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Query: UNIPROT:P06889 (Mol)
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We have studied the effects of Ca2+ antagonists and agonists on the development of choline acetyltransferase (ChAT), tyrosine hydroxylase (TOH) and acetylcholinesterase (AChE) in cultures of rat sympathetic neurons maintained for 6-9 days in low K+ (5 mM) or high K+ (35 mM) medium. Previous experiments have shown that high K+ medium increases TOH activity and TOH-mRNA level up to 3.5-fold and depresses the development of AChE, in particular of its asymmetric A12 form. Moreover, high K+ medium inhibits ChAT induction by 90% in muscle-conditioned medium (Raynaud et al., Dev. Biol., 119 (1987) 305-312; 121 (1987) 548-558). None of the Ca2+ antagonists tested affected the development of ChAT, TOH or AChE in low K+ medium. In high K+ medium, nitrendipine (3 microM) or fluspirilene (1 microM) fully restored ChAT induction by conditioned medium to the level observed in low K+ medium. Other drugs (1 microM) gave partial reversion: flunarizine greater than (+)-PN 200-110 greater than (-)-D-888 greater than cinnarizine = lidoflazine. On the other hand, ChAT induction was not restored by a calmodulin inhibitor, calmidazolium (1 microM). Fluspirilene, PN 200-110, and nitrendipine also totally abolished TOH induction by high K+ medium; fluspirilene (1 microM) suppressed the inhibitory effect of high K+ medium on AChE development and restored the development of A12 AChE. Conditioned medium also depresses AChE and blocks the development of A12 AChE (Swerts et al., Dev. Biol., 103 (1984) 230-234), but these effects were insensitive to fluspirilene. The Ca2+ agonist Bay K 8644 (1 microM) potentiated the effects of elevated K+ on both ChAT and TOH. The data suggest that the effects of long-term depolarization on ChAT, TOH and AChE are mediated by Ca2+ entry specifically through voltage-sensitive channels of the L-type. Our results on cultured sympathetic neurons raise the possibility that Ca2+ antagonists, which are widely used clinically, may affect the expression of neurotransmitter phenotypic traits in vivo and interfere with trans-synaptic induction of enzymes.
Brain Res Mol Brain Res 1989 Nov
PMID:The role of Ca2+ channels of the L-type in neurotransmitter plasticity of cultured sympathetic neurons. 257 96

The source of catecholamines in the developing chick heart was investigated by using catecholamine assays and tyrosine hydroxylase assays on hearts from normal and chemically-sympathectomized chick embryos. A biochemical index of sympathetic nerve development in the heart was obtained by monitoring the ability of sympathetic nerves in the atria to take up [3H]-norepinephrine in vitro. Specific neuronal uptake of [3H]-norepinephrine in atria was first detected on incubation day 11 and increased throughout the incubation period. High performance liquid chromatography with electrochemical detection was used to measure the norepinephrine concentration and content of embryonic hearts. The cardiac norepinephrine concentration fluctuated throughout the incubation period but was particularly low (0.01 +/- 0.005 ng/mg wet wt) on incubation days 10 to 13, coincident with the arrival of sympathetic nerves in the heart. The highest norepinephrine concentration was measured on incubation day 7 (2.09 +/- 0.50 ng/mg wet wt) prior to the arrival of sympathetic nerves in the heart. Sympathetic nerve axotomy produced by chronic treatment with 6-hydroxydopamine reduced [3H]-norepinephrine uptake in atria and norepinephrine concentration in whole hearts on incubation day 20 to 33 and 47% of control, respectively. Tyrosine hydroxylase activity was detected in normal hearts on incubation day 7, 3 to 4 days before the heart is innervated by sympathetic nerves. Tyrosine hydroxylase activity persisted in the heart on incubation day 20, despite treatment with 6-hydroxydopamine on incubation days 13 to 19. The tyrosine hydroxylase activity in 6-hydroxydopamine lesioned hearts was not significantly different from saline-treated controls. This data indicates that tyrosine hydroxylase activity is present in the immature chick heart prior to the arrival of sympathetic innervation and following chemical sympathectomy; hence, an extraneuronal source of tyrosine hydroxylase, the rate limiting enzyme for catecholamine biosynthesis, exists in the embryonic chick heart.
J Mol Cell Cardiol 1985 Apr
PMID:Endogenous tyrosine hydroxylase activity in the developing chick heart: a possible source of extraneuronal catecholamines. 286 86

A specific antiserum was used to compare phosphorylation of tyrosine hydroxylase (TH) (EC 1.14.16.2, tyrosine 3-monooxygenase) as regulated by elevated K+ and nerve growth factor (NGF) in cultured PC12 pheochromocytoma cells. Exposure of cultures to either elevated K+ or to NGF significantly enhanced the incorporation of [32P]orthophosphate into TH. The effect of elevated K+ was evident at 10 mM and was maximal by 40-80 mM. Increased phosphorylation of TH was detected at 0.1 nM (3 ng/ml) NGF and reached a maximal level by 0.3-1 nM (10-30 ng/ml) NGF. Elevated K+ showed a biphasic time course of action with one maximum of phosphorylation at about 30 sec of exposure and a second after about 10 min of exposure. In contrast, the NGF effect showed an initial lag of several minutes followed by a monophasic increase in phosphorylation to reach a plateau. Both treatments enhanced TH activity, but in each case the time courses of this did not strictly correlate with that of phosphorylation. The effect of elevated K+ on TH phosphorylation required the presence of extracellular Ca2+ and was suppressed by trifluoperazine (100 microM). N-(6-Aminohexyl)-5-(chloronaphthalene)-1-sulfonamide (W-7) (100 microM), a potent inhibitor of calmodulin activity, also blocked the enhancement of phosphorylation by elevated K+, whereas N-(6-aminohexyl)-1-(naphthalene)sulfonamide (W-5) (100 microM), a less potent analogue of W-7, did not. In contrast to these findings, the increase in TH phosphorylation brought about by NGF did not require extracellular Ca2+, and was only slightly affected by trifluoperazine or W-7. When TH phosphorylated under various conditions (control medium, elevated K+, NGF) was subjected to peptide mapping after exposure to Staphylococcus aureus protease V8, multiple phosphorylated peptides were observed. Elevated K+ and NGF each produced increases in labeling of each of the peptides. However, the relative degree of labeling of different peptides was distinct for each condition. These data suggest that elevated K+ and NGF bring about rapid enhancement of the phosphorylation of TH by means of different mechanisms.
Mol Pharmacol 1985 Aug
PMID:Regulation of tyrosine hydroxylase phosphorylation in PC12 pheochromocytoma cells by elevated K+ and nerve growth factor. Evidence for different mechanisms of action. 286 75

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.
 ...
PMID:Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells. 286 97

Intracellular activation and phosphorylation of tyrosine hydroxylase (TH; E.C. 1.14.16.2) in the median eminence of the rat brain were investigated. The in situ activity of TH was assayed by the accumulation of L-dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic fragments incubated in the presence of NSD 1015. When hypothalamic fragments were incubated with veratridine (0-10(-3) M), maximal stimulation of TH activity was observed at 10(-4) M. The mean concentration of DOPA in the median eminence of hypothalamic fragments incubated with 10(-4) M veratridine was 3 times that seen in its absence. Phosphorylation of TH in the median eminence was evaluated by autoradiographic quantification of [32P]TH in 32P-labelled median eminence tissue. The amount of [32P]TH in 32P-labelled median eminence incubated with 10(-4) M veratridine was 2 times that seen in the absence of veratridine. These data are consistent with the view that in the median eminence phosphorylation and activation of TH are linked events and that phosphorylation may be a means of regulating the biosynthesis of dopamine in this region of the brain.
Mol Cell Endocrinol 1986 Jun
PMID:In situ activity and phosphorylation of tyrosine hydroxylase in the median eminence. 287 94

Tyrosine hydroxylase isolated from striatal synaptosomes exhibits biphasic Lineweaver-Burk kinetics for its tetrahydrobiopterin cofactor, consistent with multiple Km forms of the enzyme. Incubation of striatal synaptosomes with forskolin (EC50 0.45 microM) or dibutyryl cyclic AMP (EC50 1.2 mM), results in activation of tyrosine hydroxylase, isolated from these synaptosomes via conversion of the enzyme to a single low Km form (Km 40 microM). The activation of synaptosomal tyrosine hydroxylase by forskolin or dibutyryl cyclic AMP is not additive and is similar to activation seen with cyclic AMP-dependent protein kinase phosphorylation of purified tyrosine hydroxylase. The addition of dopamine (IC50 1.0 microM) (with nomifensine and pargyline) or apomorphine (IC50 30 nM) to the synaptosomal incubation medium blocks the activation of tyrosine hydroxylase by forskolin. This effect of dopamine and apomorphine can in turn be blocked by preincubation of the synaptosomes with the dopamine receptor antagonist haloperidol (IC50 30 nM and 4.5 nM, respectively) or chlorpromazine (IC50 50 nM versus apomorphine). In contrast to the forskolin data above, dopamine failed to block the activation of tyrosine hydroxylase by dibutyryl cyclic AMP. Addition of dopamine to the tyrosine hydroxylase assay, in amounts equivalent to that carried over from the synaptosomal incubation with the tyrosine hydroxylase, had no effect on forskolin-activated enzyme. The observations that dopamine and apomorphine can block forskolin activation of tyrosine hydroxylase, that this blockade can in turn be prevented by preincubation with haloperidol or chlorpromazine, and that the amount of dopamine required for blockade of forskolin activation in synaptosomes has no effect on tyrosine hydroxylase when added to the enzyme assay constitute the first clear evidence of a presynaptic dopamine receptor (autoreceptor). This autoreceptor regulates the activity of tyrosine hydroxylase by preventing or reversing cyclic AMP-dependent activation of the enzyme, probably through a decrease in the phosphorylation state of tyrosine hydroxylase. Failure of dopamine to block dibutyryl cyclic AMP activation of tyrosine hydroxylase suggests that, if forskolin and dibutyryl cyclic AMP activate tyrosine hydroxylase through identical changes in phosphorylation state, then autoreceptor regulation of tyrosine hydroxylase must occur through a decrease in cyclic AMP levels.
Mol Pharmacol 1986 Jun
PMID:Dopamine autoreceptor regulation of the kinetic state of striatal tyrosine hydroxylase. 287 88

Incubation of rat pheochromocytoma PC12 cells with the calcium ionophore, A23187 (10(-5) M), 56 mM K+, or dibutyryl cAMP (2 mM) is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in the cells. Both the activation and the increased phosphorylation of tyrosine hydroxylase produced by A23187 and 56 mM K+ are dependent on the presence of extracellular calcium, whereas similar effects produced by dibutyryl cAMP are independent of calcium. The effects of 56 mM K+ plus dibutyryl cAMP or A23187 plus dibutyryl cAMP on the activation and phosphorylation of tyrosine hydroxylase are additive. In contrast, the effects of 56 mM K+ plus A23187 on either the activation or the phosphorylation of the enzyme are not additive. Following stimulation of intact PC12 cells with 32Pi, in order to label ATP stores, and tryptic digestion of the phosphorylated enzyme, separation of the tryptic phosphopeptides by high pressure liquid chromatography yields four distinct 32P-peptide peaks. Incubation of the cells in the presence of either 56 mM K+ or A23187 is associated with increased 32Pi incorporation into three peptides whereas, in the presence of dibutyryl cAMP, increased 32Pi incorporation is observed in only one of these peptides. When tyrosine hydroxylase purified from rat pheochromocytoma tumor is incubated in vitro with [gamma-32P]ATP and either cAMP-dependent or calcium/calmodulin-dependent protein kinase under appropriate conditions, increased phosphorylation of tyrosine hydroxylase is observed. However, even though in vitro phosphorylation by cAMP-dependent protein kinase is associated with activation of tyrosine hydroxylase, in vitro phosphorylation by calcium/calmodulin-dependent protein kinase does not lead to activation of the enzyme. Tryptic digestion of tyrosine hydroxylase phosphorylated by calcium/calmodulin-dependent protein kinase yields three distinct 32P-peptide peaks, which are identical to those phosphorylated by treatment of intact PC12 cells with either high K+ or A23187. In contrast, cAMP-dependent protein kinase phosphorylates only one peptide, which is identical to that phosphorylated by treatment of the intact cells with dibutyryl cAMP. These results indicate that tyrosine hydroxylase is activated and phosphorylated at multiple sites in PC12 cells exposed to 56 mM K+ or A23187. The results suggests that the in situ phosphorylation of these sites is catalyzed by calcium/calmodulin-dependent protein kinase; however, phosphorylation by this protein kinase is not sufficient to activate the enzyme.
Mol Pharmacol 1986 Nov
PMID:Phosphorylation of tyrosine hydroxylase on at least three sites in rat pheochromocytoma PC12 cells treated with 56 mM K+: determination of the sites on tyrosine hydroxylase phosphorylated by cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. 287 91

When rat pheochromocytoma PC18 cells are exposed to the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, there is a marked increase in the level of a single RNA species that hybridizes to the recombinant plasmid pTH.4, which contains sequences complementary to the RNA coding for tyrosine hydroxylase. This RNA species is 1800-1900 nucleotides in length and is presumably identical to an RNA species of similar size, isolated from rat pheochromocytoma PC8b cells and shown to code for tyrosine hydroxylase. Using RNA dot hybridization to quantitate the relative level of this tyrosine mRNA species, time course studies show that this mRNA increases relatively rapidly in PC18 cells treated with either 8-bromocyclic AMP or dexamethasone. A new steady state level of tyrosine hydroxylase mRNA is achieved after 6 hr or 12-24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, respectively. The changes in the level of the mRNA slightly precede the changes in the rate of synthesis of tyrosine hydroxylase in cells treated with these inducing agents. After 24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, the increases in the level of tyrosine hydroxylase mRNA are identical to the increases in the rate of synthesis of the enzyme in the cells. In cells treated simultaneously with both 8-bromocyclic AMP and dexamethasone, the increases in the enzyme level and rate of synthesis of tyrosine hydroxylase are approximately equal to the sum of the increases in these parameters observed in cells treated with either inducing agent alone. In contrast, there is not an additive increase in the level of tyrosine hydroxylase mRNA in cells treated with both inducing agents. This lack of an additive increase in mRNA for tyrosine hydroxylase is observed in total cellular RNA samples or in cytoplasmic RNA samples. Our results suggest that in cells exposed to elevated levels of either cyclic AMP or glucocorticoids, tyrosine hydroxylase is induced by a mechanism which increases the level of its mRNA, resulting in an increased rate of synthesis of the enzyme. However, in cells exposed to elevated levels of both cyclic AMP and dexamethasone, tyrosine hydroxylase enzyme levels are regulated by multiple mechanisms, one of which regulates the rate of synthesis of the enzyme without affecting the level of its mRNA.
Mol Pharmacol 1986 Nov
PMID:Induction of mRNA for tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase synthesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents. 287 92

The irreversible dopamine (DA) receptor antagonist N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine the extent of receptor reserve at DA autoreceptors regulating in vivo tyrosine hydroxylase activity. Rats were treated with vehicle or EEDQ (1 X 0.5-2 X 6 mg/kg, subcutaneously) and, 24 hr later, dose response curves were generated for DA agonist reversal of gamma-butyrolactone-induced striatal L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation. Double reciprocal plots were obtained of equieffective doses of agonist required to elicit response at several levels of effect before and after partial irreversible receptor inactivation. A pseudo-dissociation constant (pseudo-KA, in units of dose) and the fraction of receptors remaining active (q) were determined; these values were then used to calculate the relationship between receptor occupancy and response. The ED50 (1 microgram/kg) for the full DA receptor agonist N-propylnorapomorphine (NPA) was shifted 2.8, 4.8-, and 11.3-fold to the right after partial irreversible receptor blockade which left the fraction of receptors remaining active (q) at 0.37, 0.17 and 0.058, respectively. Corresponding maximal reversal of L-DOPA accumulation was 100, 77, and 58%, indicating a nonlinear relationship between receptor occupancy and response for NPA and the presence of a large receptor reserve; maximal and half-maximal responses were calculated to require occupancy of 30 and 3.8% of the total receptor pool, respectively. Dose response curves were also obtained for the DA autoreceptor-selective agents EMD 23,448 and (+)- and (-)-3-PPP before and after EEDQ treatment. In controls, EMD 23,448 and (+)-3-PPP, like NPA, completely reversed striatal gamma-butyrolactone-induced L-DOPA accumulation, whereas the maximal effect of (-)-3-PPP was 52% reversal. After EEDQ treatment (6 mg/kg), EMD 23,448 and (+)-3-PPP showed relatively small shifts in ED50 values. Furchgott analysis demonstrated that all three atypical agents are partial agonists at the DA autoreceptor with efficacies of 0.19 (EMD 23,448), 0.12 [(+)-3-PPP], and 0.05 [(-)-3-PPP] relative to NPA. The presence of a larger receptor reserve at pre-versus postsynaptic D2 DA receptors and the partial agonist character of drugs such as EMD 23,448 and the enantiomers of 3-PPP may account for their autoreceptor selectivity.
Mol Pharmacol 1987 Jun
PMID:Relationship between receptor occupancy and response at striatal dopamine autoreceptors. 288 34

A cDNA clone for rabbit tryptophan hydroxylase was used as a probe to identify human tryptophan hydroxylase gene fragments in a panel of hamster-human somatic cell hybrids and determine its chromosomal location in man. A single locus was identified for tryptophan hydroxylase on chromosome 11. Tryptophan hydroxylase is a member of the superfamily of pterin-dependent aromatic amino acid hydroxylases which includes tyrosine hydroxylase, located at 11p15.5-p15, and phenylalanine hydroxylase, located at 12q22-q24.1 in human. The locations of these genes and the evolutionary distance between their sequences suggest that at least three distinct genetic events have occurred during the evolution of the aromatic amino acid hydroxylase superfamily: two sequential gene duplications giving rise to the three distinct hydroxylase loci, and a translocation which separated the tryptophan and tyrosine hydroxylase loci on chromosome 11 from the phenylalanine hydroxylase locus on chromosome 12.
Somat Cell Mol Genet 1987 Sep
PMID:Assignment of human tryptophan hydroxylase locus to chromosome 11: gene duplication and translocation in evolution of aromatic amino acid hydroxylases. 288 73


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