Choline potentiates the trans-synaptic induction of adrenal tyrosine hydroxylase by reserpine, probably by enhancing the release of acetylcholine.

Choline or reserpine causes a trans-synaptic induction of adrenal tyrosine hydroxylase activity. To examine further the relationship between impulse flow, mediated by reserpine, and the effects of choline, we injected rats with both agents concurrently. Their simultaneous administration elevated tyrosine hydroxylase activity to a level higher than the increase caused by either agent alone. These observations suggest that reserpine acts by increasing the rate of impulse flow, while choline acts by increasing the amount of ACh released per impulse.     

ORGAN CULTURE OF THE RAT ADRENAL MEDULLA: A MODEL SYSTEM FOR THE STUDY OF TRANS-SYNAPTIC ENZYME INDUCTION

—It was the aim of the present study to develop organ culture conditions for the rat adrenal medulla which are representative for the in vivo situation. This is a prerequisite for studying the complex processes involved in trans-synaptic enzyme induction. The processes of trans-synaptic enzyme induction initiated in vivo by injecting 5 mg/kg of reserpine 2 h prior to the removal of the adrenal medulla, continued in this culture system and final levels of tyrosine hydroxylase were comparable to those seen in vivo. That these culture conditions are representative for the in vivo induction is also supported by the fact that transection of the splanchnic fibres supplying the adrenal medulla or administration of actinomycin D prior to reserpine abolished the rise in tyrosine hydroxylase activity not only in vivo, but also in culture. The findings that high concentrations (0·29 mm ) of corticosterone in the culture medium inhibited the increase in tyrosine hydroxylase activity caused by reserpine support the hypothesis that glucocorticoids act as modulatory agents in trans-synaptic enzyme induction. This inhibition was exhibited only when corticosterone was added at the initiation of the culture period. If added 2 or 4 h after the beginning of the culture period there was little or no effect on the subsequent increase of tyrosine hydroxylase.     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold

When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.     

Influence of hypothyroidism on the ontogenic development of tyrosine hydroxylase induction in the adrenal glands of the rat

The ontogenic development of the transsynaptic induction of adrenal tyrosine hydroxylase (TH), evoked by reserpine and nicotine was studied in control and hypothyroid young rats, aged 3-52 days. The enzymatic induction was measured as an increase in the enzyme activity, since this increase was shown to be impaired either by an inhibitor of RNA synthesis or by a ganglionic blocker. In the control animals, TH induction elicited by reserpine increases between 3 and 32 days of age. In the hypothyroid rats, the enzymatic induction is impaired up to 32 days; at 52 days the induction is similar in both groups of animals. When nicotine is used as a stimulating agent, hypothyroidism still impairs the enzymatic induction at 5 and 21 days, indicating that at least one of the mechanisms inhibited by hypothyroidism is localized in the adrenal chromaffin cells. The present results, taken together with previous findings dealing with adrenal epinephrine secretion, show that the thyroid hormones play a crucial role in the responses of the adrenal medulla to a stimulation in the developing rat, while they have no effect in the adult.     

The effects of reserpine and haloperidol on tyrosine hydroxylase activity in the brains of aged rats

Many neurotransmitter systems appear to be altered with aging. The effects of aging on the regulation of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines in the brain has been examined. The endogenous basal activity of tyrosine hydroxylase was lower in the hypothalamus of 24 month old Fisher 344 rats than in the hypothalamus of 3 month old or 6 month old animals. There was no difference in the basal activity of tyrosine hydroxylase in the locus ceruleus, frontal cortex, hippocampus, substantia nigra, or the striatum of rats of ages 3 months, 6 months and 24 months. Tyrosine hydroxylase activity was increased in the striatum of 3 month old (60%) and 6 month old (28%) rats after treatment with haloperidol or reserpine, whereas no change in enzyme activity followed administration of these drugs to 24 month old animals. In conclusion, increases in tyrosine hydroxylase activity in the brain that normally occur in the striatum of 3 month old rats after haloperidol or reserpine treatment are significantly decreased in 6 month old rats and not apparent in 24 month old rats.     

Molecular and physiological properties of tyrosine hydroxylase induced by reserpine in rat adrenal gland

Newly synthesized tyrosine hydroxylase (TH) induced by reserpine was compared with the enzyme in control rats in terms of the molecular and physiological properties. When repeated doses of reserpine were given at daily intervals for three days, the enzyme activity measured in homogenates of the adrenal glands was increased 3-fold. Furthermore, when TH in the adrenal glands from both control and reserpine-treated rats was purified, both total activity of the enzyme and the enzyme protein content purified from reserpine-treated rats were also about 3-fold higher than those of the control rats. The two purified enzymes revealed similar properties; a single subunit with a Mr of 60,000 was observed by SDS polyacrylamide gel electrophoresis, and the Km value for a pterin cofactor, 6-methyl-tetrahydropterin was about 300 microM. In contrast, in situ TH activity measured under physiological conditions at pH 7.2 in adrenal tissue slices was elevated 6-fold by reserpine pretreatment for 3 days, and was stimulated by carbachol (0.1 mM) and elevated K+ (52 mM) in a roughly proportional rather than additive way relative to slices from untreated rats. These results indicate that newly synthesized TH induced by reserpine in rat adrenal gland had similar properties as the enzyme in control rats and that reserpine increased not only the amount of TH molecules but also the in situ activity of TH. Since reserpine also increases the biosynthesis of tetrahydrobiopterin as demonstrated by Viveros and co-workers, this 6-fold increase in in situ TH activity may depend both upon the 3-fold increase in the amount of enzyme molecules and upon the increase of the physiologically available tetrahydrobiopterin in the adrenal gland.     

Regulation of tryptophan and tyrosine hydroxylase

The synthesis of the neurotransmitters serotonin, norepinephrine, and the dopamine is regulated by the initial amino acid hydroxylases. Little is known about the factors that regulate the level of tryptophan hydroxylase in tissue. However, the level of tyrosine hydroxylase is regulated by transsynaptic induction. Acute regulation of in vivo hydroxylase activity appears to be by substrate availability in the case of tryptophan hydroxylase and possibly by feedback inhibition with tyrosine hydroxylase. A newly described phenomenon which has been termed “receptor mediated feedback inhibition” involving neuronal feedback regulation of the activity of both tyrosine and tryptophan hydroxylase may also have an important role.     

Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

MINIMUM DURATION OF TRANS-SYNAPTIC STIMULATION REQUIRED FOR THE INDUCTION OF TYROSINE HYDROXYLASE BY RESERPINE IN THE RAT SUPERIOR CERVICAL GANGLION

—The period during which trans-synaptic stimulation is required by the rat superior cervical ganglion for induction of tyrosine hydroxylase by reserpine has been studied. Ganglia were decentralized on one side at various times before or after an injection of reserpine. The tyrosine hydroxylase activity of the denervated and control ganglia was assayed 72 h after drug treatment. When decentralization was performed 8 h after an injection of reserpine the increase in tyrosine hydroxylase activity was blocked in the denervated ganglia. Decentralization 12 h after reserpine treatment or later had no effect on the enzyme induction. The actual increase in tyrosine hydroxylase activity occurred between 24 and 48 h after injection of reserpine.     

Central Regulation of Adrenal Tyrosine Hydroxylase: Effect of Induction on Catecholamine Levels in the Adrenal Medulla and Plasma

The effect of induction of adrenal tyrosine hydroxylase (TH) by various centrally acting drugs on catecholamine levels in adrenal and plasma was investigated in rats. All the drugs tested, namely oxotremorine, Piribedil, B-HT 920, and HA-966, produced significant increases in adrenal dopamine content and plasma epinephrine level. Denervation of the adrenal abolished the increase in adrenal dopamine as it did the induction of tyrosine hydroxylase. The results suggest that the induced increase of adrenal TH activity, as mediated by certain drugs, results in an elevation of the plasma epinephrine level and that the adrenal dopamine content is a better indicator of the catecholamine-synthesizing capacity of the adrenal medulla than are the other catecholamines.     

Appearance of enkephalin-immunoreactivity in rat adrenal medulla following treatment with nicotinic antagonists or reserpine

Various neuroendocrine factors known to be important in the regulation of adrenal catecholamine biosynthesis were investigated for possible effects on enkephalin-like immunoreactivity (Enk-IR) in the adrenal medulla of the rat. In normal rats, the adrenal chromaffin cells were not stained for either methionine (met-) or leucine (leu-) Enk-IR. Staining for Enk-IR appeared in many chromaffin cells following denervation of the adrenal or treatment of rats with the nicotinic receptor antagonists chlorisondamine or pempidine. These observations suggest that splanchnic nerve activity normally depresses the levels of enkephalin-like peptides in chromaffin cells through a trans-synaptic mechanism involving acetylcholine release and nicotinic receptor stimulation. Paradoxically, treatment with reserpine also increased Enk-IR in chromaffin cells. However, this increase did not appear to result from the well known effect of reserpine to increase presynaptic nerve firing and tyrosine hydroxylase (TOH) activity, since no increase in Enk-IR was observed following treatment with phenoxybenzamine or 6-hydroxydopamine, drugs which also increase TOH activity through trans-synaptic mechanisms. The reserpine effect also did not appear to be mediated by a stress-induced increase in glucocorticoid hormones since glucocorticoid therapy alone did not increase adrenal Enk-IR. It is suggested that the increase in adrenal Enk-IR following reserpine may result from a direct action of reserpine on chromaffin cells.     

Alterations in norepinephrine,serotonin, c-AMP,and transsynaptic induction of tyrosine hydroxylase after spinal cord transection in the rat

Tyrosine hydroxylase (TH) activity and the concentrations of norepinephrine (NE), serotonin (5-HT), and cyclic adenosine 3, 5-monophosphate (c-AMP) were measured in the heart, adrenals, and brain stem of paraplegic rats. Following spinal cord transection NE concentration in the heart dropped to 30% within 24 hours and that of 5-HT decreased to 60% of control. Tyrosine hydroxylase activity and c-AMP levels in the brain stem were elevated while NE concentration remained low. At seven days, however, NE and 5-HT levels were higher than in controls while TH activity and c-AMP concentration dropped to control levels. The increase in TH activity in the brain stem may be due to curtailed end-product feedback inhibition and to reduced receptor activation. The sustained induction of the adrenal TH is probably a consequence of a continual stimulation of splanchnic nerves.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

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)     

Increase of ornithine decarboxylase activity elicited by reserpine in the peripheral and central monoaminergic systems of the rat

Ornithine decarboxylase activity was increased about tenfold in adrenal glands and in brain regions preponderantly containing aminergic neurons, by a single dose of 16 mol/kg of reserpine. Maximal enzyme activity in the adrenal glands was observed at about 8 hr after reserpine administration. The ornithine decarboxylase activity-time curves in the brain regions showed a concomitant polyphasic course, with the highest maximum at 12 hr postinjection. Ornithine decarboxylase induction is discussed as an early event in the cascade of molecular events preceding the induction of cell typic enzymes.     

INCREASED CEREBRAL EXCITABILITY CAUSED BY p-CHLOROPHENYLALANINE IN YOUNG RATS

—Single intraperitoneal doses of p-chlorophenylalanine (p-CP; 100 mg/kg or more), lowered the threshold of flurothyl-induced seizures in 1- and 2-week old rats after 24 h, but not after 4 or 72 h. In older rats there was no change in cerebral excitability after a single dose, but the seizure threshold was lowered after several daily doses. Rats given p-CP in lower doses daily from birth exhibited lowered seizure thresholds only until 3-4 weeks of age. Depletion of brain serotonin and inhibition of liver phenylalanine hydroxylase by p-CP exhibited time courses and dose responses similar to those reported for older animals. Activity of phenylalanine hydroxylase returned to normal more rapidly than the content of serotonin after a dose of p-CP, and recovery of normal seizure threshold seemed to be more closely associated with recovery of the hepatic enzyme than with restoration of the brain amine. It appears, therefore, that hyperphenylalanemia or some consequence of it may be an important factor contributing to increased cerebral excitability in p-CP intoxication and possibly in clinical phenylketonuria and that depletion of brain serotonin may not be the only or even the predominant cause of the changes in cerebral excitability in these conditions.     

Cretinism: influence on rate-limiting enzymes of amine synthesis in rat brain.

The effect of neonatal hypothyroidism on tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activity, as well as on water content, was studied in different regions of the developing rat brain. Neonatal hypothyroidism, induced by daily treatment with propylthiouracil starting at birth, led to a cretinoid syndrome with a marked impairment of body and brain growth. Compared to control littermates, 30- and 45-day-old cretinous rats had elevated levels of water in the brain stem. The activities of TH and TPH were increased in a time-dependent manner in the brain stem, basal ganglia and hypothalamus of maturing cretinous animals. The increased activity of these rate-limiting enzymes of mono-amine synthesis may account for the elevated levels of brain norepi-nephrine and serotonin in rats subjected to neonatal hypothyroidism.     

Alterations in Tyrosine Hydroxylase and Monoamine Oxidase Activity in Blood Vessels

THE enzyme tyrosine hydroxylase¹ (TH), which has been reported as the rate limiting step in noradrenaline biosynthesis, can be modified by nerve stimulation, cold^2,3, exercise⁴, reser-pine, phenoxybenzamine and monoamine oxidase inhibitors^5–7. These treatments affect not only the enzyme in vitro but also catecholamine synthesis in vivo. Much of this information has come from studies with heart, brain, adrenals and spleen, but we found that blood vessels contain appreciable concentrations of noradrenaline⁸ and synthesize it in vivo from its precursor tyrosine. We now report that blood vessels have higher tyrosine hydroxylase activity than the heart and that this activity can be modified by reserpine and L-dihydroxyphenylalanine (L-dopa). Furthermore, the activity of tyrosine hydroxylase in the blood vessels of a spontaneously hypertensive rat differs from that in its normotensive control. We also found that the activity of the enzyme monoamine oxidase in the vasculature was affected by drugs and changes in blood pressure.