Induction of ornithine decarboxylase in synchronized BHK cells by calf serum after a short period of serum-starvation

Addition of calf serum to serum-starved cultures of synchronized BHK cells induced temporary production of ornithine decarboxylase, irrespective of the phase of the cell cycle of the BHK cells. The induction depended on the duration of serum starvation and on the amount of serum added. In addition, incorporation of 3H-thymidine into the acid-insoluble fraction of BHK cells in the S phase was stimulated by this treatment.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine

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.     

Effect of growth conditions on the activity of ornithine decarboxylase in cultured hepatoma cells. II. Effect of serum and insulin

HTC cells incubated in the absence of serum for more than 14 hours have very low levels of ornithine decarboxylase, the first enzyme on the pathway of polyamine synthesis. Readdition of serum causes an increase in the activity of ODC, reaching a maximum on average 17 times above the basal level after five hours. This increase is due in part to a decrease in the apparent rate of degradation of ODC, and also to a stimulation of its synthesis. Within the first two hours the serum induction of ODC is resistant to Actinomycin D. Insulin at 5 μm/ml alsocauses an increase in ODC activity but only after a delay of two hours, in contrast to its more rapid stimulation of tyrosine transaminase activity.     

Effect of sodium arsenite on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 microM arsenite and complete inhibition was obtained at concentrations above 10 microM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half-life of ODC activity, measured after cycloheximide treatment, increased almost six-fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol-redox status in cells, also caused a similar effect on the induction and stability of ODC.     

HSG cells differentiated by culture on extracellular matrix involves induction of S-adenosylmethione decarboxylase and ornithine decarboxylase

The human salivary gland (HSG) epithelial cell line can differentiate when cultured on extracellular matrix preparations. We previously identified >30 genes upregulated by adhesion of HSG cells to extracellular matrix. In the current studies, we examined the role of one of these genes, the polyamine pathway biosynthetic enzyme S-adenosylmethionine decarboxylase (SAM-DC) and the related enzyme, ornithine decarboxylase (ODC), on HSG cell differentiation during culture on extracellular matrix. HSG cells cultured on fibronectin-, collagen I gel-, and Matrigel-coated substrates for 12-24 h upregulated SAM-DC and ODC mRNA expression and enzyme activity compared to cells cultured on non-precoated substrates. After 3-5 days, HSG cells grown on Matrigel- or collagen I gel-coated substrates acquired a differentiated phenotype: the cells showed changes in culture morphology and increased expression of salivary gland differentiation markers (vimentin, SN-cystatin, and alpha-amylase). Further, culturing the cells on substrates precoated with an anti-beta1-integrin-antibody promoted differentiation-like changes. HSG cells cultured on collagen I- or Matrigel-coated substrates rapidly entered the cell cycle but showed decreased cell proliferation at longer times. In contrast, cell proliferation was enhanced on fibronectin-coated substrates compared to cells on non-precoated substrates. Treatment with the polyamine synthesis inhibitors, difluoromethylornithine (DFMO), and methylglyoxal bis-(guanylhydrazone) (MGBG), inhibited cell proliferation and delayed (3)H-thymidine incorporation in HSG cells cultured on all of the substrates. Further, inclusion of DFMO and MGBG inhibited or delayed acquisition of the differentiated phenotype in HSG cells cultured on Matrigel- or collagen I gel-coated substrates. This suggests that the adhesion-dependent expression of SAM-DC and ODC contributes to extracellular matrix-dependent HSG cell differentiation.     

Effects of cyclic nucleotides on ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

Dibutyryl cAMP and prolactin stimulated ornithine decarboxylase activity in mouse mammary gland explants which had been preincubated with insulin and cortisol for 1 day; maximally stimulatory concentrations of dibutyryl cAMP and prolactin produced a response which was greater than the sum of the responses of prolactin and dibutyryl cAMP when tested alone. 8-Bromo-cGMP inhibited ornithine decarboxylase activity whereas other derivatives of cyclic nucleotides were without effect. Cortisol concentrations were found to be important for optimizing the dibutyryl cAMP and prolactin responses. Optimal prolactin responses were obtained with cortisol concentrations greater than 10(-7) M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10(-7) M. Despite the differing optimal cortisol concentrations for the prolactin and dibutyryl cAMP responses, it is concluded that prolactin and dibutyryl cAMP probably stimulate ornithine decarboxylase activity in the mammary gland via the same mechanism.     

Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

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.     

Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells

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.     

Changes in ornithine decarboxylase activity and cyclic adenosine-3'-5'-monophosphate concentrations during the cell cycle of synchronized BHK cells.

BHK cells were synchronized by excess thymidine treatment, which resulted in approximately 90% synchrony. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, elevated in early S phase, decreased in G2 + M and G1 phase and then increased during late G1 approximately second round of early S phase. The concentration of cyclic adenosine-3'-5'-monophosphate (cAMP) gradually decreased during S approximately G2 + M phase and then increased during late G1 approximately second round of early S phase, preceding that of ODC activity. The data suggest that ODC activity might be regulated by cellular cAMP level.     

Effect of urethan on the induction of ornithine decarboxylase in regenerating rat liver

The effect of urethan on the induction of ornithine decarboxylase in the early stage of the regeneration of rat liver was studied. The induced activity of ornithine decarboxylase was suppressed by administration of urethan immediately after partial hepatectomy. Although ornithine decarboxylase was induced biphasically by partial hepatectomy, a single intraperitoneal injection of urethan resulted in the reduction of both phases. However, the ornithine decarboxylase activity induced by glucocorticoids and growth hormone was not suppressed by urethan. The increased level of 3′,5′-cyclic adenosine monophosphate induced by partial hepatectomy was also reduced by urethan and this suppression was proportional to the suppression of ornithine decarboxylase activity. Reversal of the urethan-induced suppression of ornithine decarboxylase by administration of dibutyryl 3′,5′-cyclic adenosine monophosphate was also observed.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP

We have examined the regulation of two key enzymes that control polyamine biosynthesis-L-ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) - by agents increasing cAMP in S49 lymphoma cells. Incubation of wild type S49 cells with beta-adrenergic agonists (terbutaline or isoproterenol) inhibited ODC and SAMDC activities rapidly (less than 2 hr). more quickly than these agents arrested the cells in the G1 phase of the cell cycle. The beta-adrenergic antagonist propranolol blocked inhibition of ODC activity produced by isoproterenol, but only if added simultaneously or less than 4 hr after the agonist. Incubation of wild type S49 cells with cholera toxin or PGE1 also inhibited ODC activity. Decreases in ODC activity produced by beta-adrenergic agonists, cholera toxin, PGE1 or dibutyryl cAMP were all enhanced by the phosphodiesterase inhibitor Ro 20-1724. Results of studies of ODC and SAMDC activity in S49 variants having lesions in the pathway of cAMP generation and action were as follows: kin- cells (which lack cAMP-dependent protein kinase activity) showed no inhibition of ODC by any agent; AC- cells (which have absent nucleotide coupling units in their adenylate cyclase system) only demonstrated inhibition in response to dibutyryl cAMP; UNC cells (which have deficient coupling of hormone receptors and adenylate cyclase) only demonstrated inhibition in response to dibutyryl cAMP and cholera toxin, and beta-depleted cells (which have a decreased number of beta-adrenergic receptors) responded as did wild type cells except for absent response to isoproterenol. We conclude that inhibition of ODC and SAMDC activity in S49 cells is an early response to agents that increase cAMP and that this action occurs via the "classical" pathways of activation of adenylate cyclase and protein kinase. These results in S49 cells contrast with evidence in other systems in which cAMP has been suggested to enhance polyamine biosynthesis, perhaps through alternative mechanisms.     

Effect of urethan on the induction of ornithine decarboxylase in regenerating rat liver.

The effect of urethan on the induction of ornithine decarboxylase in the early stage of the regeneration of rat liver was studied. The induced activity of ornithine decarboxylase was suppressed by administration of urethan immediately after partial hepatectomy. Although ornithine decarboxylase was induced biphasically by partial hepatectomy, a single intraperitoneal injection of urethan resulted in the reduction of both phases. However, the ornithine decarboxylase activity induced by glucocorticoids and growth hormone was not suppressed by urethan. The increased level of 3',5'-cyclic adenosine monophosphate induced by partial hepatectomy was also reduced by urethan and this suppression was proportional to the suppression of ornithine decarboxylase activity. Reversal of the urethan-induced suppression of ornithine decarboxylase by administration of dibutyryl 3',5'-cyclic adenosine monophosphate was also observed.     

The induction of ornithine decarboxylase by L-asparagine and intracellular alkalinization

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.     

Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells

Summary The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min.1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with K_i values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a K_i = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 µM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 µM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.Abbreviations ODC Ornithine Decarboxylase - TLC Thin Layer Chromatography - DNEM Dulbecco's Modified Essential Medium - PBS Phosphate Buffered saline - PEG Polyethyleneglycol     

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for -receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic -selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific -antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, -opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.