Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Effects of imidapril on NOS expression and myocardial remodelling in failing heart of Dahl salt-sensitive hypertensive rats.

OBJECTIVES: To elucidate the relationship between renin-angiotensin system and nitric oxide in hypertensive heart failure, we evaluated the effects of long-term treatment with imidapril, angiotensin-converting enzyme inhibitor, on endothelial-cell nitric oxide synthase (eNOS) and inducible NOS (iNOS) expression in the left ventricle (LV) and its relation to myocardial remodelling in failing heart of Dahl salt-sensitive hypertensive rats (DS) fed a high-salt diet. METHODS: In DS rats fed an 8% NaCl diet after the age of 6 weeks, a stage of concentric left ventricular hypertrophy at 11 weeks (DSLVH) was followed by a distinct stage of fatal left ventricular failure with chamber dilatation at 18 weeks (DSCHF). Imidapril (DSCHF-I, n = 7, 1 mg/kg/day, subdepressor dose) or vehicle (DSCHF-V, n = 7) were given from DSLVH to DSCHF stage for 7 weeks, and age-matched (18 weeks) Dahl salt-resistant rats fed the same diet were served as control group (DR-C, n = 7). RESULTS: Markedly increased left ventricular end-diastolic diameter and reduced fractional shortening in DSCHF-V was significantly ameliorated in DSCHF-I using transthoracic echocardiography. The level of eNOS mRNA and protein in the LV was significantly suppressed in DSCHF-V compared with DR-C, and significantly increased in DSCHF-I compared with DR-C and DSCHF-V. The iNOS mRNA and protein and the fibrosis factor expression of type I collagen mRNA were significantly increased in DSCHF-V compared with DR-C, and significantly decreased in DSCHF-I compared with DSCHF-V. DSCHF-V demonstrated a significant increase in wall-to-lumen ratio, perivascular fibrosis, and myocardial fibrosis. These changes in the microvasculature were improved significantly by imidapril. CONCLUSIONS: Subdepressor dose of imidapril may ameliorate the endothelial damage not only by inhibiting production of angiotensin II but also by promoting eNOS and inhibiting iNOS mRNA and protein expression in the LV, and this increased eNOS mRNA and protein level may have a role in the improvement of congestive heart failure and myocardial remodelling.

TCV-116 stimulates eNOS and caveolin-1 expression and improves coronary microvascular remodeling in normotensive and angiotensin II-induced hypertensive rats.

Angiotensin II (Ang II) plays an important role as a modulator of vascular structure and function in arterial hypertension. This study investigated the effects of an Ang II type 1 receptor antagonist, TCV-116, on endothelial nitric oxide synthase (eNOS) mRNA and protein expression, and NOS activity and eNOS regulatory protein caveolin-1 protein expression in the left ventricle of Wistar-Kyoto rats treated for 2 weeks with Ang II (200 ng/kg/min) and evaluated these relations to myocardial remodeling. Rats given Ang II alone (ANGII) were compared with rats also receiving TCV-116 (ANGII-TCV). The eNOS mRNA and protein levels, and NOS activity and caveolin-1 protein expression in the left ventricle were significantly decreased in ANGII compared with control rats (CON), and were significantly increased in ANGII-TCV compared with ANGII. Moreover, compared with CON, the eNOS and caveolin-1 expression was significantly greater in CON treated with TCV-116. ANGII showed a significant increase of the wall-to-lumen ratio, perivascular and myocardial fibrosis, and type I collagen mRNA expression, with all these parameters being significantly improved by TCV-116. Thus, coronary microvascular and myocardial remodeling in normotensive and Ang II-induced hypertensive rats was significantly ameliorated by a subdepressor dose of TCV-116, which may be at least in part mediated by an increase in local eNOS mRNA and protein expression, and NOS activity in the left ventricle.

Celiprolol stimulates endothelial nitric oxide synthase expression and improves myocardial remodeling in deoxycorticosterone acetate-salt hypertensive rats.

OBJECTIVE: Endothelium-dependent vasodilation is attenuated in humans and experimental hypertension models, and this phenomenon may be largely due to decreased release or activity of nitric oxide (NO). However, very few studies have evaluated whether beta-adrenoceptor antagonists increase endothelial NO synthase (eNOS) expression in the left ventricle. We examined the effects of long-term treatment with celiprolol, a specific beta1-antagonist with a weak beta2-agonist action, on eNOS expression in the left ventricle and evaluated its relationship to myocardial remodeling in the left ventricle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: DOCA-salt rats (n = 18) were induced with weekly injections of DOCA (30 mg/kg) and 1% saline in their drinking water after right nephrectomy. Celiprolol (DOCA-CEL, n = 9, 10 mg/kg per day, subdepressor dose) or a vehicle (DOCA-V, n = 9) were given after induction of DOCA-salt hypertension for 5 weeks, and age-matched sham-operated rats (ShC, n = 9) served as a control group. RESULTS: Blood pressure levels in DOCA-V and DOCA-CEL were similar and significantly higher than that in ShC. The eNOS mRNA and protein levels, and NOS activity in the left ventricle significantly decreased in DOCA-V compared with ShC, and significantly increased in DOCA-CEL compared with DOCA-V. DOCA-V showed a significant increase in the wall-to-lumen ratio, perivascular fibrosis, myocardial fibrosis, and type I collagen mRNA, with all these parameters being significantly improved by celiprolol. CONCLUSIONS: Myocardial remodeling of DOCA-salt hypertensive rats was significantly ameliorated by subdepressor doses of celiprolol, which may be due to increased eNOS expression in the left ventricle.

Rabphilin-3A binds to a M(r) 115,000 polypeptide in a phosphatidylserine- and Ca(2+)-dependent manner.

Rabphilin-3A is a putative target protein for Rab3A/Smg 25A, which is a member of the Ras-related small GTP-binding protein and implicated in neurotransmitter release from the synapse. Rabphilin-3A is composed of two functionally different domains: the N-terminal Rab3A-binding and the C-terminal phosphatidylserine- and Ca(2+)-binding domains. The C-terminal domain has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2 and C-gamma 1, which are known to bind phosphatidylserine and Ca2+. In this study, we attempted to identify the Rabphilin-3A-interacting molecule in bovine brain by use of an overlay assay technique. The 32P-labeled C-terminal fragment of Rabphilin-3A (281-704 amino acids) bound to a protein molecule with a M(r) of about 115 kDa which was immobilized on a nitrocellulose sheet. This protein was highly purified and characterized. The binding of the 32P-labeled C-terminal fragment to this protein was dependent on both phosphatidylserine and Ca2+, and inhibited by an excess amount of the C-terminal fragment and the C2 domain fragment (396-704 amino acids) but not by the N-terminal fragment (1-280 amino acids). These results indicate that Rabphilin-3A binds to a protein molecule with a M(r) of 115 kDa through the C2 domain in the presence of phosphatidylserine and Ca2+.

Effects of subchronic treatment with natural human interferons on antipyrine clearance and liver function in patients with chronic hepatitis.

To determine whether natural human interferon administered under the usual therapeutic dosing scheme would inhibit the hepatic drug metabolism, we performed an antipyrine test in eight patients with chronic B or non-A, non-B hepatitis before and after a subchronic interferon therapy (6 megaunits/day for 17 +/- 4 days, mean +/- SD). Six patients received interferon-beta and 2 received interferon-alpha. To circumvent a possible influence of interferon-induced fever on the hepatic drug metabolism, the antipyrine test during the interferon therapy was performed at least 14 days after the interferon-induced fever disappeared. The kinetic parameters of antipyrine were obtained from seven saliva samples over 32 hours postdose. There were no significant differences in any kinetic parameters of antipyrine observed before and during the interferon therapy. With the sample size of the study, there was only a 20% chance (i.e., beta-power = 0.8 at alpha = 0.05) that we might have missed a 17% reduction in antipyrine clearance by the interferon therapy (type II error). On the other hand, the subchronic interferon therapy lowered serum aminotransferases and DNA polymerase activity significantly (P less than .05) compared with the respective baseline values. Our results suggest that the subchronic therapeutic dosing scheme of interferon as conducted in the present study does not cause the inhibitory effect on the oxidative drug metabolism to a statistically significant or clinically relevant degree in patients with chronic hepatitis, while it improves their liver function. Further studies are required for determining if different types of interferons administered under the different dosing schemes would alter the hepatic drug metabolism and the inhibitory effect would be time-dependent.

Computed tomography density of gallbladder stones may aid in predicting the rate of stone fragmentation with extracorporeal lithotripsy.

Thirty-six patients with gallbladder stone(s), 21 with solitary stone and 15 with multiple stones up to five in number, were treated by a single session of extracorporeal shock wave lithotripsy. In eight patients (group A), stones were gradually fragmented from the surface, producing fine powderlike fragments which made residual stones invisible, leading to reduced therapeutic efficacy. In 13 patients (group B), residual stones remained visible up to 2,400 shocks, though they showed similar fragments. Such fragments did not appear throughout treatment in 14 patients (group C). Computed tomography (CT) attenuation number, when the region of interest was settled on the whole stone, increased in the order of group A, B, and C. When the distribution of CT density on stones was analyzed, the density was diffusely lower than 50 HU in all patients in group A, higher than 50 HU only on the surface in all patients except for two in group B, and diffusely higher than 50 HU in all patients except for one in group C. CT findings may be useful for predicting the fragmentation mode of gallbladder stones by extracorporeal shock wave lithotripsy.

Three splicing variants of tomosyn and identification of their syntaxin-binding region.

We have recently isolated a neural tissue-specific syntaxin-1-binding protein, named tomosyn, which is capable of dissociating Munc18/n-Sec1/rbSec1 from syntaxin-1 to form a 10S tomosyn complex, an intermediate complex converted to the 7S SNARE complex. We isolated here two splicing variants of tomosyn: one had 36 amino acids (aa) insertion and another had 17 aa deletion. We named original one m-tomosyn, big one b-tomosyn, and small one s-tomosyn. s-Tomosyn as well as m-tomosyn was mainly expressed in brain whereas b-tomosyn was ubiquitously expressed. All the isoforms bound to syntaxin-1, but not to syntaxin-2, -3, or -4, and had a region highly homologous to VAMP, another syntaxin-binding protein. This region was necessary but not sufficient for high-affinity binding of tomosyn to syntaxin-1.

Rab3A small GTP-binding protein in Ca(2+)-dependent exocytosis.

There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in Ca(2+)-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.

Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small G protein superfamily that is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A contains at least two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal C2 domain, which interacts with both Ca2+ and phospholipid. Because Rabphilin-3A interacts preferentially with GTP-Rab3A rather than with GDP-Rab3A, we have examined here whether Rabphilin-3A affects the GTPase activity of Rab3A. Rabphilin-3A and its N-terminal fragment, but not its C-terminal fragment, very weakly stimulated the basal GTPase activity of Rab3A. However, Rabphilin-3A and its N-terminal fragment strongly inhibited the Rab3A GAP-stimulated GTPase activity of Rab3A. Ca2+ and phospholipid showed no effect on these activities of Rabphilin-3A. The physiological significance of the GAP activity of Rabphilin-3A is obscure, but it is likely that Rabphilin-3A inhibits Rab3A GAP activity and keeps Rab3A in the GTP-bound active form during its action as a target molecule for Rab3A.

Regulators of small GTPases.

Small GTPases are converted from the GDP-bound inactive form to the GTP-bound active form by a GDP/GTP exchange reaction which is regulated by GDP/GTP exchange proteins (GEPs). We have found both stimulatory and inhibitory GEPs, which we have named GDP dissociation stimulators (GDSs) and GDP dissociation inhibitors (GDIs) respectively. We have isolated Smg GDS, Rho GDI and Rab GDI, cloned them, and determined their primary structures. These GEPs are active on a group of small GTPases: Smg GDS on at least K-Ras, Rap1/Smg21, Rho and Rac; Rho GDI on at least Rho, Rac and Cdc42; Rab GDI on most of the Rab family members. These GEPs have an additional function, regulating the translocation of their substrate small GTPases between the membrane and the cytosol. The GEPs interact only with the post-translationally modified form of their substrate small GTPases.

Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase A-catalyzed phosphorylation of smg p21.

We have previously shown that cyclic AMP-dependent protein kinase (protein kinase A) phosphorylates smg p21A and -B, ras p21-like small GTP-binding proteins. In the present study, we investigated the function(s) of this phosphorylation by use of the smg p21B purified from human platelets. smg p21B bound to plasma membranes and the protein kinase A-catalyzed phosphorylation of smg p21B reduced this binding. Moreover, the phosphorylation of smg p21B enhanced the two actions of its specific GDP/GTP exchange protein, named GDP dissociation stimulator, when tested in a cell-free system: one is the action to stimulate the GDP/GTP exchange reaction of smg p21B, and the other is the action to inhibit the binding of smg p21B to membranes. Consistently, smg p21B was translocated from the membranes to the cytoplasm when it was phosphorylated by protein kinase A in intact platelets in response to prostaglandin E1 or dibutyryl cyclic AMP. The protein kinase A-catalyzed phosphorylation of smg p21B affected neither its basal GDP/GTP exchange reaction, basal GTPase activity, nor the GTPase activity stimulated by its specific GTPase activating protein. On the other hand, we have recently clarified that the structure of the C-terminal region of the post-translationally processed human platelet smg p21B is Lys-Lys-Ser-Ser-all-trans-geranylgeranyl Cys181 methyl ester, and that this modification of the C-terminal region is essential for smg p21B to bind to membranes. We furthermore determined here that protein kinase A phosphorylated Ser179 in this C-terminal region of smg p21B. These results indicate that protein kinase A-catalyzed phosphorylation of smg p21B makes smg p21B sensitive to the actions of smg p21 GDP dissociation stimulator.

Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region.

smg p21B, a member of the ras p21-like small GTP-binding protein superfamily, undergoes post-translational modifications, which are geranylgeranylation of the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids (QLL) and the subsequent carboxyl methylation of the exposed prenylated cysteine residue. smg p21B has a polybasic region upstream of the prenylated cysteine residue. We have previously proposed that these C-terminal structures of smg p21B are essential for the action of its stimulatory GDP/GTP exchange protein, named GDP dissociation stimulator (GDS). We studied here which structure of the C-terminal region of smg p21B is important for its interaction with smg p21 GDS. For this purpose, we synthesized a peptide according to the C-terminal structure of smg p21B, which was PGKARKKSSC-geranylgeranyl-carboxyl methyl, and its variously modified peptides and examined their ability to interact with smg p21 GDS and to interfere with the smg p21 GDS action to stimulate the GDP/GTP exchange reaction of smg p21B. The results indicate that the phosphorylated form of PGKARKKSSC-geranylgeranyl stoichiometrically interacts with smg p21 GDS, that the presence of the geranylgeranyl moiety is essential for, but not sufficient for, the smg p21 GDS action, and that the presence of the methyl moiety, removal of the three C-terminal amino acids, and the presence of the polybasic amino acids also affect the smg p21 GDS action. It is likely that all the steps of the post-translational processing and presence of the polybasic region in the C-terminal region of smg p21B are related to its interaction with smg p21 GDS.

Effects of angiotensin II type 1 receptor antagonist on nitric oxide synthase expression and myocardial remodeling in Goldblatt hypertensive rats.

We evaluated the effects of long-term treatment with TCV-116, an angiotensin II type 1 receptor antagonist, on endothelial-cell nitric oxide synthase (eNOS) messenger RNA (mRNA) and protein expression in the left ventricle and its relation to myocardial remodeling in Goldblatt hypertensive rats. Two-kidney, one-clip Goldblatt hypertensive rats (RHR) were assigned either to a TCV-116 treatment group (RHR-TCV, n = 8, 3 mg/kg/day, subdepressor dose) or to a group without treatment (RHR-V, n = 7) after their kidneys had been clipped for 4 weeks. TCV-116 was administered to rats in the treatment group for 6 weeks, and age-matched sham-operated rats (ShC, n = 7) served as a control group. Blood pressure in RHR-V and RHR-TCV was similar and significantly higher than that in ShC. The eNOS mRNA and protein levels and NOS activity in the left ventricle was significantly decreased in RHR-V compared with ShC, and significantly increased in RHR-TCV compared with ShC and RHR-V. RHR-V demonstrated a significant increase in fibrosis factor (type I collagen) mRNA expression, perivascular fibrosis, and myocardial fibrosis. These parameters in the microvasculature were improved significantly by TCV-116. Subdepressor dose of TCV- 116 improved pathological myocardial changes in RHR, which may be due in part to an increased eNOS mRNA and protein expression and NOS activity in the left ventricle.

Phosphorylation of Rabphilin-3A, a putative target protein for Rab3A, by cyclic AMP-dependent protein kinase.

Rab3A small GTP-binding protein and its associated proteins, such as Rabphilin-3A, a putative target protein for Rab3A, MSS4, a stimulatory GDP/GTP exchange protein for Rab3A, and Rab GDI, a translocator for the Rab family members including Rab3A, are implicated in neurotransmitter release. We have investigated here the phosphorylation of these proteins by cyclic AMP-dependent protein kinase (protein kinase A). Neither Rab3A, MSS4, nor Rab GDI was phosphorylated, but Rabphilin-3A was phosphorylated Rab GDI was phosphorylated, but Rabphilin-3A was phosphorylated at its N-terminal region. About 0.8 mol of phosphate was maximally incorporated into one mol of Rabphilin-3A. These results suggest that Rabphilin-3A is one of the targets for protein kinase A which modulates neurotransmitter release.

Identification as beta-adducin of a protein interacting with rabphilin-3A in the presence of Ca2+ and phosphatidylserine.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein implicated in neurotransmitter release. We have previously identified a Rabphilin-3A-interacting protein with a Mr of about 115 kDa in bovine brain. We have attempted here to purify this protein and to determine its primary structure. Amino acid sequence analysis has revealed that this protein is a bovine counterpart of human beta-adducin which is known to be a good substrate for protein kinase C. The Rabphilin-3A-interacting protein also binds to protein kinase C in the presence of Ca2+ and phosphatidylserine. These results indicate that Rabphilin-3A binds to beta-adducin in the presence of Ca2+ and phosphatidylserine.

Localization of rabphilin-3A, a putative target protein for Rab3A, at the sites of Ca(2+)-dependent exocytosis in PC12 cells.

Rab3A/Smg 25A, a small GTP-binding protein, is highly concentrated in presynapse of neurons and implicated in neurotransmitter release. We have recently identified a putative target protein for Rab3A, isolated its cDNA, and designated it as Rabphilin-3A. To examine whether Rabphilin-3A as well as Rab3A is localized at the sites of Ca(2+)-dependent exocytosis, we investigated here localization of Rabphilin-3A and Rab3A in comparison with the sites of exocytosis in the differentiated PC12 cells. Rabphilin-3A as well as Rab3A was highly concentrated at the tips of the neurites where Ca(2+)-dependent exocytosis took place. Inversely, neither Rabphilin-3A nor Rab3A was concentrated at the tips of the neurites where Ca(2+)-dependent exocytosis did not take place. These results suggest that Rabphilin-3A as well as Rab3A constitutes a part of the machinery necessary for neurotransmitter release.