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Biomedical subjects

M Negishi

Publications and source records attributed to M Negishi.

At least 289 records · Page 16Linked to original sources
PubMed1990

Involvement of protein kinase C in prostaglandin E2-induced catecholamine release from cultured bovine adrenal chromaffin cells.

We recently reported that prostaglandin (PG) E2 stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain induced a gradual secretion of catecholamines from the cells (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). Here we examined the involvement of two signal pathways, Ca2+ mobilization and protein kinase C activation resulting from phosphoinositide metabolism, in the PGE2-induced catecholamine release. Either the Ca2+ ionophore ionomycin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could enhance the release in the presence of ouabain, and ionomycin-induced release was additive to PGE2-induced release, but TPA-induced release was not additive. PGE2 dose-dependently stimulated the formation of diacylglycerol and caused the translocation of 4% of the total protein kinase C activity to become membrane-bound within 5 min. These effects were specific for PGE2 and PGE1 among PGs tested (PGE2 = PGE1 greater than PGF2 alpha greater than PGD2). Furthermore, the phosphoinositide-specific phospholipase C inhibitor neomycin inhibited PGE2-induced accumulation of inositol phosphates, diacylglycerol formation, translocation of protein kinase C, and also stimulation of catecholamine release. Both PGE2- and TPA-induced release were inhibited by the depletion of protein kinase C caused by prolonged exposure to TPA, but ionomycin-induced release was not inhibited. We recently found that the amiloride-sensitive Na+, H+-antiport participates in PGE2-evoked catecholamine release (Tanaka, T., Yokohama, H., Negishi, M., Hayashi, H., Ito, S., and Hayaishi, O. (1990) J. Neurochem. 54, 86-95). In agreement with our recent report, PGE2 and TPA induced a sustained increase in intracellular pH that was abolished by the protein kinase C inhibitor staurosporine but not by the calmodulin inhibitor W-7. Ionomycin also induced a marked increase in intracellular pH, but this increase was abolished by W-7 but not by staurosporine. These results demonstrate that PGE2-induced activation of the Na+, H(+)-antiport and catecholamine release in the presence of ouabain are mediated by activation of protein kinase C, rather than by Ca2+ mobilization, resulting from phosphoinositide metabolism.

Adrenal Medulla↗
PubMed1990

Two steroid 15 alpha-hydroxylase genes and a homologous gene family in mice.

Steroid 15 alpha-hydroxylase (P45015 alpha) activity is concomitant with the expression of two types of mRNA in the mouse liver. Two discrete genes, designated 15 alpha oh-1 and 15 alpha oh-2, that encode the two mRNAs were recovered from total genomic libraries of the inbred mouse strains 129/J and C57Bl/6J and identified by cDNA hybridization, restriction-site analysis and partial nucleotide sequence. Both genes are approx. 9 kb long and share significant homology, including flanking regions, over a region of at least 30 kb. The two distinct 15 alpha oh genes are members of a larger family of homologous genes and/or pseudogenes of unknown function. The most extensive sequence homology among family members in the 3' portion of the gene with progressively less homology toward the 5' end. The far 5' portions of 15 alpha oh-1 and 15 alpha oh-2 are very similar to one another but there is no observed homology with other genes of the family. The two 15 alpha oh genes and the homologous family have been localized to mouse chromosome 7 by somatic cell hybrid mapping. Analysis of a restriction fragment length polymorphism in recombinant inbred mice shows a close linkage of 15 alpha oh-1 and 15 alpha oh-2 with the Coh locus.

Animals↗
PubMed1990

Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes.

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Effects of peripheral administration of recombinant human interleukin-1 beta on feeding behavior of the rat.

This study was undertaken to investigate the changes in feeding behavior, including ambulatory activity, induced by a single injection of Interleukin-1 beta (IL-1) (2 micrograms/rat) at 18:00, just before the dark phase. For this purpose, we used the Gunma University-type automatic apparatus for continuous and direct measurement of ambulation and drinking. A significant decrease in food intake was observed for 12 hours after treatment with IL-1. Peripheral administration of IL-1 also produced a marked decrease in ambulatory activity within 3 hours which continued for 6 hours. In addition, IL-1 produced a marked decrease in drinking behavior during the first 6 hours. We reported here the changes in consummatory and ambulatory behavior of rats after acute administration of IL-1. The sickness which IL-1 produced may, at least in part, contribute to these phenomena, although precise mechanisms are still unknown.

Animals↗
PubMed1990

The significance of decreased ambulatory activity during the generation by long-term observation of obesity in ovariectomized rats.

We attempted to determine the significance of ambulatory activity as a cause of overweight in ovariectomized rats. Drinking and ambulation were measured continuously and directly for periods up to 12 months in special apparatus developed at Gunma University. In older rats, ambulatory activity decreased much more in the ovariectomy group than in the control group. There was no difference in food intake between the ovariectomized and the control group. After 2 months, the ovariectomy group increased body weight more than the control group despite no difference in food intake. The decrease in ambulatory activity was consistent in the ovariectomy group, regardless of any differences in age and body weight. These results indicate that decrease of energy expenditure by gradual decrease in ambulatory activity may be an important factor as a cause of overweight in ovariectomized, obese rats.

Animals↗
PubMed1990

Abnormal feeding behavior and insulin replacement in STZ-induced diabetic rats.

The present studies were undertaken to investigate whether or not decreased ambulatory activity, including abnormal feeding behavior in diabetic rats, will be simultaneously normalized by insulin administration. To do this, we used the Gunma University-type automatic apparatus for continuous and direct measurement of ambulation and drinking. In this study, 3 U NPH insulin were administered at 1800, just before the dark phase, and 2 U were administered at 0600, just before the light phase. With these insulin doses, we found that 5 weeks were needed to normalize ambulatory activity, 4 weeks were necessary for food intake, 6 weeks for drinking and 2 weeks for body weight. Since ambulatory activity is reported to be related to changes in dopamine turnover, further studies are in progress to determine whether or not dopamine turnover is normalized when there is no difference in ambulatory activity due to insulin replacement.

Animals↗
PubMed1990

Ambulatory activity in streptozotocin-induced diabetic rats.

This study was undertaken to investigate changes in ambulatory and drinking behavior, using the Gunma University-type special apparatus for continuous and direct measurement of ambulation and drinking in streptozotocin-induced diabetic rats (STZ, 60 mg/kg). Ambulatory activity of diabetic rats was significantly less than that of control rats during the dark phase but not during the light phase. Ambulatory activity of diabetic rats was also significantly less from 0900 to 1000 hr, but significantly more from 1400 to 1500 hr and 1700 to 1800 hr than that of control rats. On the other hand, there was significant increase in drinking behavior from DM-1W to DM-4W rats throughout the experimental time. Since diabetic animals are very sick, a wide variety of metabolic systems, including dopamine turnover, would be expected to be altered in these animals, and such alterations could also contribute to the results observed. Further studies are in progress to determine whether these abnormalities will be normalized by insulin administration.

Animals↗
PubMed1990

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

Adenine Nucleotides↗
PubMed1990

Involvement of interleukin-1-like factor(s) in type II collagen-induced arthritis in mice.

To explore the role of interleukins in development of arthritis, we induced collagen-induced arthritis in mice and examined interleukin activities in the inflamed joints. Arthritis developed in 90% of mice 4-5 weeks after primary immunization with type II collagen. Joint extracts from mice with collagen-induced arthritis contained high levels of interleukin 1 (IL-1)-like activity but not interleukin 2 (IL-2) or interleukin 4 (IL-4) activity. IL-1-like activities in the lesions were correlated with development of arthritis assessed by joint swelling and erythema. These results suggest that IL-1-like factor(s) may participate in the etiopathogenesis of collagen-induced arthritis in mice.

Animals↗
PubMed1990

Different effects of insulin administration time in streptozotocin-induced diabetic rats.

We investigated whether the difference of insulin effect existed or not when insulin was administered at different times; just before the light phase (am 6:00) (L-NPH-DM) or just before the dark phase (pm 6:00) (D-NPH-DM) in streptozotocin (60 mg/kg, ip)-induced diabetic rats. Following results were obtained 7 days after STZ administration. Body weight: A significant increase compared to (D-NPH-DM) group was observed in (L-NPH-DM) group. Food intake: Food intake gradually increased after administration of STZ. A significant decrease compared to (saline-DM) group was observed only in the (L-NPH-DM) group. Total lipid content in the liver: A significant increase compared to (saline-control) group was seen in both the (D-saline-DM) and (L-saline-DM) group. In addition, a significant decrease compared to (D-NPH-DM) was observed in the (L-NPH-DM) group. Changes in blood glucose: Different patterns in decrease of blood glucose was observed between (D-NPH-DM) and (L-NPH-DM) groups. In conclusion, the present study demonstrates for the first time that there are differences of insulin effects on body weight, food intake, total lipid content in the liver and blood glucose level with insulin administration time.

Animals↗
PubMed1990

Synergistic effect of prostaglandin E2 and ouabain on catecholamine release from cultured bovine adrenal chromaffin cells.

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.

Adrenal Medulla↗
PubMed1990

Evaluation of ZK110841 and AH6809, an agonist and an antagonist of prostaglandin DP-receptors on human platelets, with a PGD2-responsive cell line from bovine embryonic trachea.

ZK110841 and AH6809, an agonist and an antagonist of prostaglandin DP-receptors on human platelets, were evaluated with a fibroblastic cell line from bovine embryonic trachea (EBTr) which specifically responds to prostaglandin D2 (PGD2). ZK110841, equipotent to PGD2, elevated the adenosine 3':5'-cyclic monophosphate (cyclic AMP) level in EBTr cells dose-dependently, being more than 100 fold over the basal level at 1 microM. The half-maximally effective concentration of PGD2 or ZK110841 was 10-30 nM. Increasing concentrations (10(-7)-10(-4) M) of AH6809 (which possesses negligible intrinsic agonist activity) produced parallel shifts to the right of dose-response curves to PGD2 and ZK110841. Schild plots of these data were linear (correlation close to unity) and pA2 values against PGD2 and ZK110841 were calculated to be 6.36 and 6.57, respectively, indicating that AH6809 is a simple and competitive antagonist. These results demonstrate that ZK110841, AH6809 and EBTr cells will be useful for characterization of DP-receptors.

Animals↗
PubMed1990

Characterization and regulation of sex-specific mouse steroid hydroxylase genes.

We characterized the genes of the male-specific mouse steroid 16 alpha-hydroxylase (C-P-45016 alpha) and the female-specific mouse steroid 15 alpha-hydroxylase (P-45015 alpha) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15 alpha-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.

Amino Acid Sequence↗
PubMed1990

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase.

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Dexamethasone suppressible hypergonadotropism in an adolescent patient with Prader-Willi syndrome.

We report an adolescent patient with Prader-Willi syndrome accompanying suppressible hypergonadotropism. The subject is an 18-year-old female. She was obese (body mass index: 35.7) and hypomyotonic with mental retardation. On endocrinological examination, a high serum LH concentration and hyperresponsiveness of luteinizing hormone (LH) to intravenously administered LH-Releasing Hormone (LH-RH) were observed, while the basal follicle stimulating hormone level was within the normal range. In addition, serum dehydroxyepiandrosterone sulfate (DHEA-S) was also increased. Following 2 mg dexamethasone administration for 7 days, serum LH and DHEA-S were almost normalized and hyperresponse of LH to LH-RH completely disappeared. The present study provides evidence that altered responsiveness to adrenal steroid may be involved in the establishment of hypergonadotropinism in an adolescent patient with Prader-Willi syndrome.

Adolescent↗
PubMed1990

Effects of recombinant human interleukins on food intake of previously food-deprived rats.

The effects of intracerebroventricular injection of recombinant human interleukin 1 beta (rhIL-1 beta), 1 alpha (rhIL-1 alpha), and 2 (rhIL-2) on feeding behavior were examined in previously food-deprived rats for 18 hr. At doses of 2-17 ng/rat, rhIL-1 beta significantly reduced food intake in a dose-dependent manner and the feeding suppression continued about 4 hr later. Only 17 ng/rat rhIL-1 beta reduced body weight gain for 8 hr after the injection. However, rhIL-1 alpha at dose of 17 ng/rat did not show any significant change of food intake and body weight gain during the whole observation period. At both doses of 8 and 40 ng/rat, rhIL-2 also failed to suppress overfeeding after food deprivation. In adrenalectomized rats, feeding suppression by rhIL-1 beta appeared at the 1- to 2-hr time period. The present studies suggest that rhIL-1 beta may be, at least in part, involved in feeding suppression on various inflammatory processes and that adrenal hormones may not play an important role in the induction of feeding suppression by rhIL-1 beta.

Adrenalectomy↗
PubMed1990

Cyclic AMP-mediated inhibition of cell growth by prostaglandin D2 in a fibroblastic cell line (EBTr).

We have recently reported that prostaglandin D2 (PGD2) specifically elevates the intracellular cyclic AMP (cAMP) level in a fibroblastic cell line derived from bovine embryonic trachea (EBTr) (K. Sugama, T. Tanaka, H. Yokohama, M. Negishi, H. Hayashi, S. Ito, and O. Hayaishi, Biochim. Biophys. Acta, 1011: 75-80, 1989). Since the in vitro and in vivo inhibitory effects of PGD2 on cell growth are attributed to the dehydrate product 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2), the role of cAMP as a mediator of PGD2-induced cell growth has been questioned. In this study we investigated the effects of PGD2 on cAMP level, DNA synthesis, and c-myc expression using EBTr cells growth-arrested by serum starvation. Quiescent EBTr cells synchronously resumed [3H]thymidine incorporation at 12 h after the addition of serum, an incorporation which ended at 21 h. PGD2 at 1 microM inhibited approximately 30% incorporation without any delay of initiation, and it also caused a rapid increase in the cAMP level at a maximum of 10 min. The inhibition of [3H]thymidine incorporation was increased 40-45% by 5 microM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The expression of c-myc mRNA induced by serum at 1-4 h was also inhibited by PGD2. Time-course studies support the association between the reduction of c-myc expression and the successive inhibition of DNA synthesis by PGD2. The dose dependency of PGD2 for these three processes correlated well with each other, and the effects were evident at as low as 10 nM and specific for PGD2.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗