共查询到20条相似文献,搜索用时 15 毫秒
1.
R Kanamoto M Nishiyama S Matsufuji S Hayashi 《Archives of biochemistry and biophysics》1991,291(2):247-254
Asparagine stimulated the translation of ornithine decarboxylase (ODC) mRNA more than 10-fold in cultured hepatocytes which had been pretreated with glucagon in simple salt/glucose medium. Putrescine suppressed the increase in the rate of ODC synthesis caused by asparagine without significant change in the amount of ODC mRNA, suggesting that putrescine inhibited the effect of asparagine at least in part at the level of translation. Polysomal distribution of ODC mRNA was analyzed to examine the site of translational regulation by these effectors. In uninduced hepatocytes, most of the ODC mRNA was sedimented slightly after the 40 S ribosomal subunit. This ODC mRNA was sequestered from translational machinery since it was not shifted to the polysome fraction when peptide elongation was specifically inhibited by a low concentration of cycloheximide. In asparagine-treated cells, 40% of total ODC mRNA was in the polysomal fraction and formed heavier polysomes, indicating that asparagine stimulated both recruitment of ODC mRNA from the untranslatable pool and the initiation steps of translation. Putrescine did not change the distribution pattern of ODC mRNA on polysomes significantly. Thus, 30% of ODC mRNA remained on polysomes even when ODC synthesis was completely inhibited by putrescine. Paradoxically more than 70% of ODC mRNA was shifted into polysomes by putrescine in the presence of low concentrations of cycloheximide. These results, together with changes in the polysome profile, suggested that putrescine nonspecifically stimulated the recruitment of ODC mRNA from the untranslatable pool, whereas it specifically inhibited its translation at both the initiation and the elongation steps. 相似文献
2.
Ornithine decarboxylase (ODC; EC 4.1.1.17) could be induced in primary cultured hepatocytes of the frog, Xenopus laevis, by a hypotonic treatment. Addition of 10 mM putrescine caused a rapid decay of preinduced ODC after a lag period of 30 min. The putrescine-induced ODC decay was faster than the ODC decay in the presence of cycloheximide. Simultaneous addition of cycloheximide blocked the putrescine-induced acceleration of ODC decay, indicating an involvement of protein synthesis. Addition of putrescine to normal medium caused complete loss of ODC activity in 2 h and then ODC-inhibitory activity appeared and progressively increased. The inhibitory factor was non-dialysable and temperature-sensitive and showed a time-independent and stoichiometric pattern of ODC inhibition. On the basis of these observations the inhibitory factor was identified as ODC antizyme. These results indicated that in frog hepatocytes, like in mammalian cells and tissues, ODC is under negative feedback regulation mediated by antizyme. 相似文献
3.
Effects of putrescine on synthesis and degradation of ornithine decarboxylase in primary cultured hepatocytes 总被引:4,自引:0,他引:4
Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 microM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration of ODC degradation. 相似文献
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Separate mechanisms of induction for ornithine decarboxylase by triiodothyronine and aminophylline 总被引:2,自引:0,他引:2
A single dose of aminophylline (200 μmol/kg, i.p.) or triiodothyronine (T3, 300 μg/kg, i.p.) resulted in the induction of ornithine decarboxylase (ODC) in rat liver with maximal activity 10-fold and 6-fold above controls, respectively, 4 hr after the administration of the drug or hormone. After either agent, the induction of ODC was blocked by either cycloheximide or actinomycin D. The same concentrations of aminophylline and T3 administered simultaneously produced an additive 16-fold increase in ODC activity. After T3 administration, the cyclic AMP-dependent protein kinase activity ratio was unaltered at all times measured. After aminophylline, the protein kinase activity ratio was elevated by 15 min and remained elevated for 2 hr. Somatostatin administration (50 μg/100 g), which lowers plasma growth hormone to 30% of control, had no effect on the ability of T3 to induce ODC. These data suggest separate routes of induction of ODC in response to aminophylline and T3. Aminophylline induction occurs via cycyclic AMP-mediated event whereas T3 does not involve ccyclic AMP but results from a direct nuclear interaction. 相似文献
6.
The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation. 相似文献
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We investigated the mechanisms underlying the marked induction of ornithine decarboxylase (ODC) activity by hypotonic treatment and its rapid decay upon reversal to isotonicity in primary cultures of adult rat hepatocytes. Upon hypotonic treatment, ODC synthesis rate increased progressively whereas the amount of ODC mRNA increased only about twofold. In addition, ODC was stabilized severalfold. ODC activity rapidly decreased upon restoration of isotonicity, owing to immediate and nearly complete suppression of ODC synthesis and 3-6-fold stimulation of ODC decay. The stimulation of ODC decay caused by restoration of isotonicity was mostly independent of time and protein synthesis. ODC decay was also stimulated by putrescine, even under hypotonic conditions, depending on time and new protein synthesis. Restoration of isotonicity and putrescine treatment together caused a synergistic stimulation of ODC decay, confirming that these act by different mechanisms. 相似文献
9.
Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine 总被引:1,自引:0,他引:1
The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. 相似文献
10.
Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes 总被引:2,自引:0,他引:2
Y Horio H Fukui M Taketoshi T Tanaka H Wada 《Biochemical and biophysical research communications》1988,153(1):410-416
The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin. 相似文献
11.
Glucose administration inhibits the induction of ornithine aminotransferase (OAT) in both the whole animal and cultured hepatocytes. We have examined the ability of several hexoses and related molecules to inhibit the cAMP-dependent induction of OAT in primary cultures of adult rat hepatocytes. The hexoses (D-glucose, fructose, sorbitol, sorbose, and mannose) that were effective as inhibitors of OAT induction also resulted in accumulation of lactate in the culture medium, although lactate itself was not effective as an inhibitor. The hexoses and related 6-carbon structures (galactose, L-glucose, 2-deoxyglucose, 3-O-methylglucose, rhamnose, mannitol, and inositol) that were not effective as inhibitors of OAT induction did not result in accumulation of lactate in the culture medium. These results suggest that the carbohydrate repression of hepatic OAT requires metabolism of the carbohydrate by the liver cell. Upon addition to the culture medium of several compounds related to carbohydrate metabolism, many (ribose, xylitol, dihydroxyacetone, and glycerol) exhibited an inhibitory effect, with glycerol exhibiting the greatest effect. Fructose and glycerol inhibit OAT induction in the presence of 2-deoxyglucose, suggesting that the inhibitory effect of nonglucose carbohydrates is not occurring through conversion to glucose. The carbon sources observed to be most effective as inhibitors of OAT induction (glycerol, fructose, sorbitol, and sorbose result in more than 90% inhibition at 25 mM) all enter the glycolytic pathway at the triosephosphate level. The mechanism of the inhibitory effect of simple carbohydrates on OAT induction is not known but may involve an increase in certain glycolytic intermediates. Glucose and the related carbon sources exert their effect by inhibiting the cAMP-dependent increase in OAT synthesis. The cAMP-dependent increase in OAT mRNA was inhibited by fructose. These findings suggest that the carbohydrate inhibition of the cAMP-dependent increase in OAT synthesis occurs at a pretranslational level. 相似文献
12.
A Kashiwagi Y Harano K Kosugi T Nakano H Hidaka Y Shigeta 《Journal of biochemistry》1985,97(2):679-684
The specific [125I]insulin binding to primary cultured hepatocytes was significantly greater than that to freshly isolated hepatocytes. Low affinity insulin binding sites in cultured cells were 6-fold greater in number than those of freshly isolated cells without a significant change in high affinity sites. However, both sensitivity (insulin concentration for half maximum stimulation) and responsiveness (% of increase above the basal level) to insulin for the stimulation of ODC activity were similar for isolated and cultured cells indicating an important role of high affinity sites in the insulin action. On the other hand, the specific [125I]glucagon binding to cultured cells was significantly decreased. Low affinity glucagon binding sites in cultured cells decreased by about 50% in cultured cells without a significant change in high affinity sites. Both sensitivity and responsiveness to glucagon for the stimulation of ketogenesis from palmitate also decreased as compared with those of isolated cells, indicating an important role of low affinity sites in the glucagon action. These results indicate that insulin and glucagon receptors were reciprocally changed in cultured cells, as compared with isolated cells. 相似文献
13.
Dexamethasone restores hormonal inducibility of ornithine decarboxylase in primary cultures of rat hepatocytes 总被引:4,自引:0,他引:4
Induction of ornithine decarboxylase by various hormones was studied in quiescent primary cultures of adult rat hepatocytes maintained in a chemically defined medium. The following results were obtained: Enzyme activity rose transiently during the first day of cultivation in hormone-untreated cells. During this phase, insulin increased ornithine decarboxylase activity. Inducibility by insulin was maintained for more than 40 h only after pretreatment with 0.1 microM dexamethasone. Enzyme activity could be induced by 1 nM insulin and peaked after 7 h. Inducibility by glucagon and growth hormone required pretreatment with the glucocorticoid hormone. Ornithine decarboxylase activity was maximal 5 h after glucagon addition. Concentrations down to 0.1 nM were effective. Pretreatment with dexamethasone was most effective, when the hormone was present during the first 20 h of cultivation. The effect of the glucocorticoid during the pretreatment phase was diminished by colchicine and to a lesser extent by cytochalasine B. We suggest that part of the permissive effect of dexamethasone could be mediated by changes in the cytoskeleton and the function of hormone receptors. The fact that induction of ornithine decarboxylase was exerted by several hormones despite the absence of cell proliferation and DNA synthesis may indicate that polyamine biosynthesis has an important role in the quiescent hepatocyte. 相似文献
14.
Sanchez Mas J Martijnez-Esparza M Bastida CM Solano F Penafiel R Garcija-Borron JC 《Biochimica et biophysica acta》2002,1542(1-3):57-65
Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH. 相似文献
15.
The uptake of transport systems A and N amino acids, most noticeably L-asparagine, is essential for the induction of ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) in cultured cells and we have proposed that the uptake-associated pH and ionic changes might constitute part of the cell activation signal (1). In the present study, it was shown that extracellular L-asparagine caused an immediate and transient increase in intracellular pH which was continuously monitored by the fluorescence probe BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). NH4Cl and NH4OH which caused intracellular alkalinization also caused ornithine decarboxylase activity to increase. 相似文献
16.
Prolactin-stimulated ornithine decarboxylase induction in rat hepatocytes: coupling to diacylglycerol generation and protein kinase C 总被引:2,自引:0,他引:2
The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased [3H]-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle. 相似文献
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Ornithine induced more than 36-fold the ornithine decarboxylase activity in confined Ehrlich ascites tumour cells after 3.5 h of continuous perifusion with 0.5 mM ornithine; arginine and glutamine also induced the activity 3- and 4-fold, respectively. The addition of cycloheximide or actinomycin D antibiotics to the perifusion medium confirmed that the regulation of the enzyme synthesis takes place at the level of translation. Perifusion in the presence of 0.5. mM ornithine and 55, 25, and 10 μM histamine suppressed the induction by 91, 53, and 35%, respectively. Similar results were obtained in the presence of serotonin. Histidine also showed inhibitory effect but 5 mM histidine was required to produce 21% inhibition; other basic amino acids were ineffective. 相似文献