Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (-)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40-50% inactivation of Gi and Go proteins, which is evident from the reduction of ADP ribosylation with [32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.     

Presynaptic dopamine and serotonin receptors modulating tyrosine hydroxylase activity in synaptosomes of the nucleus accumbens of rats

Tyrosine hydroxylase (TH) activity was determined by measuring the formation of [3H]DOPA from [3,5-3H]tyrosine in the crude synaptosomal fraction of the nucleus accumbens under conditions preventing dopamine reuptake in 30 mM K+-containing medium. TH seems to be allosterically activated under depolarizing conditions: a 4.4 fold decrease of the Km value for tyrosine of the synaptosomal TH was observed. Synaptosomal TH activity was inhibited concentration dependently by dopamine and apomorphine resulting in IC50 values of 0.4 and 0.25 microM, respectively. The maximal inhibitory effects of dopamine as well as apomorphine were about 50% of the controls. The dopamine-induced inhibition was completely antagonized by neuroleptics. The rank order of antagonistic potencies was haloperidol greater than clozapine greater than sulpiride (with increasing EC50); methiothepine was ineffective. Moreover, synaptosomal TH activity was inhibited by serotonin in a concentration-dependent manner (IC50 = 0.8 microM). This inhibition was completely antagonized by methiothepine while, on the other hand, haloperidol was ineffective. The experimental system demonstrated here appears to be suitable for estimating the presynaptic dopamine and serotonin antagonistic potencies of drugs.     

Effects of catecholamine releasing agents on synaptosomal dopamine biosynthesis: Multiple pools of dopamine or multiple forms of tyrosine hydroxylase?

The effects of agents, which are known to induce release of catecholamines from synaptosomes, were assessed on the synthesis of dopamine from tyrosine, as reflected in the evolution of ¹⁴CO₂ from L-[1-¹⁴C]-tyrosine, in intact rat striatal synaptosomes. At a time when release had occured, whereas reserpine inhibited the synthesis of dopamine from tyrosine, with an ED₅₀ of $\text{1 × 10}{}^{\mathrm{?8}}\text{M}$, tyramine (ED₅₀ of $\text{1 × 10}{}^{\mathrm{?5}}\text{M}$) and (+)-amphetamine (ED₅₀ of $\text{1·4 × 10}{}^{\mathrm{?6}}\text{M}$) enhanced the rate of synthesis. The presence of nialamide (10^?4M) or pargyline (10^?3M) had no effect on synaptosomal dopamine synthesis in the absence or presence of amphetamine, tyramine, or reserpine. Neither reserpine, tyramine, nor amphetamine effected the activity of tyrosine hydroxylase or DOPA decarboxylase in the absence of synaptosomal structural integrity. Nor did these drugs effect the accumulation of [³H]-tyrosine into synaptosomes. The data are consistent with the existence of at least two pools of synaptosomal dopamine, one of which can interact with tyrosine hydroxylase. Two hours after pretreatment of rats with 5 mg/kg (+)-amphetamine, the level of synaptosomal dopamine biosynthesis was decreased by 39%. The rate of dopamine synthesis in synaptosomes from amphetamine-pretreated rats was assessed in the presence of reserpine and tyramine. The data are not consistent with alterations in pool size being the only mechanism affecting synaptosomal dopamine synthesis. A mechanism is discussed involving an equilibrium of tyrosine hydroxylase between active and inactive conformers in the presence of an inhibitory pool of dopamine.     

Striatal dopamine D1-like receptors have higher affinity for dopamine in ethanol-treated rats.

Experiments were carried out with brain tissues of ethanol-experienced (long-term ethanol intake but withdrawn) vs. ethanol-naive animals. The in vitro 3H antagonist binding of [3H]SCH 23390 and of [3H]spiperone to striatal dopamine D1- and D2-like receptors revealed no significant changes in KD and Bmax values. Displacement of the 3H antagonist binding by dopamine indicated high- and low-affinity states, which also showed no significant alteration at the D2-like receptor but a 5-fold increase of dopamine affinity at the high-affinity state of the D1-like receptor of the ethanol-experienced rats.     

Mouse neuroblastoma clone N1E-115: A suitable model for studying the action of dopamine agonists on tyrosine hydroxylase activity

The DOPA-content in neuroblastoma clone N1E-115 is higher than the dopamine or noradrenaline content. Blockade of tyrosine hydroxylase by ifα-methyl-p-tyrosine (1 × 10 su?3 M) resulted in a decrease of cellular DOPA-content to 24.9% after 4 hr. The accumulation of DOPA in these cells which is probably due to limited activity of l-aromatic amino acid decarboxylase led us to use DOPA-content as a measure of tyrosine hydroxylase (TH) activity. Dopamine and especially apomorphine were effective at low concentrations (dopamine ic₅₀1 × 10^?5 M, apomorphine 2 × 10^?7 M); lisuride had no effect on TH-activity. The low effective dose of apomorphine and the failure of lisuride to influence TH-activity are comparable to the observations in striatal synaptosomal preparations and make the N1E-115 clone a suitable model for studying the mechanism of TH-regulation. However, since haloperidol (1 × 10^?5 M) did not reverse the apomorphine-induced blockade of TH, a receptor-mediated blockade of TH seems to be improbable.     

Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels.

In previous reports, methamphetamine was shown to depress tyrosine hydroxylase (TH) activity in the rat corpus striatum. To evaluate further the mechanism of this decrease in TH activity, enzyme activity was measured in the rat corpus striatum and substantia nigra after repetitive and single-dose methamphetamine administration. Following repeated doses of methamphetamine, nigral TH activity decreased and reached 45% of controls at 12 hr and returned to normal at 60 hr. Striatal TH activity decreased to 40% of control at 36 hr and returned toward normal at 60 hr. When methamphetamine was administered every 6 hr for 30 hr and then discontinued, nigral TH activity returned toward control levels 4 days prior to recovery of striatal TH activity. Methamphetamine initially increased striatal dopamine levels at 6 hr (170% of control). Dopamine levels then decreased in parallel with striatal TH activity but failed to increase as the enzyme recovered. Concurrent administration of chlorpromazine with methamphetamine prevented the methamphetamine-induced decrease in nigral and striatal TH activity and striatal dopamine levels. The results indicate that the methamphetamine-induced depression of striatal and nigral TH activity may be related to increased stimulation of dopamine receptors in the striatum.     

Role of dopamine storage function in the control of rat striatal tyrosine hydroxylase activity

Summary Pretreatment of rats with reserpine prevents and post-treatment with RO4-1284 depletes the -butyrolactone (GBL)-induced increase of striatal dopamine (DA) levels. This suggests that the accumulation of DA in striatal nerve endings that normally follows GBL-induced cessation of nigrostriatal impulse flow is in reserpine-sensitive sites. Three days after a single injection of reserpine, the ability of either haloperidol, a DA receptor blocker, or GBL to enhance DA synthesis is greatly reduced and these responses recover slowly over a two week period. Similarly, the ability of haloperidol to elevate striatal DA metabolite concentrations shows a similar pattern of inhibition. The rate of recovery after reserpine of haloperidol effects on DA metabolite concentrations and the activation of striatal tyrosine hydroxylase (measured in vivo by the 30 min l-DOPA accumulation after decarboxylase inhibition with NSD-1015) after either haloperidol or GBL parallels the rate of recovery of basal DA levels. The accumulation of DA after GBL proceeds for 60 min before beginning to plateau in normal rats, but 3 days after reserpine the DA elevation stops after 15 min and lasts for only 30 min in 10 day reserpinized animals. The initial 15 min accumulation of DA after GBL is the same in normal, 3 day and 10 day reserpinized rats, indicating that the initial enzymic rate of activity is the same, but the duration of activation is less. Thus, inhibition of DA storage function by reserpine alters the coupling of DA autoreceptor activity with tyrosine hydroxylase activity. It is suggested that DA storage function modulates tyrosine hydroxylase activity by controlling the amount of DA available for attachment to and inhibition of tyrosine hydroxylase enzyme. This hypothesis is consistent with recent immunocytochemical observations which suggest an association of tyrosine hydroxylase with synaptic vesicles in DA neuronal terminal areas.     

Influence of dopamine synthesis on methamphetamine-induced changes in striatal and adrenal tyrosine hydroxylase activity

Summary Methamphetamine in large doses decreases striatal tyrosine hydroxylase activity. This effect is prevented by neuroleptic agents such as chlorpromazine and haloperidol which would suggest that released dopamine may be involved in the response. To test this hypothesis, we have altered dopamine synthesis with -methyl-p-tyrosine and l-Dopa and found that dopamine synthesis is necessary for the observed depression of striatal TH activity by methamphetamine. In the adrenal gland, however, the increase in TH activity by methamphetamine is not prevented by inhibition of catecholamine synthesis. It is possible that released dopamine may be inhibiting TH activity by activation of pre- or postsynaptic dopamine receptors in the neostriatum resulting in activation of the neuronal feedback pathway or released dopamine may act on dendrodendritic autoreceptors in the substantia nigra.     

Striatal vs extrastriatal dopamine D2 receptors in antipsychotic response--a double-blind PET study in schizophrenia.

Blockade of dopamine D2 receptors remains a common feature of all antipsychotics. It has been hypothesized that the extrastriatal (cortical, thalamic) dopamine D2 receptors may be more critical to antipsychotic response than the striatal dopamine D2 receptors. This is the first double-blind controlled study to examine the relationship between striatal and extrastriatal D2 occupancy and clinical effects. Fourteen patients with recent onset psychosis were assigned to low or high doses of risperidone (1 mg vs 4 mg/day) or olanzapine (2.5 mg vs 15 mg/day) in order to achieve a broad range of D2 occupancy levels across subjects. Clinical response, side effects, striatal ([11C]-raclopride-positron emission tomography (PET)), and extrastriatal ([11C]-FLB 457-PET) D2 receptors were evaluated after treatment. The measured D2 occupancies ranged from 50 to 92% in striatal and 4 to 95% in the different extrastriatal (frontal, temporal, thalamic) regions. Striatal and extrastriatal occupancies were correlated with dose, drug plasma levels, and with each other. Striatal D2 occupancy predicted response in positive psychotic symptoms (r=0.62, p=0.01), but not for negative symptoms (r=0.2, p=0.5). Extrastriatal D2 occupancy did not predict response in positive or negative symptoms. The two subjects who experienced motor side effects had the highest striatal occupancies in the cohort. Striatal D2 blockade predicted antipsychotic response better than frontal, temporal, and thalamic occupancy. These results, when combined with the preclinical data implicating the mesolimbic striatum in antipsychotic response, suggest that dopamine D2 blockade within specific regions of the striatum may be most critical for ameliorating psychosis in schizophrenia.     

Effect of adrenalectomy on tyrosine hydroxylase activity

Bilateral adrenalectomy produced a fall in blood pressure and an increase in tyrosine hydroxylase activity in the superior cervical ganglion in the rat. The fall in blood pressure and the increase in tyrosine hydroxylase activity in the superior cervical ganglion were prevented by giving the adrenalectomized animals 0.9% saline as their only drinking fluid. The increase in tyrosine hydroxylase activity was also prevented by decentralization (severing the preganglionic fiber) of the superior cervical ganglion. These results suggest that the induction of tyrosine hydroxylase activity results from a reflexly mediated increase in nerve impulse traffic that results from the adrenalectomy-induced fall in blood pressure. Further characterization of this response showed that the glucocorticoid, dexamethasone, did not cause a further induction of enzyme in adrenalectomized rats whereas, treatment with epinephrine as well as dexamethasone, did result in an augmentation of the enzyme activity above that seen in the already induced adrenalectomized animals.     

Effect of triamterene on tyrosine hydroxylase activity

Summary Triamterene, which is structurally similar to the natural cofactor of tyrosine hydroxylase, (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, inhibited tyrosine hydroxylase in rat adrenal gland homogenates and was found to be a competitive inhibitor of the synthetic cofactor 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin. When triamterene (30 mg/kg i.p.) was administered to rats, a significant decrease in the adrenal, whole brain and kidney enzyme activities was observed after 90 min; if the drug was given orally, the diuretic affected only adrenal tyrosine hydroxylase. In both cases the drug decreased potassium excretion to 1/5 of control values and increased the excretion of sodium. Catecholamine levels in atria, kidneys, adrenal glands and whole brain were not affected in acute experiments. Chronic treatment (triamterene 30 mg/kg p.o. twice daily during 4 days) inhibited tyrosine hydroxylase and decreased catecholamine levels in the kidneys. Low potassium excretion was observed throughout the 4 days of treatment. In these chronic experiments the inhibition of the adrenal enzyme seen in acute treatments was not observed. This recovery cannot be explained by increase in the adrenal biopterin levels. It could be mediated by an induction of the adrenal tyrosine hydroxylase.     

Inhibition of tyrosine hydroxylase activity by methyl bromide exposure.

Rats were exposed to methyl bromide gas (16-250 ppm) for 8 hr, and tyrosine hydroxylase (TH) activity in the striatum, hypothalamus, frontal cortex, midbrain, and medulla oblongata was measured in brain homogenates from exposed rats, and in vivo following administration of decarboxylase inhibitor. Exposure to methyl bromide dose-dependently inhibited both in vitro and in vivo TH activity. Of the five brain areas, TH activity in the hypothalamus was most sensitive to methyl bromide. The time course of enzyme inhibition after exposure was similar to those of decreases in catecholamine concentrations, locomotor activity change, and body temperature reported previously. These results suggest methyl bromide reduces catecholaminergic neuronal activity in the brain via inhibition of TH activity.     

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.