hiromi Shimomura, Mitsunori FukudaAbstract:Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine Cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland Acinar Cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid Acinar Cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid Acinar Cells.
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roles of munc18 3 in amylase release from rat parotid Acinar Cells
Archives of Biochemistry and Biophysics, 2004Co-Authors: Akane Imai, Tomoko Nashida, Hiromi ShimomuraAbstract:Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid Acinar Cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid Acinar Cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid Acinar Cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid Acinar Cells. Furthermore, stimulation of the Acinar Cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the Acinar Cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.
Wei Wei - One of the best experts on this subject based on the ideXlab platform.
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trypsinogen activation peptide induces hmgb1 release from rat pancreatic Acinar Cells
Open Medicine, 2017Co-Authors: Guoliang Wang, Yan Liu, Danhua Dui, Liang Bai, Yao Liu, Fei Tian, Wei WeiAbstract:AbstractBackgroundThe development of acute pancreatitis (AP) is associated with intracellular events in pancreatic Cells, as well as with early and late inflammatory responses; however, their underlying mechanisms remain unclear. This study investigated trypsinogen activation peptide (TAP)-induced release of high mobility group box-l (HMGB1) from pancreatic Acinar Cells and how ethyl pyruvate (EP) affects this release.MethodologyPancreatic Acinar Cells from Sprague Dawley rats were divided into control, TAP (administered TAP), and EP (administered TAP and EP) groups. Cells were collected at 3, 6, 12, and 24 hours after TAP administration to detect HMGB1 mRNA and protein levels using quantitative PCR (qPCR) and Western blotting, respectively.ResultsThe TAP and EP groups exhibited higher levels of HMGB1 mRNA and protein expression (P<0.05) than the control group. The HMGB1 mRNA and protein expression levels also increased with prolonged TAP activity (P<0.05)–especially at 12 and 24 hours (P<0.01)–and showed positive correlations with TAP activity duration (3, 6, 12, and 24 hours) (r=0.971, P<0.01; r=0.966, P<0.01, respectively).ConclusionTAP induces HMGB1 release from pancreatic Acinar Cells. A positive temporal link exists between early TAP activity and late HMGB1 expression in AP, and EP inhibits HMGB1 release.
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Trypsinogen activation peptide induces HMGB1 release from rat pancreatic Acinar Cells
De Gruyter, 2017Co-Authors: Wang Guoliang, Liu Yan, Dui Danhua, Bai Liang, Liu Yao, Tian Fei, Wei WeiAbstract:The development of acute pancreatitis (AP) is associated with intracellular events in pancreatic Cells, as well as with early and late inflammatory responses; however, their underlying mechanisms remain unclear. This study investigated trypsinogen activation peptide (TAP)-induced release of high mobility group box-l (HMGB1) from pancreatic Acinar Cells and how ethyl pyruvate (EP) affects this release
Akane Imai - One of the best experts on this subject based on the ideXlab platform.
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characterization of cysteine string protein in rat parotid Acinar Cells
Archives of Biochemistry and Biophysics, 2013Co-Authors: Hiromi Shimomura, Akane Imai, Tomoko NashidaAbstract:Abstract Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid Acinar Cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid Acinar Cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized Acinar Cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11–82, containing the J-domain only, nor GST-CSP183–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in Acinar cell exocytosis.
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exocyst subunits are involved in isoproterenol induced amylase release from rat parotid Acinar Cells
European Journal of Oral Sciences, 2012Co-Authors: Akane Imai, Sumio Yoshie, Tomoko Nashida, Maiko Hagatsujimura, Hiromi ShimomuraAbstract:Exocytosis of secretory granules in parotid Acinar Cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian Cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid Acinar Cells. The expression of mRNA for all eight exocyst subunits was detected in parotid Acinar Cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid Acinar Cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid Acinar Cells.
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relation of rab26 to the amylase release from rat parotid Acinar Cells
Archives of Oral Biology, 2006Co-Authors: Tomoko Nashida, Akane Imai, Hiromi ShimomuraAbstract:Summary Amylase secretion from rat parotid Acinar Cells is induced by the accumulation of cAMP in response to β-adrenergic agonists as well as by the elevation of intracellular Ca 2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised Acinar Cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca 2+ -induced release of amylase. Finally, in the early stage of β-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon β-adrenergic stimulation.
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the small gtpase rab27b regulates amylase release from rat parotid Acinar Cells
Journal of Cell Science, 2004Co-Authors: Akane Imai, Sumio Yoshie, Tomoko Nashida, Hiromi Shimomura, Mitsunori FukudaAbstract:Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine Cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland Acinar Cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid Acinar Cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid Acinar Cells.
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roles of munc18 3 in amylase release from rat parotid Acinar Cells
Archives of Biochemistry and Biophysics, 2004Co-Authors: Akane Imai, Tomoko Nashida, Hiromi ShimomuraAbstract:Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid Acinar Cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid Acinar Cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid Acinar Cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid Acinar Cells. Furthermore, stimulation of the Acinar Cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the Acinar Cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.
