The Receptor‐Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

The rate of dopa synthesis in the rat superior cervical ganglion was increased 4-to 6-fold during continuous electrical stimulation of the cervical sympathetic trunk at 10 Hz for 30 min. This increase was only partially blocked by 3 mM hexamethonium and was not significantly affected by 6 FiM atropine. In the presence of both hexamethonium and atropine, nerve stimulation still produced a 2-to 4-fold increase in dopa synthesis. Physostigmine increased dopa synthesis in both control and stimulated ganglia. This effect of physostigmine was completely blocked by hexamethonium and atropine. Dopa synthesis was also significantly increased when ganglia were incubated in a medium containing an elevated concentration of K+ (55 mM). This stimulatory effect of high K+ was totally dependent on the presence of Ca2+ in the medium, was decreased by 60% by prior decentralization of the ganglion, and was unaffected by hexamethonium and atropine. The data demonstrate that tyrosine hydroxylase activity is rapidly increased after preganglionic nerve stimulation and suggest that this increase is mediated in part by acetylcholine and in part by a second (noncholinergic) transmitter.The effects of an elevated K+ concentration may be mediated both by the release of a noncholinergic transmitter from the preganglionic nerve terminals and by direct depolarization of the ganglionic neurons.The ability to block the postsynaptic consequences of presynaptic nerve stimulation with a specific pharmacological antagonist is a crucial piece of evidence in identifying which transmitter or transmitters act at a particular synapse. Thus, the finding in bullfrog paravertebral ganglia that nicotinic and muscarinic antagonists block most, but not all, of the postsynaptic electrophysiological changes produced by preganglionic nerve stimulation indicates that acetylcholine is not the sole preganglionic neurotransmitter in these ganglia (1).Data presented in the present report indicate that a similar situation holds in the rat superior cervical ganglion (SCG). In this study we examined a postsynaptic biochemical consequence of preganglionic nerve stimulation-namely, the acute increase in the activity of tyrosine 3-monooxygenase (tyrosine hydroxylase; TyrOHase; EC 1.14.16.2). Regulation of this enzyme is of interest because it catalyzes the rate-limiting step in norepinephrine biosynthesis. We have shown previously that pharmacological activation of either nicotinic or muscarinic receptors in the SCG leads to arapid increase in TyrOHase activity (2). We now report that preganglionic nerve stimulation also causes a rapid elevation of enzyme activity and that this increase is mediated in part via a cholinergic (nicotinic) mechanism and in part via a mechanism that is insensitive to both nicotinic and muscarinic antagonists. A preliminary report of this work has been presented (3). METHODSAdult male Sprague-Dawley rats (175-200 g) were housed in individual cages under controlled lighting (12 hr light:12 hr dark) with ad lib access to food and water fo...

Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Acute transsynaptic regulation of tyrosine 3-monooxygenase activity in the rat superior cervical ganglion: evidence for both cholinergic and noncholinergic mechanisms.

Ip¹,

Perlman²,

Zigmond³

1983

“…7 and references therein). Analogues of cAMP have been reported to accelerate catecholamine biosynthesis in intact tissue preparations (8)(9)(10)(11). In broken cell preparations, tyrosine hydroxylase has been shown to be activated, in the presence of ATP and Mg2', by cAMP or cAMP-dependent protein kinase (e.g., see refs.…”

mentioning

confidence: 99%

Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

Albert¹,

Helmer-Matyjek²,

Nairn³

et al. 1984

189

Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the K. for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the K; for dopamine from 4.2 jIM to 47.5 pjM, and produced no change in the Km for tyrosine. Little or no change in apparent V. was observed. These kinetic changes are similar to those seen upon activation of tyrosine hydroxylase by cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps of tyrosine hydroxylase were identical whether the phosphorylation was catalyzed by protein kinase C or by the catalytic subunit of cAMP-dependent protein kinase. Both protein kinases phosphorylated serine residues. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase and activate tyrosine hydroxylase by the same mechanism.Nerve stimulation and neurotransmitters have been demonstrated to accelerate catecholamine biosynthesis in central and peripheral nervous tissue, adrenal medulla, and pheochromocytoma cells (1-4). This acceleration is thought to result from an increase in the catalytic activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) (cf. ref. 5), the rate-limiting enzyme in the biosynthesis of catecholamines (6). Several lines of evidence have suggested that cAMP-dependent phosphorylation may be one means of activation of tyrosine hydroxylase in situ (cf. ref. 7 and references therein). Analogues of cAMP have been reported to accelerate catecholamine biosynthesis in intact tissue preparations (8-11). In broken cell preparations, tyrosine hydroxylase has been shown to be activated, in the presence of ATP and Mg2', by cAMP or cAMP-dependent protein kinase (e.g., see refs. 8 and 12). Finally, purified tyrosine hydroxylase has been shown to be phosphorylated and activated by purified cAMP-dependent protein kinase (13)(14)(15).Recent evidence has suggested a role for calcium as well as cAMP in the activation of tyrosine hydroxylase in situ (16,17 Other reagents and enzymes were obtained from the following commercial sources: H1 histone (III-S), catalase, bovine serum albumin, bovine brain L-a-phosphatidyl-L-serine, diolein, dithioerythritol, EGTA, Tris, and ATP (Sigma); leupeptin (Chemicon, El Segundo, CA); 6,7, Purification of Tyrosine Hydroxylase. Tyrosine hydroxylase was partially purified as described (13) Abbreviation: 6-MePH4, 6-methyltetrahydropterine. 7713The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…The effect of preganglionic stimulation on tyrosine hydroxylase activity appears to be mediated in part by a nicotinic mechanism and in part by a noncholinergic mechanism (16). Nicotinic agonists, muscarinic agonists, and agents that raise ganglionic levels of cAMP all increase tyrosine hydroxylase activity in the ganglion (18)(19)(20). We have recently found that all of these agents also increase the phosphorylation of tyrosine hydroxylase in the SCG (21).…”

mentioning

confidence: 99%

Electrical stimulation increases phosphorylation of tyrosine hydroxylase in superior cervical ganglion of rat.

Cahill¹,

Perlman²

1984

Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) in this tissue. Ganglia were incubated with [32pJp1 for 90 min and were then electrically stimulated via the preganglionic nerve. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labeled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32p into the enzyme was quantitated by densitometry of the radioautograms. Stimulation of ganglia at 20 Hz for 5 min increased the incorporation of 32p into tyrosine hydroxylase to a level 5-fold that found in unstimulated control ganglia. The increase in phosphorylation of tyrosine hydroxylase was dependent on the duration and frequency of stimulation. Preganglionic stimulation did not increase the phosphorylation of tyrosine hydroxylase in a medium that contained low Ca2+ and high Mg2 . Increases in phosphorylation were reversible; within 30 min after the cessation of stimulation, the incorporation of 32p into tyrosine hydroxylase decreased to the level found in unstimulated ganglia. The nicotinic antagonist hexamethonium reduced the increase in 32p incorporation into tyrosine hydroxylase by about 50%, while the muscarinic antagonist atropine had no effect. Thus, preganglionic stimulation appeared to increase the phosphorylation of tyrosine hydroxylase in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Antidromic stimulation of ganglia also increased the phosphorylation of tyrosine hydroxylase. Two-dimensional gel electrophoresis revealed that electrical stimulation also increased the incorporation of 32p into at least six other phosphoproteins in the ganglion.Electrical stimulation of sympathetic nerves increases the rate of catecholamine biosynthesis in these nerves (1). This acceleration of catecholamine synthesis was initially thought to be due to the release of catecholamines from the nerve terminals, resulting in a decrease in intraneuronal catecholamine levels and a relief of the feedback inhibition of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) by catecholamines (2). It is now clear, however, that stimulation of both peripheral and central adrenergic nerves causes a stable activation of tyrosine hydroxylase and a change in the kinetic properties of the enzyme (most commonly, a decrease in the apparent Km for pteridine cofactor and an increase in the Ki for catecholamines) (3-6). There is evidence that the activation of tyrosine hydroxylase may be mediated by phosphorylation of the enzyme. Tyrosine hydroxylase is a phosphoprotein (7) and can be activated in vitro by phosphorylation (8-12). Moreover, phosphorylation of tyrosine hydroxylase is increased in situ in adrenal chromaffin cells by stimuli that increase the rate of catecholamine synthesis in these cells (13,14).Preganglionic stimulation increases tyrosine hydroxylase activity in the...