Modulatory role of catecholamines on tyrosine hydroxylase induction

Summary The possible modulatory role of cytoplasmic catecholamines on tyrosine hydroxylase induction was studied. Rat superior cervical ganglia were kept in organ culture and after 48 h tyrosine hydroxylase activity was determined. Exposure to 10^–4 M carbachol during 4 h almost doubled the control activity. Incubation with 10^–5 M noradrenaline or 10^–5 M dopamine impaired the carbachol-mediated induction of the enzyme. This effect was not blocked by 10^–7 M propranolol, 2.4×10^–6 M haloperidol or 3.1×10^–6 M phentolamine. Inhibition of monoamine oxidase activity by 5.1×10^–4 M pargyline inhibited the effect of carbachol. When the pool of endogenous catecholamines was decreased by -methyl-p-tyrosine, carbachol induced tyrosine hydroxylase to the same extent as in non-depleted ganglia. It is suggested that the long-term regulation of tyrosine hydroxylase is modulated by a strategic cytoplasmic pool of catecholamines.     

Trans-synaptic regulation of tyrosine hydroxylase activity in adrenergic neurones: Effect of potassium concentration on cultured sympathetic ganglia

Summary Superior cervical ganglia from 6-day and 4-week-old mice showed increased tyrosine hydroxylase activity when cultured for 48 h in media containing 5 or 15 times the normal potassium concentration. Increased sodium concentration had no such effect. These results suggest that depolarisation of adrenergic neurones may be important in the trans-synaptic regulation of enzyme activity in both adult and neonatal ganglia.     

Neurally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit?

Summary Cold-exposure of rats for 1–4 days leads to a gradual increase in tyrosine hydroxylase and dopamine -hydroxylase activity in superior cervical ganglia and adrenals. This increase is neurally mediated, since it can be abolished by decentralization of the ganglia and transsection of the splanchnic fibres supplying the adrenal medulla. Enzyme kinetic data suggest that the increased enzyme activity results from an increased synthesis of enzyme protein. The activity of dopa decarboxylase, the third enzyme involved in the synthesis of norepinephrine, remains unchanged both in ganglia and in adrenals. In the adrenals, the gradual rise in tyrosine hydroxylase and dopamine -hydroxylase activity is virtually parallel and reaches more than twice the control values. In the superior cervical ganglion, however, there is a marked difference between the rise in activity of the two enzymes. The increase in tyrosine hydroxylase activity amounts to 217% after 4 days of cold-exposure, that of dopamine -hydroxylase to 131% of controls (100%) only. The determination of enzyme turnovers by measuring the decay in activity after inhibition of protein synthesis by cycloheximide was attempted but the turnover of tyrosine hydroxylase both in adrenals and superior cervical ganglia was too slow to be determined by this method. The same was true for dopamine -hydroxylase in the adrenal medulla. However, in the superior cervical ganglion the turnover of the latter enzyme could be determined; the half-life amounts to 13 h. The fact that the turnover of dopamine -hydroxylase in the superior cervical ganglion is more rapid than that of tyrosine hydroxylase could explain the difference in the increase in enzyme activity in spite of a similar rise in the rate of synthesis. It is concluded that the enzymes involved in the synthesis of norepinephrine are not regulated as an operational unit, but that the results are compatible with the assumption that the synthesis of the two enzymes, tyrosine hydroxylase and dopamine -hydroxylase, which are specifically located in adrenergic neurons, may be regulated by a common mechanism.     

Tyrosine hydroxylase regulation in rat striatal and olfactory tubercle slices--I. Activation induced by exposure to a calcium-free and high-magnesium medium.

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca²⁺-free KRP medium both in the presence and absence of high Mg²⁺ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca²⁺ from the incubation medium resulted in a moderate increase in the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, and a significant increase in the activity of tyrosine hydroxylase isolated from olfactory tubercle slices. The presence of Mg²⁺ (12 mM) in the Ca²⁺-free KRP medium produced a further increase in the activity of the enzyme isolated both from striatal and olfactory tubercle slices. The per cent of stimulation of enzyme activity induced by incubating the slices in a Ca²⁺-free, high-Mg²⁺ KRP medium was maximal when assayed at pH 7.0. The Mg²⁺-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca²⁺ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg²⁺ (5–20 mM) to striatal and olfactory tubercle homogenates also resulted in an increase in the activity of tyrosine hydroxylase. The addition of high concentrations of Ca²⁺ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg²⁺ (10 mM). Incubation of striatal slices in a Ca²⁺-free, high-Mg²⁺ KRP medium produced changes in the kinetic properties of tyrosine hydroxylase. The apparent _m of the enzyme for 6-methyl-5,6,7,8-tetrahydropterine HCl (6-MPH₄) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the V_max. However, the K_i of the enzyme for dopamine (DA) was unchanged. Magnesium ions (12 mM) added to KRP-high K⁺ (55 mM) medium blocked the activation of tyrosine hydroxylase induced by K⁺-depolarization of striatal slices. However, Mg²⁺ (12 mM) addition to the incubation medium did not block but actually further increased the tyrosine hydroxylase activation that results after incubating striatal slices in a KRP medium enriched with dibutyryl cAMP (1 mM). Moreover, the stimulating effect on tyrosine hydroxylase observed when assays were conducted in the presence of Mg²⁺, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca²⁺-free, high-Mg²⁺ KRP medium. The results reported in this work clearly demonstrate that a kinetic activation of tyrosine hydroxylase in dopaminergic nerve terminals can occur under conditions of diminished extracellular Ca²⁺ and blockade of transmitter release. Cyclic AMP does not seem to play a role in the tyrosine hydroxylase activation induced by incubation of slices in Ca²⁺-free and high-Mg²⁺ medium. The results support the hypothesis that the increase in DA biosynthesis observed in dopaminergic neurons after inhibition of impulse flow may result primarily as a consequence of a diminished entrance of Ca²⁺ into the nerve terminal.     

Cyp3a11‐mediated testosterone‐6β‐hydroxylation decreased,while UGT1a9‐mediated propofol O‐glucuronidation increased,in mice with diabetes mellitus

The db/db mouse is one of the most popular animal models for type 2 diabetes mellitus, but changes in the activities of important P450s and UGTs are still not completely clear. This study was designed to investigate the alterations of major hepatic cytochrome P450s and UDP‐glucuronyltransferase enzymes in db/db mice. Mouse liver microsomes (MLMs) were obtained from male db/db mice and their wild type littermates. After incubation of the substrates separately with MLMs, the samples were pooled and analysed by high‐throughput liquid chromatography–tandem mass spectrometry system for the simultaneous study of nine phase I metabolic reactions and three glucuronidation conjugation reactions to determine the activity of the metabolic enzymes. Compared with normal controls, the Cl_int estimate for testosterone‐6β‐hydroxylation was lower (46%) (p < 0.05), while the V_max and Cl_int estimates for propofol O‐glucuronidation were 5‐fold higher (p < 0.01) in the liver microsomes from db/db mice. There was no significant difference in phase I metabolic reactions of phenacetin‐O‐deethylation, coumarin‐7‐hydroxylation, bupropion‐hydroxylation, omeprazole‐5‐hydroxylation, dextromethorphan‐O‐demethylation, tolbutamide‐4‐hydroxylation, chlorzoxazone‐6‐hydroxylation and midazolam‐1‐hydroxylation and in glucuronidation reactions of estradiol 3‐O‐glucuronidation, and 3‐azido‐3‐deoxythymidine glucuronidation. The data suggest that, in db/db mice, the activity of Cyp3a11, catalysing testosterone‐6β‐hydroxylation, decreased, while the activity of UGT1a9, catalysing propofol O‐glucuronidation, increased. Copyright © 2016 John Wiley & Sons, Ltd.     

Dependence of 5-HT and catecholamine synthesis on concentrations of precursor amino-acids in rat brain

Summary The short-term influence of varying concentrations of the precursors tryptophan and tyrosine on the formation of 5-hydroxytryptophan and Dopa, respectively, in three different rat-brain regions was investigated. The concentrations of the precursors were either increased by the intraperitoneal administration of the respective precursor or decreased by loading with large neutral amino acids competing with the precursors for the same carrier mechanisms.The formation of 5-hydroxytryptophan was found to depend on the level of tryptophan in the brain in a manner predicted from published kinetic data, except when large doses of non-precursor amino acids had been given (>300 mg/kg). In the latter case the hydroxylation of tryptophan was less rapid than expected. The apparent K _m of tryptophan hydroxylase calculated from these data was about 25 M, which is in reasonably good agreement with earlier published in-vitro and in-vivo data.The formation of Dopa likewise depended on the level of the precursor tyrosine in a predictable manner, in this case without any anomalous results after large doses of non-precursor amino acids. The apparent K _m of tyrosine hydroxylase was calculated from these invivo observations to be about 25 M, which is in fairly close agreement with published in-vitro data.It is concluded that under normal conditions tryptophan hydroxylase in the rat brain is about half-saturated with its amino-acid substrate, whereas tyrosine hydroxylase appears to be about 75% saturated.     

Dissociation between changes in cyclic AMP and subsequent induction of TH in the rat superior cervical ganglion and adrenal medulla

Summary Changes in the levels of cyclic AMP were studied in the superior cervical ganglion and adrenal medulla of male Sprague-Dawley rats under experimental conditions which lead to a trans-synaptic induction of tyrosine hydroxylase (TH) in these organs.In the superior cervical ganglion an intermittent 2-hour swimming stress or treatment with reserpine (5 or 10 mg/kg s.c.) failed to change cyclic AMP levels significantly (P>0.1) while 48 h later TH was markedly (P<0.001) increased. In the adrenal medulla the increase in cyclic AMP occurring during cold exposure was much greater than that observed during swimming stress. However, the increase in TH activity resulting from swimming stress was markedly greater then that resulting from cold exposure. The same inverse relationship holds true when comparing the effect of reserpine with that of cold stress. Moreover, during cold exposure cyclic AMP levels were maximal (700%) within 20 min and had fallen almost to control levels (180%) after 60 min. However, no significant increase in TH (P>0.1) was observed 48 h after a 1-hour cold stress whereas a 2-hour cold stress produced a marked (P<0.001) increase in TH activity. Thus, the second hour of cold stress during which cyclic AMP was only marginally elevated seems to be essential for initiating the biochemical events ultimately leading to an increased synthesis of TH.It is concluded that either cyclic AMP is not involved in trans-synaptic induction of TH or that changes in a small pool are essential and are overshadowed by a much larger pool of cyclic AMP not reacting in the same way.     

Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization--effect of ethanol.

Slices from rat corpus striatuum were incubated for 10 min at 37° in freshlyy oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K⁺ (55mM) media both in the presence and absence of ethanol (0.2 to 0.8%, w/v). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K⁺ (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K⁺ media also produced changes in the kinetic properties of tyrosine hydroxylase. The K_^m of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH₄) was decreased from 0.82 to 0.09 mM and the K_i of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K⁺ media markedly blocked the K⁺-induced activation of tyrosine hydroxylase as well as the kinetic alterations in the enzyme observed after K⁺-depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The results reported in this work suggest that the increase in DA synthesis observed in striatal slices after K⁺-depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appears to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. Also, the blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K⁺-depolarization seems to offer a likely explanation for the inhibitory effect of ethanol on K⁺-induced increase in DA synthesis reported recently by Gysling et al. (Biochem. Pharmac.25, 157 (1976)).     

Regulation of adrenal tyrosine hydroxylase activity: neuronal versus local control studied with apomorphine.

Administration of apomorphine to rats leads to a temporary decrease in adrenal catecholamines and a long-term increase in adrenal tyrosine hydroxylase activity. The kinetic characteristics of tyrosine hydroxylase of the apomorphine-treated and control rats were determined. A significant increase in V_max was observed in the treated group as compared to controls; however, there was no change in the K_m for the cofactor DMPH₄ or tyrosine between the two groups in undialyzed or dialyzed preparations, indicating that the increase in tyrosine hydroxylase activity was probably due to an increase in enzyme protein. The temporary decrease in adrenal catecholamines was found to be due to increased secretion after apomorphine treatment, even though the concentration of injected apomorphine appeared to be sufficient to inhibit adrenal tyrosine hydroxylase. This was further substantiated by the fact that the decrease in adrenal catecholamines was prevented by adrenal denervation. The delayed increase in tyrosine hydroxylase activity after apomorphine treatment was observed in hypophysectomized rats; however, it was abolished after splanchnic nerve transection. A relationship was sought between the decrease in adrenal catecholamines and increase in adrenal tyrosine hydroxylase activity. When apomorphine and L-3,4-dihydroxyphenylalanine (L-DOPA) were administered simultaneously, there was no short-term decrease in adrenal catecholamine content but the increased tyrosine hydroxylase activity was still observed. Also, the administration of α-methyl-p-tyrosine to rats decreased the concentration of adrenal catecholamines and yet did not affect adrenal tyrosine hydroxylase activity. The results suggest that increased nerve activity, and not adrenal catecholamine concentrations, regulates the induction of adrenal tyrosine hydroxylase. Furthermore, regulation would be by way of some central mechanism involving dopamine-sensitive receptors.     

Tyrosine hydroxylase:--examination of conditions influencing activity in pheochromocytoma, adrenal medulla and striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum was studied with regard to factors influencing enzyme activity. The pH optimum was different for tyrosine hydroxylase from the three tissues examined: pheochromoeytoma, pH 7.8; adrenal medulla, pH 6.8; and striatum, pH 6.0. In preparations of tyrosine hydroxylase from each tissue, the presence of Mg²⁺. ATP and cyclic AMP resulted in a significant activation of the enzyme. On the other hand, addition of catecholamines to the reaction mixture produced a marked inhibition which became more pronounced with increasing pH. In all three tissues, the pH for maximum per cent stimulation by Mg²⁺, ATP and cyclic AMP was different from the pH optimum for tyrosine hydroxylase. In addition, the magnitude of this stimulation, as well as the basal activity, was dependent on the buffer present in the reaction mixture.     

Absence of increased tyrosine hydroxylation after induction of brain tyrosine hydroxylase following reserpine administration.

Reserpine was administered to rats by i.p. injection, and seven days later, with a control group, crude synaptosomal suspensions were prepared from hippocampus and cerebellum for the estimation of rates of synaptosomal tyrosine hydroxylation; additionally, samples of hippocampus, cerebellum and hypothala- mus were homogenized in a hypotonic buffer containing Triton for the 171 in vitro estimation of tyrosine hydroxylase. In the samples from reserpine pretreated animals, in vitro tyrosine hydroxylase activities were considerably elevated compared to controls (234 per cent of controls in the cerebellum, 154 per cent in hippocampus and 181 per cent in hypothalamus). However, the synaptosomal tyrosine hydroxylation rates were not elevated in the drug-treated group. The rate of product formation with time was linear in preparations from both control and reserpine pretreated animals. The results suggests that enzyme induction leads to an increased potential for tyrosine hydroxylation which may not be expressed due to interaction with regulatory mechanisms operating on the enzyme.     

Effects of some psychotropic drugs on tyrosine hydroxylase activity in different structures of the rat brain

M.J. BESSON, A. CHERAMY, C. GAUCHY and J. MUSACCHIO, Effects of some psychotropic drugs on tyrosine hydroxylax activity in different structures of the rat brain, European J. Pharmacol. 22 (1973) 181–186.The effect of amphetamine, rcserpins and thioproperazine treatments on CA synthesis and tyrosine hydroxylase activity were investigated in some structure of the rat brain. Striatal and cortical CA synthesis was estimated on dices incubated with a physiolosical medium containing 1–3,5-³H-tyrosine; ³H-H₂O formed during the hydroxylatioa of ³H-tyrosine to ³H-DOPA was measured. Striatal DA synthesis was decreased in all cases 24 hr after the last injection of a chronic treatment with reserpine, thioproperazine or amphetamine; cortical CA synthesis was decreased only after reserpine treatment. Tyrosine hydroxylase activity was measured on a partially purified enzyme preparation. Enzyme activity was not affected in the striatum by repeated treatments with these different drugs. Repeated treatment with reserpine increased tyrosine hydroxylase activity in the brain stem and the cortex; in contrast, repeated treatment with thioproperazine decreased the enzyme activity in these two structures.     

Drug-induced changes in brain tyrosine hydroxylase activity in vivo.

Brain tyrosine hydroxylase activity was determined in vivo by estimating the concentration of dihydroxyphenylalanine (DOPA) following the inhibition of DOPA decarboxylase. The effects of a number of centrally acting drugs from different therapeutic classes were studied on whole brain tyrosine hydroxylase activity.It was found that chlorpromazine and haloperidol, which block central catecholaminergic receptors, increased tyrosine hydroxylase activity, whereas apomorphine, a central dopamine agonist, decreased the activity of this enzyme. Piribedil, a dopamine agonist which has a different spectrum of activity to apomorphine, did not affect hydroxylase activity. Clozapine slightly increased the enzyme activity.The amphetamines, many of which release catecholamines in vivo, decreased hydroxylase activity. Antidepressants, which differed in terms of their basic structures and biochemical modes of action were without effect as were the β-adrenergic blocking drugs propranolol and the α-blocker phenoxybenzamine. Reserpine caused an increase in hydroxylase activity which reached a peak 2 hr after injection and then returned to control levels 2 hr later. Both the tyrosine hydroxylase inhibitor, α-methyl-p-tyrosine, and the tryptophan hydroxylase inhibitor, p-chlorophenylalamine, markedly decreased tyrosine hydroxylase activity in vivo.It is concluded that a dopamine receptor mediated feedback mechanism may play a contributory role in the control of brain tyrosine hydroxylase activity in vivo.     

Tyrosine hydroxylase: Allosteric activation induced by stimulation of central noradrenergic neurons

Summary Electrical stimulation of the rat locus coeruleus causes about a 300% increase in the activity of the tyrosine hydroxylase prepared from the hippocampus on the stimulated side and assayed in the presence of subsaturating concentrations of tyrosine and pteridine cofactor. Addition of calcium or cAMP to soluble preparations of tyrosine hydroxylase isolated from the hippocampus produces a similar activation of tyrosine hydroxylase. The activation of tyrosine hydroxylase produced by calcium is reversed by addition of the calcium chelator, EGTA, while the activation produced by cAMP addition or by electrical stimulation of the locus coeruleus is unaffected by addition of EGTA to the assay medium. The activation of tyrosine hydroxylase produced by electrical stimulation or by addition of calcium or cAMP to the assay medium appears to be mediated in part by alterations in the kinetic properties of the enzyme. All treatment causes the enzyme to have an increased affinity for substrate and pteridine cofactor and a decreased affinity for the endproduct inhibitor, norepinephrine. These results are suggestive that the activation of tyrosine hydroxylase which occurs during periods of increased impulse flow in noradrenergic neurons may be initiated by alterations in calcium fluxes or by changes in the steady state levels of cAMP which accompany neuronal depolarization.A preliminary account of this work was reported in the fall meeting of the American Society for Pharmacology and Experimental Therapeutics held, August 18–22, 1974, at Université de Montreal, Montreal, Quebec, Canada.     

The mycotoxin ochratoxin A is a substrate for phenylalanine hydroxylase in isolated rat hepatocytes and in vivo

Ochratoxin A (OTA), is a mycotoxin contaminating food and feed stuffs, consisting of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group tol-phenylalanine by an amide bond. When OTA (0.12–1.4 mM) is incubated with freshly isolated rat hepatocytes, it inhibits both the hydroxylation of phenylalanine (0.05 mM) to tyrosine, catalyzed by phenylalanine hydroxylase and the subsequent metabolism of tyrosine as measured by homogentisate oxidation. The IC₅₀ of OTA for phenylalanine hydroxylation is 0.43 mM. OT, (0.5–1.0 mM), the dihydroisocoumarin moiety of OTA, does not inhibit phenylalanine hydroxylase activity under these conditions. During incubations of hepatocytes with uniformly labelled [³H]-OTA and unlabelled phenylalanine, tyrosine-ochratoxin A is formed (up to 6% of the total mycotoxin added), indicating that ochratoxin can act as a substrate for phenylalanine hydroxylase. In vivo tyrosine-OTA is also found in liver of poisoned animals.     

Is inhibitio of striatal synaptosomal tyrosine hydroxylation by dopamine agonists a measure of dopamine autoreceptor function?

Sumamry Rat striatal synaptosomal tyrosine hydroxylation was inhibited dose- and pH dependently by a number of dopamine agonists. The catecholic agonists apomorphine and (–)N-n-propylnorapomorphine inhibited synaptosomal tyrosine hydroxylase completely, with IC₅₀ values of around 0.3 mol/l at pH 6.6. The noncatechol agonists pergolide and bromocriptine and the putative dopamine autoreceptor agonists 3-PPP(–), 3-PPP(+), HW-165 and B-HT 920 produced only partial inhibition of synaptosomal tyrosine hydroxylation at high concentrations. Comparison of the inhibition of synaptosomal and soluble tyrosine hydroxylase indicated that the inhibition produced by apomorphine could be ascribed to a direct effect on the enzyme, whereas this was not the case for the noncatechol agonists. The inhibition produced by pergolide and 3-PPP(–) was not antagonised by either dopamine receptor or alpha-adrenoceptor antagonists. The present results have been compared with results reported in the literature for inhibition of synaptosomal tyrosine hydroxylation and for two other tests of dopamine autoreceptor agonist activity (inhibition of dopamine release from striatal slices in vitro, and inhibition of the gamma-butyrolactone induced increase in dopamine synthesis in vivo). It is concluded that inhibition of striatal synaptosomal tyrosine hydroxylation by dopamine agonists does not fulfil the criteria required for it to be considered as a useful measure of dopamine autoreceptor function.     

Activation and inactivation of striatal tyrosine hydroxylase: the effects of pH, ATP and cyclic AMP, S-adenosylmethionine and S-adenosylhomocysteine

The activity of tyrosine hydroxylase (TH) in the corpus striatum of haloperidol treated and control rats has been examined. The activation of TH by haloperidol caused a decrease in the Km for tetrahydrobiopterin but no change in the Vmax. This effect was totally abolished when homogenates were prepared at high values of pH. A similar activation could be produced in vitro by preincubating with S-adenosylmethionine; conversely, enzyme activity was reduced by preincubating with S-adenosylhomocysteine. ATP and cyclic AMP activated the enzyme when incubated together with TH in vitro but the activity was reduced when the enzyme was preincubated with these substances. A possible role for carboxymethylation in controlling tyrosine hydroxylase activity is discussed.     

Regulation of tyrosine hydroxylase activity by prostaglandin E1 in guinea pig adrenal gland.

The action of prostaglandin E1 on tyrosine hydroxylase activity in adrenal slices of guinea pig was studied. The activity of tyrosine hydroxylase and was decreased by the incubation of adrenal slices with prostaglandin E1 at concentrations beyond 2 mug per ml for 2 hours. The activity of tyrosine hydroxylase was stimulated by dibutyryl adenosine 3',5'-monophosphate in slices of guinea pig adrenal glands. Incubation of adrenal slices wtth the combination of PGE1 and DBc-AMP lead to a tyrosine hydroxylase activity higher than that with PGE1 alone, but not as great as DBc-AMP alone. It was suggested that PGE1 inhibited the enzyme activity independently of the cyclic AMP level. Other prostaglandins such as PGA1 and PGB1 were deficient to the extent that the tyrosine hydroxylase activity was decreased. PGE1 inhibited the enzyme activity much to the same extent seen with protein synthesis inhibitors such as cycloheximide and actinomycin D. However, PGE1 did not influence the incorporation of L-leucine-14C into acid insoluble protein. The studies reported here showed that PGE1 inhibited the synthesis of tyrosine hydroxylase.     

Effects of morphine, in vitro and in vivo, on thyrosine hydroxylase activity in rat brain

The effect of morphine on the synthesis of catecholamines was determined in rat brain. In agreement with other studies, morphine produced a dose-dependent increase in the biosynthesis of the catecholamines. To assess whether morphine might enhance the synthesis of norepinephrine and dopamine by a direct chemical interaction with tyrosine hydroxylase, the rate-limiting step in their biosynthesis, the effects of the narcotic on the activity of the enzyme were determined in several regions of rat brain. Morphine, in vitro, from 10^?8 to 10^?2 M had no effect on the activity of tyrosine hydroxylase in any region examined. Moreover, morphine (10^?5 and 10^?3 M) had no effect on the apparent K_m of tyrosine hydroxylase for either substrate or cofactor (6-7-dimethyl-5, 6,7,8-tetrahydropterine). In addition, morphine (10^?5 and 10^?3 M) failed to block the inhibition of tyrosine hydroxylase, in vitro, by norepinephrine and dopamine in the hypothalamus and caudate respectively. The effects of morphine treatment, in vivo, on enzyme activity were also examined. The results of these studies indicated that acute injections of morphine had no effect on tyrosine hydroxylase activity, in vitro, indicating that the drug did not alter the level of an endogenous activator or inhibitor of tyrosine hydroxylase. Further studies indicated that development of tolerance to and physical dependence on morphine was not associated with an increase in the activity of brain tyrosine hydroxylase activity. The results of these studies suggest that morphine does not enhance the biosynthesis of catecholamines by a direct effect of tyrosine hydroxylase and that tolerance to the narcotic is not characterized by an induction of this enzyme.     

The long-term regulation of tyrosine hydroxylase activity in cultured sympathetic ganglia: Role of ganglionic noradrenaline content

1 An organ culture system is described for the in vitro maintenance of superior cervical sympathetic ganglia taken from mice of any age. The relation of tyrosine hydroxylase (T-OH) activity to ganglionic noradrenaline (NA) content has been investigated under various culture conditions.2 Depolarizing stimuli such as raised extracellular potassium and ouabain evoked increases of approximately 70% in the T-OH activity of cultured ganglia over a 48 h period. Exposure to a high concentration of potassium (high K(+)) for 30 min at the start of a 48 h culture was sufficient to elicit significant increases in T-OH activity.3 Depolarization-induced rises in T-OH activity were observed after culture in the presence or absence of nerve growth factor.4 The NA content of ganglia, cultured for 48 h in the presence of high K(+), ouabain, reserpine, clorgyline and alpha-methyl-p-tyrosine, showed no constant relation to their T-OH activity.5 Dibutyryl cyclic adenosine 3'-5'-monophosphate (dibutyryl cyclic AMP) mimicked high K(+) in its effect on ganglionic T-OH activity and NA content. Theophylline enhanced the potassium effects.6 Rises in the T-OH activity of ganglia cultured in the presence of high K(+) and dibutyryl cyclic AMP were abolished if the protein synthesis inhibitors cycloheximide or actinomycin D were present in the culture medium.7 It is concluded that the link between prolonged depolarization and rises in T-OH activity does not seem to depend upon changes in ganglionic NA content. In the intact animals, trans-synaptic modulation may take the form of a depolarization-induced rise in the cyclic AMP content of sympathetic ganglionic neurones leading to nuclear mediated synthesis of T-OH.