Inactivation of phosphorylated rat tyrosine hydroxylase by ascorbate in vitro.

Tyrosine hydroxylase activity is reversibly controlled by the actions of several protein kinases. Previous studies showed that, following phosphorylation by protein kinase A, physiological concentrations of ascorbate irreversibly inactivate tyrosine hydroxylase. Several studies were performed to establish the mechanism of inactivation. We found that inactivation occurred under oxygen-free conditions. The results of this and other experiments suggest that oxygenated species such as superoxide or hydrogen peroxide were not required for inactivation by ascorbate. Inhibition of tyrosine hydroxylase by low concentrations of ascorbate raised the question concerning the mechanism for maintaining enzyme activity under physiological conditions. We report that tyrosine, N alpha-methyl tyrosine, 3-iodotyrosine, and phenylalanine protected the phosphorylated enzyme against ascorbate inactivation. Catecholamines (dopamine, norepinephrine, and some of their analogues) also protected the enzyme against ascorbate inactivation. We performed studies to assess conformational changes of tyrosine hydroxylase by measuring the extrinsic fluorescence using 8-anilino-1-naphthalenesulfonic acid as a reporter group. Phosphorylation of tyrosine hydroxylase by protein kinase A decreased the extrinsic fluorescence. Treatment of tyrosine hydroxylase with ascorbate produced a further decrease in fluorescence. These results provide evidence for conformational changes following these treatments. In contrast to extrinsic fluorescence, the circular dichroic spectrum of tyrosine hydroxylase failed to change following phosphorylation by protein kinase A or inhibition by ascorbate. The spectrum was consistent with a secondary structure of tyrosine hydroxylase with 55% alpha helix, 20% beta sheet, 2% beta turn, and 23% random coil.

Regulation of tyrosine hydroxylase activity in rat PC12 cells by neuropeptides of the secretin family.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by the cAMP as well as the calcium and cGMP second messenger systems. Treatment of intact rat PC12 cells with neuropeptides including secretin and vasoactive intestinal polypeptide (VIP) stimulated tyrosine hydroxylase activity 2 to 3-fold in vitro. Secretin (EC50 = 10 nM) was about 3 orders of magnitude more potent than VIP (EC50 = 3 microM). A combination of several protease inhibitors failed to enhance the potency of either peptide. Other members of the secretin family including glucagon and peptide histidine isoleucine (PHI) stimulated tyrosine hydroxylase activity to a lesser extent. Somatostatin, which is not homologous to secretin, was ineffective. The maximal response of tyrosine hydroxylase activation to 1 microM secretin occurred within 6-15 sec. Secretin, VIP, and forskolin also enhanced tyrosine hydroxylase activity (3,4-dihydroxyphenylalanine production) in intact cells, as determined by high performance liquid chromatography and electrochemical detection. Secretin, VIP, PHI, and glucagon increased the levels of cAMP in PC12 cells more than 10-fold, as determined by radioimmunoassay. We also demonstrated that cAMP is released from the cells into the incubation medium following secretin treatment. Secretin and VIP treatment also enhanced the activity of cAMP-dependent protein kinase in a concentration-dependent fashion, as measured subsequently in vitro. Based on the greater potency of secretin in comparison with VIP, PHI, and glucagon, we suggest that the PC12 cells contain a secretin-preferring receptor that increases cAMP levels and brings about an activation of tyrosine hydroxylase activity through the stimulation of cAMP-dependent protein kinase.

Adenosine receptor activation and the regulation of tyrosine hydroxylase activity in PC12 and PC18 cells.

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Degradation of rat brain cholinergic muscarinic receptors in vitro: enhancement by agonists and inhibition by antagonists.

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.

Human brain and placental choline acetyltransferase: purification and properties.

Choline acetyltransferase (EC 2.3.1.6) catalyzes the biosynthesis of acetylcholine according to the following chemical equation: acetyl-CoA + choline in equilibrium to acetylcholine + CoA. In addition to nervous tissue, primate placenta is the only other animal source which contains appreciable acetylcholine and its biosynthetic enzyme. Human brain caudate nucleus and human placental choline acetyltransferase were purified to electrophoretic homogeneity using ion-exchange and blue dextran-Sepharose affinity chromatography. The molecular weights determined by Sephadex G-150 gel filtration and sodium dodecyl sulfate gel electrophoresis are 67000 plus or minus 3000. N-Ethylmaleimide, p-chloromercuribenzoate, and dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Dithiothreitol reverses the inhibition produced by the latter two reagents. The pKa of the group associated with N-ethylmaleimide inhibition is 8.6 plus or minus 0.3. A chemically competent acetyl-thioenzyme is isolable by Sephadex gel filtration. The enzymes from the brain and placenta are thus far physically and biochemically indistinguishable.