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M Negishi

Publications and source records attributed to M Negishi.

At least 217 records · Page 12Linked to original sources
PubMed1993

Activation of heat shock transcription factors by delta 12-prostaglandin J2 and its inhibition by intracellular glutathione.

We recently showed that delta 12-prostaglandin (PG) J2 bound to the thiol groups of nuclear proteins and stimulated the synthesis of a 67-kDa heat shock protein (HSP) in porcine aortic endothelial cells, and that intracellular glutathione (GSH) blocked this binding and HSP induction (Koizumi et al., Biochem Pharmacol 44: 1597-1602, 1992). In the present study, we examined the molecular mechanism underlying the induction of HSP by delta 12-PGJ2. Treatment of cells with delta 12-PGJ2 induced the activation of heat shock transcription factors (HSF) in a time- and concentration-dependent manner. Cycloheximide pretreatment inhibited this activation. Treatment of cells with buthionine sulfoximine, an inhibitor of GSH synthesis, depleted the intracellular GSH and enhanced the activation of HSF by delta 12-PGJ2, but treatment with GSH increased the intracellular GSH level and thus reduced the activation. Moreover, the thiol-reactive agents arsenite and diethylmaleate also induced the activation of HSF, and this activation was inhibited by GSH treatment and enhanced by buthionine sulfoximine treatment. These results taken together suggest that delta 12-PGJ2 binds to the thiol groups of nuclear proteins and activates HSF, leading to the synthesis of the 67-kDa HSP.

Animals↗
PubMed1993

Two isoforms of prostaglandin E receptor EP3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization.

We recently identified two isoforms of mouse prostaglandin (PG) E receptor EP3 subtype, EP3 alpha and EP 3 beta, which are produced by alternative splicing and different only in the carboxyl-terminal domain (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, Y., T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). We examined here agonist-induced desensitization of the two isoforms using Chinese hamster ovary cells stably expressing these isoforms. Exposure of the EP3 alpha isoform to PGE2 for 30 min did not change maximal response but increased PGE2 concentration needed to inhibit forskolin-induced cAMP accumulation in the cells. Further exposure of this isoform to PGE2 suppressed the maximal response as well as sensitivity to PGE2 in a time-dependent manner; after 24-h exposure, it elicited only 50% of the maximal response of the control cells. Consistent with these results, short term exposure sequestered the EP3 alpha isoform away from the cell surface and long term incubation decreased the total receptor number in the cells. In contrast, exposure of the EP3 beta isoform to PGE2 did not affect its dose-response curve for PGE2, and no sequestration or decrease in the receptor number was observed in this isoform. Thus, alternative splicing produced the two isoforms with different carboxyl-terminal domains, which are different in sensitivity to agonist-induced desensitization.

Alternative Splicing↗
PubMed1993

Site-directed mutagenesis of mouse steroid 7 alpha-hydroxylase (cytochrome P-450(7) alpha): role of residue-209 in determining steroid-cytochrome P-450 interaction.

We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae. Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4). The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone. The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable. The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu. The mutant P450(7) alpha displays a 17-fold lower Vmax. value than the wild-type enzyme. Unexpectedly, it also has 3-fold lower Km and Kd values. Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket. Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha. Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold. Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 >> iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

Amino Acid Sequence↗
PubMed1993

Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M(r) = approx. 130 kDa, corresponding to the M(r) of the complex of EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Adenosine Diphosphate↗
PubMed1993

Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

Adenylyl Cyclases↗
PubMed1993

Enhancement by protein kinase C of prostacyclin receptor-mediated activation of adenylate cyclase through a calmodulin/myristoylated alanine-rich C kinase substrate (MARCKS) system in IC2 mast cells.

The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly enhanced cAMP formation induced by carbacyclin, a stable prostacyclin analogue, in cultured mast cells (IC2 cells), but did not enhance basal or NaF plus AlCl3-induced cAMP formation. On the other hand, W-7, a calmodulin (CaM) inhibitor, almost completely suppressed the enhancing activity of TPA, suggesting the involvement of CaM in the enhancement by TPA of carbacyclin-induced cAMP formation. The enhancing activity of TPA disappeared in TPA-treated cells permeabilized with saponin in the presence of Ca2+, but reconstitution with CaM in the permeable cells resulted in remarkable restoration of the action of TPA. On the other hand, TPA treatment induced the phosphorylation and translocation of myristoylated alanine-rich C kinase substrate (MARCKS) from the membrane to the cytosol. Exogenously added protein kinase C (PKC) also phosphorylated MARCKS and induced its translocation in the cells permeabilized with saponin. Whereas the addition of CaM did not enhance the carbacyclin-stimulated GTPase activity and adenylate cyclase activity in the control permeable cells, in which MARCKS bound to the membrane, CaM markedly enhanced those activities in the PKC-treated permeable cells, which lost endogenous membrane-bound MARCKS. When MARCKS was added to the PKC-treated permeable cells, MARCKS bound to the membrane and inhibited the effects of CaM. These results suggest that activation of PKC enhances the prostacyclin-activated adenylate cyclase through a CaM/MARCKS system.

Adenylyl Cyclases↗
PubMed1993

Engineering mouse P450coh to a novel corticosterone 15 alpha-hydroxylase and modeling steroid-binding orientation in the substrate pocket.

The F209L mutation alters specificity of P450coh from coumarin 7-hydroxylation to 15 alpha-hydroxylation of 11-deoxysteroids such as testosterone and 11-deoxycorticosterone. Neither the wild-type nor F209L exhibits activity toward 11 beta-hydroxysteroids including corticosterone. Mutation of Phe-209 to Asn, however, confers on mutant F209N a high corticosterone 15 alpha-hydroxylase activity. F209V also exhibits low corticosterone 15 alpha-hydroxylase activity; Km and Vmax are 10-fold higher and lower, respectively, than for F209N. The results are consistent with the hypothesis that direct interaction of Asn-209 with 11OH is responsible for high corticosterone 15 alpha-hydroxylase activity. To support this hypothesis, a possible steroid-binding orientation is modeled in the substrate pocket of P450cam. Our weighted homology and constrained alignments map residue 209 of P450coh to Met-184 and Met-191 of P450cam. Energy minimization of corticosterone in the substrate pocket results in the 11OH of the steroid directed toward Met-184 (7 A) and Met-191 (16 A), and in C15 located near the sixth axial position of the heme. The steroid-binding model suggests that the P450cam's substrate pocket may be conserved in the mammalian P450 and can accommodate a steroid molecule, and that residue 209 appears to be located at the critical site that determines the steroid-substrate specificity of a P450 depending on the type of group at the 11-position of steroid molecule.

Amino Acid Sequence↗
PubMed1993

Alpha-melanocyte stimulating hormone (MSH) antagonizes the anorexia by corticotropin releasing factor (CRF).

We examined a hypothesis that alpha-Melanocyte Stimulating Hormone (MSH) can antagonizes the anorexia induced by intracerebroventricular injection of corticotropin releasing factor (CRF). Food intake of the rats deprived of food for 18 h was significantly inhibited by 4 h after intracerebroventricular injection of CRF. alpha-MSH also suppressed food consumption by 4 h after the treatment. However, simultaneous administration of alpha-MSH attenuated the anorexic effect of CRF at 0-2 h period and food consumption for 4 h was equal to controls. In addition, simultaneous administration of alpha-MSH deleted the stimulatory effects of CRF on serum corticosterone levels. The present experiment demonstrated that alpha-MSH can antagonizes the anorexia and activation of hypothalamo-pituitary-adrenal axis by CRF, suggesting that the processing step of proopiomelanocortin may be important for the induction of anorexia elicited by CRF.

Animals↗
PubMed1993

Two types of prostacyclin receptor coupling to stimulation of adenylate cyclase and phosphatidylinositol hydrolysis in a cultured mast cell line, BNu-2cl3 cells.

Prostacyclin (PGI2)-mediated signal transduction was examined in interleukin 3 (IL-3)-dependent BNu-2cl3 mast cells. Iloprost, a stable PGI2 analogue, induced the accumulation of intracellular cAMP and IP3, and an increase in the intracellular Ca2+ concentration. Pretreatment of the cells with a protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate, suppressed the iloprost-induced IP3 accumulation and Ca2+ mobilization, but inversely potentiated the cAMP accumulation, suggesting that neither of these signal transduction pathways of iloprosts is the result of a secondary effect of activation of the other. Removal of IL-3 from the culture medium reduced the iloprost-induced IP3 accumulation and Ca2+ mobilization, while it had no effect on the iloprost-induced cAMP accumulation at all. These results taken together suggest that BNu-2cl3 cells express two types of PGI2 receptor; one couples to stimulation of adenylate cyclase, its expression being independent of IL-3, while the other couples to phosphatidylinositol hydrolysis, its expression being dependent on IL-3.

Adenylyl Cyclases↗
PubMed1993

Phase I/II trial of didanosine (2',3'-dideoxyinosine) in hemophiliac patients with AIDS or AIDS-related complex.

Forty-three hemophiliacs with AIDS or ARC received a daily dose of 334 or 500 mg didanosine (2',3'-dideoxyinosine or ddI) orally in 2 divided doses in phase I/II, open-label clinical trial conducted in Japan. Twenty-eight patients completed 6 months of therapy. There was an increase in circulating CD4(+) cells in 19 valuable patients from 91 +/- 25 (mean +/- SE) at entry to 131 +/- 38 at 24 weeks of therapy P = 0.01; Wilcoxon signed rank). Fourteen of 37 patients met the criteria for CD4 rise >/= 50/mm3 rise or >/= 50% increase from entry values) for more than 4 consecutive weeks. Twenty patients were p24 positive at entry. Nine out of the 10 evaluable patients (90%) showed a decline in p24 antigen at weeks 20-24 (P = 0.02). Thirty-five patients had symptoms related to HIV-1 infection at entry. Twenty-seven patients reported improvements in constitutional symptoms during therapy. Nine patients presented with possible drug-related adverse effects, and didanosine was discontinued in 6 patients (one each with edema; abdominal pain with anorexia; hematuria with edema and rash; sense of abdominal distension with anorexia; diarrhea and abdominal pain; and irritability). One patient had a transient increase in serum amylase level to twice the upper limit of normal, but he continued to receive the drug. These data suggest that didanosine was generally well tolerated in hemophiliacs with AIDS or ARC, and its administration correlated with improvement in constitutional symptoms and laboratory findings. The adverse effects of didanosine seen in this population were moderate to mild, and no complications related to hemorrhagic diathesis were observed, although the relative risk of acute pancreatitis in this population (while not seen in the present study to date) requires more study.

Journal Article↗
PubMed1993

Arachidonic acid activates cation channels in bovine adrenal chromaffin cells.

Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that approximately 70% of the cells responded to arachidonic acid in increasing the intracellular Ca2+ concentration. Because this increase was markedly less in the absence of external Ca2+, we examined the effect of arachidonic acid on Ca2+ influx electrophysiologically. Bath application of 10 microM arachidonic acid induced a long-lasting inward current when the cell was clamped at -50 mV. Other fatty acids, such as oleic acid, linoleic acid, eicosatrienoic acid, and eicosapentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (> or = 2 microM) activated a single-channel current in the inside-out patch, even in the presence of inhibitors of cyclooxygenase and lipoxygenase, possibly suggesting that arachidonic acid could activate channels directly. The onset delay of the inward channel current in the outside-out patch configuration (54.2 +/- 63.5 s; mean +/- SD) was significantly shorter than that in the inside-out patch one (197.3 +/- 177.7 s). Washout of arachidonic acid decreased the probability of channel openings in the outside-out patch but not in the inside-out one. These results suggest that arachidonic acid activates channels reversibly from outside of the plasma membrane. The unitary conductance for Ca2+ of arachidonic acid-activated channel was approximately 17 pS. The arachidonic acid-activated channel was permeable to Ba2+, Ca2+, and Na+ but not to Cl-. The opening probability of the arachidonic acid-activated channel did not depend on membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1993

A trans-acting locus regulates transcriptional repression of the female-specific steroid 15 alpha-hydroxylase gene in male mice.

Steroid 15 alpha-hydroxylase (P450(15 alpha)) is a female-specific enzyme in the livers of many inbred mice including DBA/2J. Run-on assays using liver nuclei from GH-deficient Little mice indicated that the P450(15 alpha) gene is transcriptionally repressed by GH in male mice. BALB/cJ is a variant strain in which the gene is expressed in the males as well as in the females. Genetic crosses between DBA/2J and BALB/cJ indicated that expression of the P450(15 alpha) gene in BALB/cJ males is inherited as a recessive trait and is regulated by a single locus. The parental origin of the P450(15 alpha) genes, determined using restriction site polymorphism in the exons of the P450(15 alpha) genes, divided the F2 males expressing the P450(15 alpha) gene into three phenotypes at a ratio of 1:1:2, the individuals expressing the gene from only BALB/cJ or DBA/2J and the individuals expressing the genes from both parents respectively. The results indicate that the repression of the P450(15 alpha) gene in male mice is regulated by a trans-acting regulatory locus between the DBA/2J and BALB/cJ pairs. Because hypophysectomy derepressed the P450(15 alpha) gene in F1 males and GH repressed the gene in hypophysectomized F1 males, the hormone appears to regulate gene repression through a trans-acting locus, named GH-dependent repression, GDR.

Animals↗