Dopamine Synthesis In Synaptosomes Research Articles

Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinson's disease with L-DOPA.

Reduction of dopamine synthesis inhibition by dopamine autoreceptor activation in striatal synaptosomes with in vivo reserpine administration.

In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D2 DA agonists bromocriptine, lisuride, and pergolide, and the D1-D2 DA agonist apomorphine were studied in rat striatal synaptosomes, in which the rate of DA synthesis (formation of 14CO2 from L-[1-14C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP-dependent, but possibly a Ca(2+)-dependent mechanism was involved. The DA agonists showed a concentration-dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D2 DA antagonist (-)-sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration-response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P1 + P2 fraction obtained from the reserpine-treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (-)-Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of synaptosomal dopamine synthesis by corticotropin-releasing factor in rat striatum: role of Ca 2+-dependent mechanisms

Corticotropin-releasing factor (CRF) stimulated synaptosomal dopamine (DA) synthesis in rat striatal homogenates. The stimulatory effect of CRF was antagonized by α-helical CRF 9–41 and was dependent on the extracellular Ca 2+ concentration, being absent after removal of Ca 2+ from the incubation medium and maximal at about 1.2 mM extracellular Ca 2+. The stimulation of DA synthesis by forskolin (FSK) was less sensitive to a change of extracellular Ca 2+ concentration. 3-Isobutyl-1-methylxanthine potentiated the FSK effect but did not affect the response to CRF. CRF stimulation was additive to that produced by 40 mM KCl and was little affected by the calmodulin inhibitor, W-7, which markedly suppressed the veratridine-induced stimulation of DA synthesis. Polymyxin B antagonized the CRF stimulation while it had a weaker effect on FSK-stimulated DA synthesis. Tolbutamide reduced the FSK stimulation more effectively than the CRF response. CRF elicited a non-significant increase of cyclic AMP formation by striatal membranes. These results indicate that CRF stimulates striatal DA synthesis by a Ca 2+ -dependent mechanism possibly involving the activation of the protein kinase C pathway.

Evidence that adenosine A 2 and dopamine autoreceptors antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes

Incubation of rat striatal synaptosomes with the adenosine receptor agonist 2-chloroadenosine (2-CADO) produced a concentration-dependent increase of dopamine (DA) synthesis (about 50% of control value). The effect was not additive with the stimulation produced by either 10 μM forskolin or 2 mM dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with 2-CADO produced an activation of tyrosine hydroxylase (TH) which withstood washing and lysing of the tissue. This activation was largely independent of the presence of Ca 2+ ion in the preincubation medium and, when analyzed as a function of different concentrations of the protein cofactor 6-methyl-5,6,7,8-tetrahydropterin (0.08–0.4 mM), itv was associated with an apparent increase in the V max of the enzyme. Quinpirole, a selective D 2DA receptor agonist, reduced control synaptosomal DA synthesis and caused a persistent inhibition of TH activity. When added together with 2-CADO, quinpirole depressed the stimulation of DA synthesis and TH activity produced by the adenosine analog. The effect of quinpirole was stereospecifically antagonized by the D 2 DA antagonist sulpiride. Quinpirole also inhibited the activation of TH elicited by a submaximal concentration of forskolin, but not that produced by dibutyryl cyclic AMP. The inhibitory effect of quinpirole on basal and 2-CADO-stimulated TH activities was mimicked by DA. These results indicate that presynaptic DA autoreceptors and adenosine A 2 receptors interact antagonistically in controlling DA synthesis in rat striatal synaptosomes presumably by exerting opposite inputs on a presynaptic adenylate cyclase system.

Corticotropin-releasing factor activates tyrosine hydroxylase in rat and mouse striatal homogenates

Corticotropin-releasing factor (CRF) caused a concentration-dependent increase in dopamine (DA) synthesis measured in rat striatal synaptosomes. The concentrations of CRF producing half-maximal and maximal stimulation were 40 and 300 nM, respectively. The maximal effect corresponded to a 34% increase from the control level of DA synthesis. CRF 1 μM increased DA synthesis in synaptosomes of mouse striatum by 48%. Pretreatment of striatal synaptosomes with CRF produced a persistent activation of tyrosine hydroxylase assayed in extracts of lysed synaptosomes. These results suggest that CRF may play a modulatory role in striatal DA synthesis by acting on receptors located on DA terminals.

Cholecystokinin stimulates dopamine synthesis in synaptosomes by a cyclic AMP-dependent mechanism.

The effects of different fragments of cholecystokinin (CCK) on dopamine synthesis were studied in synaptosomal preparations from the striatum, substantia nigra, and frontal cortex. In striatal synaptosomes, dopamine synthesis rate measured by dopamine accumulation was 12.5% lower than that measured by 3,4-dihydroxyphenylalanine (DOPA) accumulation; however, K+-accelerated synthesis was the same for both methods. Synthesis rate was independent of exogenous tyrosine levels. In the three regions studied, the combined stimulatory effects of 8-Br-cyclic AMP and high K+ were additive. CCK-5, CCK-3, CCK-27-33, and CCK-8 (sulphated) enhanced synthesis, CCK-5 being the most potent fragment. The nonsulphated octapeptide had no effect. In all three regions, CCK-5 and high K+ had an additive effect on dopamine synthesis; CCK-5 and 8-Br-cyclic AMP together produced the same enhancement of synthesis as CCK-5 alone. CCK-5 produced similar dose-dependent increases in dopamine synthesis and cyclic AMP accumulation in striatal synaptosomes, and both effects were blocked by the CCK antagonist proglumide.

Evidence that amphetamine and Na + gradient reversal increase striatal synaptosomal dopamine synthesis through carrier-mediated efflux of dopamine

Amphetamine (AMPH) releases dopamine (DA) from striatal synaptosomes and concomitantly increases DA synthesis. Since AMPH may release DA through carrier-mediated diffusion via reversal of the DA uptake system, the increase in DA synthesis might depend on a functioning uptake carrier. Consistent with such a mechanism, the uptake inhibitors nomifensine (NMF) and benztropine (BZT) completely prevented the AMPH-induced increase in DA synthesis at concentrations known to inhibit DA uptake. Changes in the Na + gradient across the synaptosomal membrane also promote DA release, since DA and Na + are cotransported by the neuronal uptake carrier. Incubation of synaptosomes in medium containing decreasing Na + increased DA synthesis inversely proportional to Na + over the range 128 to 20 mM. Similarly, incubations in the presence of 10 −4 M ouabain to inhibit Na +, K +-ATPase and allow intracellular accumulation of Na + also increased DA synthesis. These changes in DA synthesis could also be prevented by BZT and were non-additive with the AMPH-induced increase in DA synthesis. However, a concentration of ouabain (10 −6 M) which by itself did not increase DA synthesis, and does not promote DA release, potentiated the AMPH-induced increase in DA synthesis. Further, the increased DA synthesis promoted by all three manipulations was only marginally dependent on the presence of Ca 2+ in the incubation medium. However, at 5 and 10 mM Na +, a second component of increased DA synthesis was observed which was insensitive to BZT, but was prevented by Ca 2+ removal. These results suggest that the increase in DA synthesis, and presumably DA release promoted by AMPH, lowered Na +, and ouabain, depend on the availability of the DA carrier at the internal face of the neuronal membrane and the intracellular content of Na +. The second component of increased DA synthesis which is evident at 5 and 10 mM Na + is discussed in terms of a possible Ca 2+-mediated change in DA synthesis which is independent of the DA carrier.

Striatal synaptosomal dopamine synthesis: Evidence against direct regulation by an autoreceptor mechanism

Regulation of the rate-limiting step in dopamine (DA) synthesis was estimated in striatal synaptosomes by measuring the rate of hydroxylation of L-4-[ 3H]phenylalanine, a substrate of tyrosine hydroxylase (TH). DA inhibited hydroxylation with an IC 50 of 0.2 μM. The concentration-response curve of DA-induced inhibition was not affected by the presence of 1 μM chlorpromazine, a phenothiazine DA antagonist. Sulpiride and haloperidol, DA antagonists of the benzamide and butyrophenone classes respectively, also failed to alter the inhibition of substrate hydroxylation by 1 μM DA, even at cocnentrations up to 10 μM. In contrast, a parallel 15 fold shift to the right in the concentration-response curve of DA-induced inhibition of hydroxylation was obtained when 10 μM nomifensine, a competitive DA uptake inhibitor, was added. Even in the presence of nomifensine, 1μM chlorpromazine had no effect on the DA concentration-response curve. The addition of DMPH 4, an artificial cofactor for TH, completely blocked DA-induced inhibition of enzymatic activity. These data suggest that direct autoreceptor control of synaptosomal TH activity does not exist in vitro, and that DA-induced inhibition of TH occurs subsequent to reuptake via classical feedback inhibition, presumably by competitive displacement of the necessary endogenous cofactor.

Reduced sensitivity of synaptosomal dopamine autoreceptors following increase in synaptosomal dopamine synthesis

Reduced sensitivity of synaptosomal dopamine autoreceptors following increase in synaptosomal dopamine synthesis

Inhibition of dopamine synthesis in striatal synaptosomes by lisuride: stereospecific reversal by (-)-sulpiride.

Lisuride, an ergot D2 dopamine receptor agonist inhibited dopamine synthesis in striatal synaptosomes concentration-dependently. Significant inhibition was detected at 10(-8) M, and the inhibition by 10(-4) M lisuride was 50%. The inhibitory effect of lisuride was reversed by more than 50% not only by the D1-D2 dopamine receptor blocker haloperidol but also by the D2 dopamine receptor blocker(-)-sulpiride. The effect of sulpiride was stereospecific. Under the same test conditions a similar inhibition of dopamine synthesis by apomorphine was reversed by the neuroleptics almost completely. The results suggest that there are dopamine autoreceptors controlling dopamine synthesis in synaptosomes and these receptors resemble D2 dopamine receptors according to the nomenclature of Kebabian and Calne (1979).

Influence of calcium on dopamine synthesis and tyrosine hydroxylase activity in rat striatum.

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium‐induced activation of tyrosine hydroxylase. Calcium (12.5–200 μM) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 mM) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μM) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μM) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM) decreased cell‐free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.

Long-term effect of kainic acid on striatal dopaminergic neurons

Summary The effect of intrastriatally injected kainic acid (KA) on dopamine (DA) and DOPAC content, synaptosomal DA synthesis and soluble tyrosine hydroxylase (TH) activity in the caudate nucleus (CN) was studied at 40 and 150 days after lesion. Since tissue weight decreased to 70 and 30% of the control side, respectively, at these time intervals, the results were expressed per whole CN. The following changes were observed: a) DA levels were decreased to 70 and 45% of the untreated side at 40 and 150 days after KA administration, respectively; b) both DOPAC content and soluble TH activities were reduced to approximately 50 and 30% of the control at 40 and 150 days after treatment, respectively; c) synaptosomal DA synthesis was reduced to 30% of the control at 40 days and remained so at 150 days after KA. KA failed to modify the K m of TH, whereas the V max was decreased to 45 and 25% of controls at 40 and 150 days after treatment respectively. The results indicate that, after a long lag period, KA produces a reduction in striatal dopaminergic neurons.

Stimulation of synaptosomal tyrosine hydroxylation by phencyclidine in vitro

Phencyclidine (PCP), a potent psychoactive drug, produces some animal behavior that are belived to be mediated by dopaminergic and/or cholinergic neurons in the basal ganglia. In this study, we have monitored the effects of PCP in vitro on the synthesis, uptake, and release of dopamine (DA) in rat striatal synaptosomes. Using tyrosine hydroxylation as an index of DA synthesis, we observed a concentration-dependent stimulation of DA synthesis by PCP. The stimulatory effect was antagonized by reserpine (1 μM) and was observed only when synaptosomes were preincubated under conditions which prevented the spontaneous release of [ 3H]DA. Two hydroxylated metabolites of PCP were also tested and found to have little effect on tyrosine hydroxylation. Like PCP these metabolites are potent inhibitors of synaptosomal [ 3H]DA uptake, but they apparently lack PCP's ability to release synaptosomal DA. Taken together, these results support our hypothesis that PCP stimulates synaptosomal DA synthesis by releasing DA from an inhibitory pool.

Divergent reserpine effects on amfonelic acid and amphetamine stimulation of synaptosomal dopamine formation from phenylalanine

Some effects of a non-amphetamine central stimulant, amtbnelic acid, on the synaptosomal (P 2) formation and release of dopamine being produced continuously from [ 14C]phenylalanine substrate have been determined. How reserpine action may modify the effects of amfonelic acid and those of amphetamine (the latter was included for comparative purposes) was also examined. For these studies, P 2 preparation from rat caudate nuclei was incubated with [ 14C]phenylalanine with and without various drug additions, and the particulates were separated from the medium after incubation. The separated fractions were analyzed for labeled dopamine and for the synaptosomal content of the labeled substrate. Of the total labeled dopamine formed, the fraction that was present in the medium was determined and taken as a measure of the spontaneous release (no drug addition) or its enhancement by any addition. Amfonelic acid and amphetamine (0.91–18.2 μM) comparably stimulated the synthesis and release of [ 14C]dopamine. The addition of reserpine (1.8 μM) alone had an inhibitory effect on total synthesis and a stimulatory one on release. In the presence of reserpine, the synthesis stimulation by amphetamine was maintained or accentuated, but amfonelic acid induced an inhibitory effect additive to that due to reserpine alone. The release stimulations by amphetamine and amfonelic acid were comparable in the absence as well as in the presence of reserpine. Following reserpine pretreatments at 24 and 2 hr, amphetamine (9.1 μM) markedly stimulated, but AA (9.1 μM) affected nonsignificantly, the dopamine synthesis. The pretreatments did not abolish the release-enhancing effect of either stimulant. None of the drug additions resulted in a significant alteration of the paniculate [ 14C]phenylalanine substrate level. In summary, the results show that amfonelic acid, like amphetamine, may release continuously forming synaptosomal dopamine and stimulate dopamine synthesis. However, the synthesis stimulation by amfonelic acid, but not that by amphetamine, may he abolished by reserpine action, either in vitro or in vivo. The results suggest that. although amphetamine may stimulate by releasing the newly forming dopamine pool, a significant amfonelic acid action may he on the catecholamine storage system, and synaptosomal dopamine synthesis may be under the controlling influence of both the newly forming amine and the vesicular stores.

PH-induced alterations in dopamine synthesis regulation in rat brain striatal synaptosomes.

Abstract: Dopamine synthesis regulation as a function of pH has been examined in rat brain striatal synaptosomes. Synthesis stimulation produced by lowering the incubation pH from 7.2 to 6.2 is accompanied by a significant increase in apparent A'm for tyrosine and in apparent Vmax. While these kinetic alterations are similar to those produced by the depolarizing agent veratridine, it does not appear that synthesis is stimulated at pH 6.2 via synaptosomal depolarization since (1) synthesis stimulation still occurs at pH 6.2 in a calcium‐free medium in contrast to the calcium‐dependency of veratridine‐ induced stimulation and (2) tyrosine uptake is not inhibited by incubation at pH 6.2, but is markedly inhibited by veratridine. In order to study how the regulatory properties of synaptosomal preparations vary according to pH, the ability of synaptosomal dopamine synthesis to respond to various agents was tested between pH 7.2 and 6.2. The stimulatory effects of veratridine, amphetamine, phenylethylamine and dibutyryl cyclic AMP at pH 7.2 were significantly diminished at pH 6.2. In addition, incubation at pH 6.2 antagonized the veratridine‐induced inhibition of tyrosine uptake, suggesting an interference with the depolarization process. The inhibitory effects of dopamine and tyramine at pH 7.2 were also antagonized at pH 6.2. In contrast to the effects of pH 6.2 buffer, incubation at pH 6.6 does not markedly alter responses to the various drugs. The results suggest that, although basal dopamine synthesis rates can be increased by lowering the pH, synaptosomal regulatory properties are significantly altered as the pH is lowered below 6.6.

Lack of effect of gamma-butyrolactone on amphetamine-induced decrease in striatal synaptosomal dopamine synthesis.

Lack of effect of gamma-butyrolactone on amphetamine-induced decrease in striatal synaptosomal dopamine synthesis.

Effect of ethanol on dop amine synthesis in rat striatal synaptosomes

We have examined the effects of acute and chronic ethanol treatment and of ethanol withdrawal on dopamine synthesis in rat striatal synaptosomes in vitro. Our studies showed that acute exposure to ethanol produces a dose-related decrease in dopamine synthesis in synaptosomes. This effect of acute ethanol treatment is time-dependent, since there is an initial stimulation of dopamine synthesis, which then is followed by a depression of synthesis. Chronic exposure to ethanol in the form of a liquid diet for 2 weeks does not alter dopamine synthesis in striatal synaptosomes. After withdrawal from ethanol, dopamine synthesis in striatal synaptosomes is decreased.

Differential effects of D- and L-amphetamine and methylphenidate on rat striatal dopamine biosynthesis

Methylphenidate and the optical isomers of amphetamine have differential effects, in vivo and in vitro on rat striatal synaptosomal dopamine (DA) biosynthesis. Whereas the systemic administration of D- or L-amphetamine produces a dose-dependent decrease in synaptosomal DA synthesis, with ED 50's of 1.1 and 3.5 mg/kg, respectively, methylphenidate, at doses which produce comparable degrees of stereotyped behavior, has no effect on this measure of DA biosynthesis. In vitro, D- and L-amphetamine exhibit a three-fold difference in ED 50's (1 × 10 −6 M and 3 × 10 −6 M, respectively) with respect to both activation of striatal synaptosomal DA synthesis, and to reversal of reserpine-induced inhibition of DA synthesis. In contrast, although in vitro methylphenidate can partially activate DA synthesis, it does not overcome the reserpine-induced inhibition. The data are discussed in terms of the possible differential mechanisms by which these two types of stimulants may exert their effects on stereotypy.

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 14CO 2 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 ED 50 of 1 × 10 −8 M , tyramine (ED 50 of 1 × 10 −5 M ) and (+)-amphetamine (ED 50 of 1·4 × 10 −6 M ) enhanced the rate of synthesis. The presence of nialamide (10 −4 M) or pargyline (10 −3 M) 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.

Stimulation of synaptosomal dopamine synthesis by veratridine

ISOLATED synaptosomal preparations maintain many of the functional properties of intact neurones1 and the uptake of putativebiogenicamine neurotransmitters such as noradrenaline, dopamine and serotonin, is well documented2–5. In addition, stimulation of synaptosomal catecholamine release has been demonstrated in response to deplolarising concentrations of potassium4,6 or veratridine6, an alkaloid able to produce depolarisation in intact neurones by increasing sodium permeability7,8. In intact adrenergic neurones stimulation of transmitter release is often accompanied by a concurrent increase in the rate of transmitter synthesis, possibly through a reduction in feedback inhibition of tyrosine hydroxylase9–13. The demonstration of a link between stimulated neurotransmitter release and stimulated synthesis in synaptosomes would be an important indication of the validity of studies of synaptosomal processes in order to gain insight into the functioning of intact neurones. We report here that striatal synaptosomes maintain the ability to respond to depolarising concentrations of veratridine with a calcium-dependent tetrodotoxin-sensitive increase in dopamine synthesis.