Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : II. Initial Studies on Catecholamine Metabolism

Initial studies are reported on the catecholamine metabolism of low-density cultures of dissociated primary sympathetic neurons. Radioactive tyrosine was used to study the synthesis and breakdown of catecholamines in the cultures. The dependence of catecholamine synthesis and accumulation on external tyrosine concentration was examined and a concentration which is near saturation, 30 µM, was chosen for further studies. The free tyrosine pool in the nerve cells equilibrated with extracellular tyrosine within 1 h; the total accumulation of tyrosine (free tyrosine plus protein, catecholamines, and metabolites) was linear for more than 24 h of incubation. Addition of biopterin, the cofactor of tyrosine hydroxylase, only slightly enhanced catecholamine biosynthesis by the cultured neurons. However, addition of reduced ascorbic acid, the cosubstrate for dopamine β-hydroxylase, markedly stimulated the conversion of dopamine (DA) to norepinephrine (NE). Phenylalanine, like tyrosine, served as a precursor for some of the DA and NE produced by the cultures, but tyrosine always accounted for more than 90% of the catecholamines produced. The DA pool labeled rapidly to a saturation level characteristic of the age of the culture. The NE pool filled more slowly and was much larger than the DA pool. The disappearance of radioactive NE and DA during chase experiments followed a simple exponential curve. Older cultures showed both more rapid production and more rapid turnover of the catecholamines than did younger cultures, suggesting a process of maturation.     

Synergistic effects of muscarinic agonists and secretin or vasoactive intestinal peptide on the regulation of tyrosine hydroxylase activity in sympathetic neurons

Cholinergic agonists and certain peptides of the glucagon-secretin family acutely increase tyrosine hydroxylase activity in the superior cervical ganglion in vitro. The present study was designed to investigate possible interactions between these two classes of agonists in regulating catecholamine biosynthesis. Synergistic effects were found between carbachol and either secretin or vasoactive intestinal peptide in the regulation of DOPA (dihydroxyphenylalanine) synthesis. In addition, synergism was found at the level of the accumulation of cyclic adenosine monophosphate, the likely second messenger in the peptidergic regulation of tyrosine hydroxylase activity. The synergism seen with carbachol was blocked by a muscarinic, but not by a nicotinic, antagonist. Synergism was also found between bethanechol, a muscarinic agonist, and secretin, but not between secretin and dimethylphenylpiperazinium, a nicotinic agonist. Since previous immunohistochemical results suggest that vasoactive intestinal peptide and acetylcholine are colocalized in some preganglionic sympathetic neurons, the present data raise the possibility that the two might act synergistically in vivo in regulating catecholamine biosynthesis. Synergistic postsynaptic actions may be a common feature at synapses where peptides of the secretin-glucagon and acetylcholine are colocalized.     

Effects of cytoskeleton-disrupting agents on tyrosine transport into cultured bovine adrenal chromaffin cells

The effects of various cytoskeleton-disrupting agents on tyrosine transport into chromaffin cells were examined to assess the possibility of cytoskeleton involvement in the regulation of precursor supply for catecholamine synthesis. Tyrosine transport was markedly increased by cytochalasin B. Vinblastine also stimulated tyrosine transport, although its effect was less pronounced than that of cytochalasin B. While colchicine failed to cause any significant increase in the transport under the same conditions. These results therefore suggest a possible role of microfilaments as a factor regulating tyrosine transport into chromaffin cells.     

Effects of Pseudorabies Virus on the Neuronal Properties of PC 12 Cells

The effect of pseudorabies virus on neuronal functions was investigated in PC12 cells. During the period investigated, choline acetyltransferase was not affected, while the acetylcholinesterase activity declined steadily starting at 12 h post infection (p.i.), reaching its minimal level of 40% of the control value at 24 h p.i. In contrast, the activity of tyrosine hydroxylase, the key enzyme in catecholamine synthesis, increased to 150% of the control level by 15 h p.i., dropping off slowly with the appearance of viral cytopathology. In parallel, the infection induced, by a process independent of the extracellular Ca2+, an increased release of dopamine at 11 h p.i., followed by noradrenaline at 20 h p.i. In the infected cells, the intracellular content of catecholamine was maintained only in the presence of a high amount of catecholamine precursors in the culture medium. Three plaque-forming units per cell was the minimal multiplicity of infection required to obtain the maximal changes in enzyme activities; higher multiplicities induced more rapidly the maximal effects on tyrosine hydroxylase and acetylcholinesterase. Inhibition of DNA synthesis did not prevent the increase in tyrosine hydroxylase activity; however, protein synthesis was required. In conclusion, infection of the PC12 cells with pseudorabies virus induced significant changes in catecholaminergic and cholinergic metabolism, indicating the ability of this virus to interfere selectively with specialized neuronal functions.     

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.     

Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro

Summary In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently of the action of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.     

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.     

Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.     

A rapid histidine decarboxylase assay

Histidine decarboxylase (EC 4.1.1.22) catalyzes the conversion of histidine to histamine. Because current assays for enzyme activity are time consuming and require additional enzymes or large amounts of tissue, a rapid radioisotopic assay was devised. Using commercially available radioactive histidine (without additional purification), the enzyme mediates the formation of histamine. The product is resolved from precursor by paper electrophoresis in a formic acid-acetic acid solution for 15 min. After drying and ninhydrin staining, radioactive histamine is measured by liquid seintillation spectrometry. This assay procedure is sensitive enough to measure decarboxylase activity in milligram quantities of rat brain.     

Suppressive effect of endogenous regucalcin on deoxyribonuclic acid synthesis in the nuclei of rat renal cortex

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on deoxyribonucleic acid (DNA) synthesis activity in the nuclei isolated from rat renal cortex was investigated. The addition of calcium chloride (10-100 microM) in the reaction mixture containing the nuclei caused a significant decrease in DNA synthesis activity. Nuclear DNA synthesis activity was significantly raised in the presence of EGTA (1 mM), a chelator of Ca2+, indicating that nuclear Ca2+ has an inhibitory effect. Regucalcin (0.1-0.5 microM) added in the reaction mixture in the presence of either EGTA (1 mM) or calcium chloride (50 microM) had a significant inhibitory effect on nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity. This increase was completely abolished by the addition of regucalcin (0.5 microM). The effect of anti-regucalcin monoclonal antibody in increasing DNA synthesis was enhanced in the presence of EGTA. Additionally, an inhibitory effect of calcium chloride (10 or 50 microM) was enhanced in the presence of anti-regucalcin monoclonal antibody (25 ng/ml). The present study demonstrates that endogenous regucalcin has a suppressive effect on DNA synthesis in the nuclei of rat renal cortex.     

Oral branched-chain amino acid supplements that reduce brain serotonin during exercise in rats also lower brain catecholamines

Exercise raises brain serotonin release and is postulated to cause fatigue in athletes; ingestion of branched-chain amino acids (BCAA), by competitively inhibiting tryptophan transport into brain, lowers brain tryptophan uptake and serotonin synthesis and release in rats, and reputedly in humans prevents exercise-induced increases in serotonin and fatigue. This latter effect in humans is disputed. But BCAA also competitively inhibit tyrosine uptake into brain, and thus catecholamine synthesis and release. Since increasing brain catecholamines enhances physical performance, BCAA ingestion could lower catecholamines, reduce performance and thus negate any serotonin-linked benefit. We therefore examined in rats whether BCAA would reduce both brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Sedentary and exercising rats received BCAA or vehicle orally; tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis rates were measured 1 h later in brain. BCAA reduced brain tryptophan and tyrosine concentrations, and serotonin and catecholamine synthesis. These reductions in tyrosine concentrations and catecholamine synthesis, but not tryptophan or serotonin synthesis, could be prevented by co-administering tyrosine with BCAA. Complete essential amino acid mixtures, used to maintain or build muscle mass, were also studied, and produced different effects on brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Since pharmacologically increasing brain catecholamine function improves physical performance, the finding that BCAA reduce catecholamine synthesis may explain why this treatment does not enhance physical performance in humans, despite reducing serotonin synthesis. If so, adding tyrosine to BCAA supplements might allow a positive action on performance to emerge.     

Dynamics of tyrosine hydroxylase mediated regulation of dopamine synthesis

Tyrosine hydroxylase's catalysis of tyrosine to dihydroxyphenylalanine (DOPA) is the highly regulated, rate-limiting step catalyzing the synthesis of the catecholamine neurotransmitter dopamine. Phosphorylation, cofactor-mediated regulation, and the cell's redox status, have been shown to regulate the enzyme's activity. This paper incorporates these regulatory mechanisms into an integrated dynamic model that is capable of demonstrating relative rates of dopamine synthesis under various physiological conditions. Most of the kinetic equations and substrate parameters used in the model correspond with published experimental data, while a few which were not available in literature have been optimized based on explicit assumptions. This kinetic pathway model permits a comparison of the relative regulatory contributions made by variations in substrate, phosphorylation, and redox status on enzymatic activity and permits predictions of potential disease states. For example, the model correctly predicts the recent observation that individuals with haemochromatosis and having excessive iron accumulation are at increased risk for acquiring Parkinsonism, a defect in neuronal dopamine synthesis (Bartzokis et al., 2004; Costello et al., 2004). Alpha synuclein mediated regulation of tyrosine hydroxylase has also been incorporated in the model, allowing an insight into the over-expression and aggregation of alpha synuclein in Parkinson's disease. Action Editor: Upinder Bhalla     

Regulation of Nerve Growth Factor Action on Nsp100 Phosphorylation in PC12h Cells by Calcium

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.     

Neuroendocrine changes in male hamsters following photostimulation

Transfer of gonadally regressed male golden hamsters from a short (5 L:19 D) to a stimulatory (14 L:10 D) photoperiod elicits, within 24 hr, significant changes in hypothalamic dopamine, serotonin, and possibly norepinephrine metabolism. Hypothalamic LHRH content was significantly elevated in short-photoperiod animals, but within 24 hr of transfer to a 14:10 photoperiod, LHRH declined to levels not different from those in hamsters maintained continuously in a long photoperiod. Plasma FSH levels were also significantly elevated within 24 hr of transfer, but increases in plasma LH were somewhat slower. Chronic treatment with the tyrosine hydroxylase inhibitor, alpha-methyl tyrosine (alpha MPT), which inhibits catecholamine synthesis, blocked the effect of a stimulatory photoperiod on plasma FSH levels, while treatment of 5:19 hamsters with the catecholamine precursor, L-dopa, mimicked the effects of photostimulation on plasma FSH levels. Testicular weights were not affected by alpha MPT or L-dopa treatment for 1 week. From these data, it appears that endocrine events associated with photoperiod-induced testicular recrudescence are under the control of hypothalamic neurotransmitters.     

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-l-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6-8 h after an initial 2-3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.