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1.
Arch Oral Biol ; 117: 104775, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32512258

ABSTRACT

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.


Subject(s)
Apoptosis , Chronic Periodontitis/pathology , Salivary Glands/pathology , Animals , B-Lymphocytes/cytology , Rats , Tumor Necrosis Factor-alpha/blood
2.
J Membr Biol ; 203(3): 119-26, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15986091

ABSTRACT

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.


Subject(s)
Aquaporins/metabolism , Membrane Proteins/metabolism , Parotid Gland/physiology , Secretory Vesicles/metabolism , Water-Electrolyte Balance/physiology , Animals , Aquaporin 5 , Male , Rats , Rats, Sprague-Dawley
3.
J Immunol ; 167(10): 5814-23, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11698455

ABSTRACT

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.


Subject(s)
Membrane Microdomains/metabolism , Membrane Microdomains/physiology , Phosphotyrosine/metabolism , Receptors, IgG/metabolism , Receptors, IgG/physiology , Superoxides/metabolism , Ubiquitin-Protein Ligases , beta-Cyclodextrins , Cells, Cultured , Cyclodextrins/pharmacology , Detergents/chemistry , Enzyme Inhibitors/pharmacology , HL-60 Cells , Humans , Phosphorylation/drug effects , Protein Transport , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Signal Transduction , Tretinoin/pharmacology , src-Family Kinases/metabolism
4.
Cell Calcium ; 30(2): 107-16, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11440468

ABSTRACT

In rabbit parotid acinar cells, the muscarinic cholinergic agonist methacholine induced an increase in the intracellular Ca(2+) concentration and provoked nitric oxide (NO) generation. Ca(2+)-mobilizing reagents such as thapsigargin and the Ca(2+) ionophore A23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the removal of extracellular Ca(2+). Immunoblot analysis indicated that the antibody against the neuronal type of nitric oxide synthase (NOS) cross-reacted with NOS in the cytosol of rabbit parotid gland cells. Immunofluorescence testing showed that neuronal NOS is present in the cytosol of acinar cells but less in the ductal cells. NOS was purified approximately 8100-fold from the cytosolic fraction of rabbit parotid glands by chromatography on Sephacryl S-200, DEAE-Sephacel, and 29,59-ADP-Sepharose. The purified NOS was a NADPH- and tetrahydroxybiopterin-dependent enzyme and was activated by Ca(2+) within the physiological range in the presence of calmodulin. These results suggest that NO is generated by the activation of the neuronal type of NOS, which is regulated in rabbit parotid acinar cells by the increase in intracellular Ca(2+) levels induced by the activation of muscarinic receptors.


Subject(s)
Calcium/metabolism , Nitric Oxide/metabolism , Parotid Gland/cytology , Parotid Gland/metabolism , Animals , Calcimycin/pharmacology , Calmodulin/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Immunoblotting , Ionophores/pharmacology , Kinetics , Methacholine Chloride/pharmacology , Microscopy, Fluorescence , Muscarinic Agonists/pharmacology , NADP/pharmacology , Nitric Oxide Synthase/metabolism , Protein Binding , Rabbits , Rats , Thapsigargin/pharmacology , Time Factors
5.
Biochemistry ; 40(4): 888-95, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11170409

ABSTRACT

Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.


Subject(s)
Antigens, Differentiation/metabolism , Enzyme Inhibitors/pharmacology , Gangliosides/pharmacology , N-Glycosyl Hydrolases/antagonists & inhibitors , NAD+ Nucleosidase/metabolism , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Antigens, CD/metabolism , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Membrane/metabolism , Enzyme Inhibitors/metabolism , Extracellular Space/drug effects , Extracellular Space/enzymology , Extracellular Space/metabolism , Flow Cytometry , Gangliosides/metabolism , Gangliosides/physiology , HL-60 Cells , Humans , Hydrolysis , Membrane Glycoproteins , N-Glycosyl Hydrolases/metabolism , Oligosaccharides/metabolism , Time Factors , Tumor Cells, Cultured
6.
J Theor Biol ; 204(2): 165-77, 2000 May 21.
Article in English | MEDLINE | ID: mdl-10887899

ABSTRACT

Amylase secretion from parotid acinar cells results from stimulus-regulated fusion of apical membrane and secretory granules that contain amylase. The time course of amylase secretion induced by various secretagogues has been reported. Calcium-mobilizing agonists such as carbamylcholine and substance P induce rapid and transient secretion while cAMP-mobilizing agonists such as isoproterenol cause long-term secretion. Combination of these two types of agonists results in a rapid and high rate of secretion. To explain the various time courses of these stimulations, it was assumed that amylase secretion is a consecutive reaction that consists of two first-order reactions. It was postulated that secretory granules were classified into three states: (A) pre-docked, (B) docked, and (C) fusion. The simple simulation could explain the time course of amylase secretion induced by various secretagogues by simply changing the rate constants for docking (reaction A to B) and fusion (reaction B to C) steps. It was also found that calcium mainly enhances the last fusion step and that cAMP activates the docking step. The amount of docked granules is estimated to be quite small, which accounts for why amylase secretion is regulated mainly by cAMP. The effects of the two types of secretagogues were synergistic, meaning that their intracellular signaling pathways are independent. At the same time, this also suggests that basal and enhanced secretion induced by two types of agonists have the same exocytotic process and that two stimuli independently activate the same machinery that mediates docking or fusion. This simulation is useful in analysis of the effects of secretion modulators and the molecular mechanism of amylase secretion.


Subject(s)
Amylases/metabolism , Computer Simulation , Cyclic AMP/pharmacology , Exocytosis/drug effects , Parotid Gland/metabolism , Calcium Signaling/drug effects , Cytoplasmic Granules/drug effects , Humans , Models, Biological , Parotid Gland/cytology , Parotid Gland/drug effects , Secretory Rate , Stimulation, Chemical
7.
J Biol Chem ; 274(33): 23642-6, 1999 Aug 13.
Article in English | MEDLINE | ID: mdl-10438547

ABSTRACT

Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Membrane Proteins/metabolism , Parotid Gland/metabolism , Vesicular Transport Proteins , Animals , Calcium/metabolism , Cell Membrane/metabolism , Cyclic AMP/metabolism , Enzyme Activation , Parotid Gland/cytology , Precipitin Tests , Protein Binding , Qa-SNARE Proteins , R-SNARE Proteins , Rats , SNARE Proteins
8.
Eur J Morphol ; 36 Suppl: 46-9, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9825892

ABSTRACT

Rat parotid acinar cells secrete amylase through the stimulation of beta-adrenoceptors followed by accumulation of intracellular cAMP. However, it remains unclear at the molecular level how secretory granules fuse with the apical membranes. We have examined whether SNARE proteins are involved in exocytosis in the salivary glands, and have found that one of the SNARE proteins, VAMP-2, is localized at the secretory granule membrane of rat parotid acinar cells. Moreover, botulinum neurotoxin B, which has endoprotease activity that cleaves VAMP-2, inhibited cAMP-dependent amylase release but did not inhibit basal secretion in the absence of cAMP. These results suggest that VAMP-2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. In contrast, both neurotoxins A and C1 (endoproteases that cleave SNAP-25 and syntaxin 1 respectively) failed to inhibit cAMP-dependent amylase release. Therefore, neither SNAP-25 nor syntaxin 1 are involved in amylase secretion in the parotid glands. Clarification of the mechanism of secretion will require the identification of proteins that interact and function cooperatively with VAMP-2. This approach may also reveal details of the molecular mechanism by which the cAMP facilitates secretion in other systems, including neurotransmission.


Subject(s)
Cyclic AMP/physiology , Exocytosis/physiology , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Salivary Glands/chemistry , Vesicular Transport Proteins , Animals , R-SNARE Proteins , SNARE Proteins , Salivary Glands/cytology , Salivary Glands/physiology , Syntaxin 1
9.
Eur J Morphol ; 36 Suppl: 186-9, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9825919

ABSTRACT

ADP-ribosylation factor (Arf) is a 20 kDa polypeptide that is a member of the Ras superfamily of small molecular mass GTP-binding proteins. In addition to an essential role of Arf1 in vesicle budding, recent observations suggest a role for Arf6 in calcium-dependent exocytosis in bovine adrenal chromaffin cells. In rat parotid acinar cells, exocytosis is cAMP-dependent and our findings suggest an interaction of Arf1 with the secretory granules. We describe here the structural and functional background to the Arf proteins focusing on their role in rat parotid acinar cells.


Subject(s)
Adenosine Diphosphate/metabolism , GTP-Binding Proteins/metabolism , Parotid Gland/cytology , Parotid Gland/metabolism , ADP-Ribosylation Factor 1 , ADP-Ribosylation Factors , Animals , Exocytosis/physiology , Rats
10.
Eur J Morphol ; 36 Suppl: 194-7, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9825921

ABSTRACT

Guanosine 3',5'-cyclic monophosphate (cGMP) is a second messenger generated in response to hormones or neurotransmitters in various tissues and cells. In parotid acinar cells, the activation of muscarinic cholinergic and beta-adrenergic receptors induces an increase in intracellular cGMP. However, the mechanism of cGMP production in parotid acinar cells has not been well elucidated. cGMP production is induced by the activation of guanylyl cyclases, which are directly activated by nitric oxide (NO). NO plays an important role as an inter- and intracellular signal molecule in various organs and cells. Biosynthesis of NO is catalyzed by NO synthase (NOS), and NO generation is controlled by the regulation of NOS activity, for example by Ca2+. We have studied the regulation of NOS activity, NO generation and cGMP production in rabbit parotid acinar cells, and have demonstrated a functional Ca2+-NO-cGMP signaling pathway.


Subject(s)
Calcium Signaling/physiology , Cyclic GMP/metabolism , Nitric Oxide/metabolism , Parotid Gland/cytology , Parotid Gland/physiology , Animals , Rabbits
11.
Cell Signal ; 10(6): 371-5, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9720759

ABSTRACT

The process of membrane fusion is separated into three steps: docking, priming and fusion. The last fusion step of most regulated exocytosis is triggered by cytosolic free calcium (Ca2+). However, enzyme secretion from the parotid salivary glands is regulated by the accumulation of intracellular cAMP, although Ca2+ does augment the cAMP-induced secretion. The difference of the regulatory mechanisms is thought to be due to the brake points that will be passed upon stimulations. Vesicles of Ca2+-regulated exocytosis such as neurotransmission and norepinephrine release from chromaffin cells are waiting for the stimulation docked to the plasma membrane, and Ca2+ triggers the membrane fusion after the priming. In contrast, secretory granules of parotid acinar cells begin exocytosis with the docking step that may be regulated by cAMP. After the start of the docking, the exocytotic process of enzyme release runs a similar course to that of the neurotransmission: the priming and the Ca2+-enhanced fusion steps. Therefore, there are probably some common mechanisms involving the SNARE proteins both in Ca2+-regulated exocytosis and cAMP-dependent secretion.


Subject(s)
Cyclic AMP/physiology , Exocytosis/physiology , Parotid Gland/metabolism , Vesicular Transport Proteins , Amylases/metabolism , Animals , Membrane Proteins/physiology , SNARE Proteins
12.
Arch Biochem Biophys ; 357(1): 147-54, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9721194

ABSTRACT

We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20. 5-kDa small-molecular-mass GTP-binding protein in the cytosolic fraction of rat parotid acinar cells was identified as ADP-ribosylation factor1 by using a pan-Arf monoclonal antibody and isotype-specific polyclonal antibodies for Arf proteins 1, 3, 5, and 6. Incubation of the cytosolic fraction with isolated secretory granule membranes in the presence of GTPgammaS resulted in the translocation of Arf1 from the cytosolic fraction to the secretory granule membranes. The translocation was not observed in the presence of GDPbetaS in place of GTPgammaS, indicating that the process is GTP-dependent. The immunoelectron microscopy experiment confirmed Arf1 is translocated to the secretory granules. A prior treatment of the granule membranes with trypsin inhibited the translocation of Arf1 at 2 mM Mg2+, but had no effect in the absence of Mg2+ (condition of spontaneous conversion of Arf-GDP to Arf-GTP). Thus, the trypsin-sensitive nucleotide exchange activity for Arf1 is probably associated with the secretory granule membranes. These results demonstrate Arf1 translocates to the secretory granules in rat parotid acinar cells.


Subject(s)
Carrier Proteins/metabolism , Cytoplasmic Granules/metabolism , GTP-Binding Proteins/metabolism , Parotid Gland/metabolism , ADP-Ribosylation Factor 1 , ADP-Ribosylation Factors , Animals , Biological Transport , Carrier Proteins/chemistry , Cattle , Cell Fractionation , Coatomer Protein , Cytoplasmic Granules/chemistry , GTP-Binding Proteins/chemistry , Guanine Nucleotides/metabolism , Humans , Male , Membrane Proteins/metabolism , Microscopy, Immunoelectron , Parotid Gland/chemistry , Parotid Gland/cytology , Rats , Rats, Sprague-Dawley
13.
Cell Calcium ; 23(6): 405-12, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9924632

ABSTRACT

We investigated the mechanism of guanosine 3',5'-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.


Subject(s)
Calcium/metabolism , Cyclic GMP/biosynthesis , Nitric Oxide/biosynthesis , Parotid Gland/metabolism , Aminoquinolines/pharmacology , Animals , Arginine/metabolism , Calcimycin/pharmacology , Calcium/pharmacology , Citrulline/metabolism , Enzyme Inhibitors/metabolism , Guanylate Cyclase/pharmacology , Methacholine Chloride/pharmacology , Models, Biological , NG-Nitroarginine Methyl Ester/pharmacology , Rabbits , Receptors, Muscarinic/metabolism , Thapsigargin/pharmacology , Time Factors
14.
J Biol Chem ; 271(22): 13130-4, 1996 May 31.
Article in English | MEDLINE | ID: mdl-8662834

ABSTRACT

Amylase exocytosis of the parotid gland is mediated by intracellular cAMP. To investigate whether cAMP-dependent secretion has a mechanism similar to that of regulated neuroexocytosis, we examined the expression of synaptosome-associated proteins. In rat parotid acinar cells, we found 25 (p25) and 18 kDa (p18) proteins reacted with antibodies against Rab3A and vesicle-associated membrane protein 2 (VAMP-2), respectively. On the other hand, syntaxin 1 and SNAP-25, which interact with VAMP-2 at synapses, were undetectable. Rab3A-like p25 and VAMP-2-like p18 were also expressed in other exocrine acinar cells. The latter was localized at secretory granule membranes, and the former was detected in secretory granule and cytosolic fractions. The antibody against VAMP-2 used in this study did not react with cellubrevin, and p18 was cleaved with botulinum neurotoxin B. Thus, we identified p18 as VAMP-2. Botulinum neurotoxin B inhibited the cAMP-induced amylase release from streptolysin O-permeabilized acinar cells. Therefore, VAMP-2 is required for cAMP-regulated amylase release in rat parotid acinar cells. This is the first report that VAMP-2 is involved in regulated exocytosis that is independent of Ca2+.


Subject(s)
Amylases/antagonists & inhibitors , Botulinum Toxins/pharmacology , Cyclic AMP/metabolism , Exocytosis , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Parotid Gland/drug effects , Amylases/metabolism , Animals , Cell Membrane/metabolism , Cytosol/metabolism , GTP-Binding Proteins/metabolism , Hydrolysis , Male , Parotid Gland/cytology , Parotid Gland/metabolism , R-SNARE Proteins , Rats , Rats, Sprague-Dawley , rab3 GTP-Binding Proteins
15.
Oncogene ; 10(6): 1119-23, 1995 Mar 16.
Article in English | MEDLINE | ID: mdl-7700637

ABSTRACT

During screening for inhibitors of ras-mediated differentiation of PC12 cells, trichostatin A (TSA) was isolated from the metabolites of Streptomyces as a potent inhibitor. TSA blocked both oncogenic ras- and NGF-induced neurite outgrowth from PC12 cells. However, addition of TSA 1 h after NGF-stimulation did not inhibit neuronal differentiation, suggesting that TSA affects an early step in the NGF-signaling pathway mediated by ras. Northern blotting analysis showed that TSA prolonged the maximum expression period of c=fos mRNA triggered by NGF and delayed its return to the basal level. TSA reduced c-jun mRNA induction by NGF but greatly enhanced c-myc mRNA induced by NGF. Yoshida et al. (J. Biol. Chem, 265, 17174-17179, 1990) showed that TSA inhibits histone deacetylation, which might influence the gene expression involved in cellular differentiation. In this study, we also found that TSA prevents histone deacetylation in PC12 cells as well as other cell lines, suggesting that inhibition of histone deacetylation by TSA might affect the expression of early-response genes. We also demonstrated that TSA induced reversion of oncogenic ras-transformed NIH3T3 cells to a normal morphology, suggesting that inhibitors of ras-mediated differentiation of PC12 cells may be effective as anticancer agents.


Subject(s)
Cell Transformation, Neoplastic/drug effects , Genes, ras , Hydroxamic Acids/pharmacology , Neurites/drug effects , 3T3 Cells , Acetylation , Animals , Cell Differentiation , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation/drug effects , Histones/metabolism , Mice , Nerve Growth Factors/antagonists & inhibitors , Nerve Growth Factors/pharmacology , PC12 Cells , Rats , Time Factors
16.
J Biol Chem ; 270(9): 4661-7, 1995 Mar 03.
Article in English | MEDLINE | ID: mdl-7876237

ABSTRACT

The "switch I" region (Asp30-Asp38) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called "effector region." As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Ras failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, M., Koide, H., Nishimura, S., and Yokoyama, S. (1991) FEBS Lett. 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys42-Ile55 of Ras and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP-->GTP exchange. So, this region is referred to as a "constitutive" effector (Ec) region, distinguished from switch I, a "switch" effector (Es) region. The Ec region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the Ec region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S. (1994) Oncogene 9, 2153-2157).


Subject(s)
Mitogen-Activated Protein Kinases , ras Proteins/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Differentiation/genetics , DNA Mutational Analysis , Enzyme Activation , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Kinetics , Mitogen-Activated Protein Kinase 3 , Mutagenesis, Site-Directed , Neurites , Oncogene Proteins v-raf , PC12 Cells , Protein Binding , Protein Structure, Tertiary , Proteins/metabolism , Rats , Retroviridae Proteins, Oncogenic/genetics , ras GTPase-Activating Proteins , ras Proteins/chemistry , ras Proteins/genetics
17.
Oncogene ; 9(8): 2153-7, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8036000

ABSTRACT

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.


Subject(s)
Genes, ras , Mutation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cells, Cultured , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Mice , PC12 Cells , Proto-Oncogene Proteins c-raf , Rats
18.
Biochemistry ; 33(1): 65-73, 1994 Jan 11.
Article in English | MEDLINE | ID: mdl-8286364

ABSTRACT

The Tyr residues in positions 32 and 40 of human c-Ha-Ras protein were replaced by site-directed mutagenesis (Y32F, Y32W, Y40K, and Y40W) to examine their roles in the signal-transducing activity and the sensitivity to the GTPase activating protein (GAP). The signal-transducing activity of the oncogenic Ras protein in PC12 cells was lost upon mutations Y32F and Y40K, but retained upon mutations Y32W and Y40W. These results suggest that residues 32 and 40 are both required to have aromatic groups and residue 32 is further required to have a hydrogen donor. On the other hand, three mutations (Y32F, Y32W, and Y40W) caused no appreciable reduction in either GAP-binding affinity or GAP sensitivity. By the Y40K mutation, GAP-binding affinity was slightly lowered, while GAP sensitivity was drastically impaired. Therefore, for residues 32 and 40 of Ras, interactions with GAP appear to be different from those with the target of signal transduction in the PC12 cell. As for the Y32W-Ras protein bound with an unhydrolyzable GTP analogue (GMPPNP), the Trp32 fluorescence is appreciably red-shifted, weaker, and more susceptible to KI quenching as compared to that of the GDP-bound form. Two-dimensional NMR spectroscopy with selectively deuterated Ras proteins revealed fewer and weaker nuclear Overhauser effects on the aromatic protons of Trp32 in the GMPPNP-bound form than in the GDP-bound form. This indicates that the side chain of Trp32 is more exposed to the solvent in the GMPPNP-bound form than in the GDP-bound form.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Proto-Oncogene Proteins p21(ras)/chemistry , Amino Acid Sequence , Base Sequence , DNA Primers , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/metabolism , Humans , Magnetic Resonance Spectroscopy/methods , Molecular Sequence Data , Mutagenesis, Site-Directed , Proto-Oncogene Proteins p21(ras)/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Restriction Mapping , Signal Transduction , Spectrometry, Fluorescence/methods
19.
Cancer Lett ; 69(3): 161-5, 1993 May 14.
Article in English | MEDLINE | ID: mdl-8513441

ABSTRACT

Protease-inhibitory activity of recombinant Ha-ras gene products (Ras) toward papain and cathepsins B and L was investigated. v-Ha-Ras showed more potent inhibitory activity toward cathepsin B as compared with c-Ha-Ras. We have also investigated protease-inhibitory activity of c-Ha-Ras mutants with point mutations in amino acids between positions 23 and 50. Inhibitory activity of Ras toward papain and cathepsin L was not largely altered among mutants. However, the inhibitory activity toward cathepsin B was significantly impaired by a mutation at position 43, 44, 45 or 48. These results suggest that 43Gln-Val-Val sequence plays an important role at least to inhibit cathepsin B.


Subject(s)
Cathepsin B/metabolism , Cathepsins/metabolism , Endopeptidases , Genes, ras/genetics , Oncogene Protein p21(ras)/metabolism , Papain/metabolism , Point Mutation , Amino Acid Sequence , Cathepsin L , Cysteine Endopeptidases , Molecular Sequence Data
20.
J Protein Chem ; 11(6): 731-9, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1466766

ABSTRACT

A truncated human c-Ha-Ras protein that lacks the C-terminal 18 amino acid residues and the truncated Ras protein with the amino acid substitution Gly-->Val in position 12 were prepared by an E. coli overexpression system. The truncated Ras protein showed the same guanine-nucleotide binding activity and GTPase activity as those of the full-length Ras protein. Further, the same extent of GTPase activity enhancement due to GTPase-activating protein was observed for the truncated and full-length Ras proteins. In fact, two-dimensional proton NMR analyses indicated that the tertiary structure of the truncated Ras protein (GDP-bound or GMPPNP-bound) was nearly the same as that of the corresponding catalytic domain of the full-length Ras protein. Moreover, a conformational change around the effector region upon GDP-->GMPPNP exchange occurred in the same manner for both proteins. These observations indicate that the C-terminal flanking region (18 amino acid residues) of the Ras protein does not appreciably interact with the catalytic domain. Therefore, the truncated Ras protein is suitable for studying the molecular mechanism involved in the GTPase activity and the interaction with the GTPase-activating protein. On the other hand, an active form of the truncated Ras protein, unlike that of the full-length Ras protein, did not induce neurite outgrowth of rat pheochromocytoma PC12 cells. Thus, membrane anchoring of the Ras protein through its C-terminal four residues is not required for the interaction of Ras and GAP, but may be essential for the following binding of the Ras-GAP complex with the putative downstream target.


Subject(s)
Guanine Nucleotides/metabolism , Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Animals , Binding Sites , Catalysis , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/metabolism , Humans , Magnetic Resonance Spectroscopy , Neurites/drug effects , Neurites/physiology , PC12 Cells , Protein Conformation , Proto-Oncogene Proteins p21(ras)/pharmacology , Rats , Signal Transduction , Solutions , ras GTPase-Activating Proteins
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