Effects of phencyclidine on synaptosomal dopamine continuously appearing from phenylalanine: Sensitivity to reserpine

In the present study some effects of phencyclidine on synaptosomal (P₂) synthesis and release of labelled dopamine, continuously appearing from [¹⁴C]phenylalanine, were investigated in vitro. Also examined was the sensitivity of such effects of phencyclidine to reserpine, acting either in vitro or in vivo. Synaptosomal (P₂) preparations from rat caudate nuclei were incubated with the drugs added at various concentrations in the presence of [¹⁴C]phenylalanine substrate. The particulates were quickly separated from the medium after incubation and the separated fractions were analyzed for labelled dopamine and the synaptosomal uptake of [¹⁴C]phenylalanine. Of the total labelled dopamine formed, the fraction that was present in the medium was determined and taken as a measure of the spontaneous or basal release and its enhancement by any drug addition. Phencyclidine (3.1–36.4 μM) stimulated both the total (medium plus particulates) formation and the synaptosomal release of labelled dopamine, while reserpine (0.09–1.80 μM) inhibited the formation and enhanced the release. A coaddition of reserpine (0.09–1.80 μM) and phencyclidine to the incubation medium blocked the stimulating effect of the latter drug on the labelled dopamine synthesis; the same experiments revealed furthermore, an inhibitory effect of phencyclidine (0.91–36.4 μM) on the synthesis, an effect that was additive to the inhibition due to reserpine alone. The enhancing effect of phencyclidine on labelled dopamine release was however maintained in the presence of reserpine. Phencyclidine (0.22–36.4 μM) also inhibited the formation of labelled dopamine and enhanced its release when the P₂ fraction was prepared from reserpine-pretreated rats (5.0 and 2.5 mg/kg, i.p., at 24 and 2 hr before the experiment). Reserpine (1.80 μM) itself, added in vitro to the same P₂ preparation, was without any significant effect. The phencyclidine congener Ketamine, with or without reserpine, had little effect either on the synthesis or the release. In none of the incubations did the addition of drugs affect the synaptosomal uptake of labelled phenylalanine.     

Characteristics of dopamine release from isolated nerve endings of the tuberoinfundibular neurones

In the present study the release of dopamine (DA) from a synaptosomal preparation obtained from the nerve endings of the tuberoinfundibular dopaminergic nerves has been analysed, utilizing a superfusion technique.The exposure of median eminence synaptosomes to increasing concentrations of K⁺ ions produced a dose-dependent efflux of preaccumulated [³H]DA. The removal of Ca²⁺ ions from the superfusion medium completely prevented the high K⁺-induced release of [³H]DA. Removal of Na⁺ ions from the superfusion medium, a condition creating a favourable outward Na⁺ gradient, produced a marked release of [³H]DA. In order to establish whether this efflux of dopamine was a carrier-mediated process nomifensine, a dopamine transport inhibitor devoid of dopamine releasing effect in the median eminence, was added to the superfusion medium. Nomifensine largely counteracted the release of dopamine induced by the lack of Na⁺.d-Amphetamine produced a dose-related stimulation of [³H]DA in synaptosomes from the median eminence.The possible existence of a direct control of dopamine release mediated by presynaptic receptors was tested by studying the effect of apomorphine on the efflux of preaccumulated [³H]DA. Apomorphine was unable to modify both the spontaneous and the K⁺-evoked release of dopamine.The present results give support to the idea that dopamine released from the nerve endings of tuberoinfundibular neurones into the primary plexus acts as a neurotransmitter. The release of dopamine in these nerves does not seem to be regulated by autoreceptors.     

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 (P₂) 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, P₂ 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.     

Effects of omega-amino acids on tritiated dopamine release from rat striatum: evidence for a possible glycinergic mechanism.

The effects of GABA and glycine on the release of tritiated dopamine from prelabelled slices of rat striatum have been compared. Both GABA (>50 μM) and glycine (>200 μM) released tritiated dopamine, but had no effect on the release of radiolabelled 5-hydroxytryptamine and GABA. The GABA antagonist picrotoxin (50 μM) markedly reduced the ability of GABA to release [³H]dopamine, but had no effect on the glycine response. Conversely strychnine (0.5 μM), a specific glycine receptor antagonist at low concentrations, abolished both the GABA and the glycine response on [³H]dopamine release. Two other ω-amino acids, β-alanine and taurine, both at 500 μM, had no effect on [³H]dopamine release from rat striatal slices.In additional experiments, release of radioactivity was demonstrated from neonatal rat spinal cord and striatal slices after prelabelling with [³H]glycine. This release was calcium-dependent. The possibility that glycine may function as a neurotransmitter substance within the striatum is considered, and the hypothesis that GABA may partially exert some of its pharmacological effects through the glycine receptor is discussed.     

Inhibitory effects of brotizolam,a new thienodiazepine,on limbic forebrain and neostriatal dopaminergic systems in vivo and in vitro

Studies were performed to elucidate the effects of brotizolam, a newly synthesized thienodiazepine, chemically related to the benzodiazepines. on dopamine turnover in the limbic forebrain and neostriatum. Intraperitoneally administered brotizolam retarded the rate of α-methyl-p-tyrosine-induced depletion of dopamine in the olfactory tubercle (OT), nucleus accumbens (NA) and caudate nucleus (CN). Significant retardation was observed with brotizolam in doses ranging from 0.1–10 mg/kg in the olfactory tubercle, and from 1–10 mg/kg in the nucleus accumbens and caudate nucleus. These inhibitory effects of brotizolam were antagonized by bicuculline, a GABA antagonist, in all of the regions examined. Using slices of the olfactory tubercle, nucleus accumbens and caudate nucleus, the effects of brotizolam on dopaminergic nerve terminals were examined _{in vitro}. Basal release of dopamine was not affected by brotizolam in concentrations up to 10^?6 M; however. K⁺-stimulated release of dopamine was significantly reduced by brotizolam at 10^?7M or above. The reduction of K⁺-stimulated release of dopamine was antagonized by bicuculline, added in the superfusion medium. These data suggest that brotizolam inhibits the release of dopamine in the limbic forebrain and neostriatal systems probably through mechanisms including a facilitation of GABAergic action on dopaminergic nerve terminals.     

Interactions of dopamine and the release of [3H]-taurine and [3H]-glycine from the isolated retina of the rat

1 The dose-related, calcium-dependent, potassium-stimulated release of preloaded [³H]-dopamine from the superfused rat retina has been demonstrated.

2 A high-affinity uptake system for dopamine exists in rat retina in vitro; K_m value was calculated as 1.89 μM, V_max value as 1.4 nmol g^-1 tissue h^-1.

3 Dopamine (0.8 and 4 mM) inhibited the spontaneous release of [³H]-glycine from retina, and in the case of 0.8 mM dopamine this inhibitory effect was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

4 The potassium-evoked (25 mM) release of [³H]-glycine from rat retina was similarly inhibited by dopamine (0.4-4 mM) in a dose-related manner when added to the superfusate with the potassium. The effect of 0.8 mM dopamine was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

5 Dopamine (4 mM) significantly reduced the spontaneous release of [³H]-taurine from rat retina.

6 The potassium-stimulated (25 mM) release of [³H]-taurine occurred after the cessation of the depolarizing stimulus. This delayed release of [³H]-taurine was unaffected if dopamine was applied to the superfusate at the same time as the potassium, but it was significantly reduced if dopamine (0.8 and 4 mM) was applied after the depolarizing stimulus had been removed and during the actual amino acid release phase.

7 The inhibition of K⁺-stimulated (25 mM) delayed release of [³H]-taurine by applying dopamine (0.8 mM) after the depolarizing stimulus was blocked by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

8 The results are discussed with respect to the possible neurotransmitter role for dopamine within the rat retina, and its possible interaction with glycine and taurine.

     

Inhibition of K+-stimulated [3H]dopamine and [14C]acetylcholine release by the putative dopamine autoreceptor agonist,B-HT 920

Summary The inhibition of K¹-stimulated [³H]dopamine and [¹⁴C]acetylcholine release from preloaded rat striatal slices was used to examine the presynaptic selectivity of the putative dopamine autoreceptor agonist, B-HT 920. In the micromolar range, B-HT 920 caused a concentration-dependent inhibition of the release of both labeled neurotransmitters as evoked by 20 mM K⁺. The effect of B-HT 920 on both [³H]dopamine and [¹⁴C]acetylcholine release was completely blocked by (+) butaclamol but not by (–) butaclamol. Sulpiride, a selective D₂ antagonist, similarly blocked the inhibitory effect of B-HT 920 on the release of both labeled neurotransmitters indicating both responses were mediated by D₂ receptors. (+) Butaclamol alone elevated stimulated [³H]dopamine release suggesting a significant amount of autoreceptor occupancy by endogenously released dopamine. Experiments with tolazoline and the alpha₂ agonist, B-HT 933, did not suggest any involvement of alpha-adrenoceptor activity in the inhibitory effects of B-HT 920 on the release of either transmitter. Inhibition of release was a selective effect of B-HT 920 as the drug was without effect on the K⁺-stimulated release of [³H]serotonin. The results indicate that in vitro B-HT 920 is active of both pre-and postsynaptic dopamine receptors in contrast to the pattern of effects observed after its in vivo administration.     

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max values of [³H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K⁺-dependency of the [³H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [³H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [³H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [³H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

Neurotensin effect on dopamine release and calcium transport in rat striatum: interactions with diphenylalkylamine calcium antagonists

Summary The release of dopamine was investigated in rat striatal slices exposed in vitro to neurotensin. This peptide increased basal and K⁺-evoked dopamine release. Moreover neurotensin antagonized the flunarizine-induced inhibition of K⁺-stimulated dopamine release. The K⁺-evoked ⁴⁵Ca²⁺ accumulation was also inhibited by flunarizine. This effect was antagonized by neurotensin. The results suggest that dopamine release in rat striatum is regulated by different molecular events also of peptidergic nature having as possible mechanism of action an influence on calcium ion movements.     

Effect of metabolic inhibitors and ouabain on amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue

In experiments on isolated rat cerebral cortex, it was observed that d-amphetamine released norepinephrine from cerebral cortex into the incubation medium. Amphetamine-induced release of norepinephrine was markedly potentiated in the presence of the metabolic inhibitors sodium cyanide, 2,4-dinitrophenol and iodoacetate. Release of norepinephrine by the metabolic inhibitors in the absence of amphetamine was minimal. Amphetamine-induced release of dopamine from corpus striatum was also potentiated by sodium cyanide. The EC₅₀ for amphetamine-induced release of dopamine was 20μM with amphetamine alone and 0.58 μM in the presence of sodium cyanide. Release of norepinephrine and dopamine by elevated concentrations of potassium chloride was again potentiated by the metabolic inhibitors. Similarly, ouabain (10–25 μM) produced minimal release of norepinephrine but potentiated amphetamine- and potassium-induced release of catecholamines. The metabolic inhibitors markedly reduced the ATP content of the chopped tissue during the incubation while ouabain had no effect. The results suggest that the potentiation of amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue is not simply due to depletion of tissue ATP levels but may be related to sodium or potassium transport. The Na⁺ , K⁺ -ATPase sodium pump is inhibited either: (1) when ATP levels are reduced by metabolic inhibitors, or (2) when Na⁺ , K⁺ -ATPase is inhibited by ouabain. Inhibition of the sodium pump would lead to an increase in the intraneuronal sodium concentration. A model is described in which norepinephrine and sodium are co-transported across the membrane during amphetamine-induced release of the biogenic amine. Elevation of the intraneuronal sodium concentration would provide increased levels of the co-substrate for transport and could account for the potentiation of release produced by metabolic inhibitors and ouabain.     

Selectivity of release of norepinephrine, dopamine and 5-hydroxytryptamine by amphetamine in various regions of rat brain

Tissues from various regions of rat brain were incubated with [³H]norepinephrine, [³H]dopamine or [³H]5-hydroxytryptamine; excess amine was washed from the tissues and the [³H]amines present in the incubation medium and tissues were measured. The release of the accumulated [³H]amine by amphetamine was related to the nature of the neurons in the several regions and the release of each of the [³H]amines was quite selective for neurons which contain the corresponding endogenous amine. The release of [³H]norepinephrine from norepinephrine neurons is most sensitive to amphetamine, while the release of [³H]dopamine from dopamine neurons requires higher concentrations of amphetamine. Release of [³H]5-hydroxytryptamine from 5-hydroxytryptamine neurons appears to be least sensitive to amphetamine. This selective effect of amphetamine on neurons containing biogenic amines may help to explain the behavioral changes which occur at different doses of amphetamine. The selectivity of release of the [³H]-amines was also examined by observing the accumulation and release of [³H]norepinephrine and [³H]5-hydroxytryptamine in the presence of five-fold higher concentrations of the unlabeled amines. There was relatively little effect of unlabeled norepinephrine on the accumulation and release of [³H]5-hydroxytryptamine or of unlabeled 5-hydroxytryptamine on [³H]norepinephrine accumulation and release. Unlabeled dopamine reduced the accumulation and altered the release of [³H]norepinephrine and [³H]5-hydroxytryptamine, but this was much less marked than the effect of the unlabeled analogues of the [³H]amines. The release of each of the [³H]amines by its unlabeled analogue occurred with concentrations as low as, or lower than, the release produced by amphetamine or the other unlabeled amines. The release of the [³H]amines by a five-fold higher concentration of the unlabeled analogues was so high that it was not increased further by very high concentrations of amphetamine (10^?3 M). These results suggest that, under the conditions of this study, the [³H]amines are accumulated within specific neurons of brain tissue and that they are selectively released by amphetamine.     

Dopamine-induced inhibition of endogenous acetylcholine release from the isolated ileal synaptosomal preparations of guinea-pig mediated via α-adrenoceptors

1 The effect of exogenous dopamine on the release of endogenous acetylcholine (ACh) from isolated ileal synaptosomal guinea-pig preparations was examined by means of high pressure liquid chromatography with electrochemical detection. 2 Release of ACh was induced by substance P or by depolarization with high potassium (50 mM) in a medium containing atropine, propranolol and naloxone. 3 Dopamine produced a concentration-dependent inhibition of the evoked ACh release induced by substance P or in samples depolarized by high potassium. This action of dopamine was not reversed by the dopamine receptor antagonists either for the DA2 subtype, domperidone, or for the DA1 subtype, SCH23390. Fenoldopam, the agonist of dopamine DA1 receptors, or quinpirole, the agonist of dopamine DA2 receptors, reduced the evoked ACh release, although only in high, non-dopamine-specific concentrations. 4 Failure of guanethidine or desipramine to inhibit this effect of dopamine ruled out mediation by endogenous noradrenaline. 5 Idazoxan and yohimbine reversed this dopamine-induced inhibition at concentrations sufficient to abolish the action of clonidine. Influx of ⁴⁵Ca stimulated by substance P or high potassium into synaptosomal preparations was attenuated in the presence of dopamine. This inhibition by dopamine was also reversed by idazoxan or yohimbine but not by dopamine receptor antagonists. Moreover, the dopamine-induced inhibitions of both the ACh release and the influx of ⁴⁵Ca disappeared in the samples treated with pertussis toxin at a dose sufficient to abolish the action of clonidine. 6 It is concluded that dopamine suppresses the influx of calcium ions into cholinergic nerve terminals via an activation of α₂-adrenoceptors coupled with a pertussis toxin-sensitive GTP-binding protein, resulting in the decrease of ACh release from ileal synaptosomes of guinea-pigs.     

Direct dopamine D2-receptor-mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+-mobilizing agent

1. In CHO cells transfected with the rat dopamine D₂ receptor (long isoform), administration of dopamine per se elicited a concentration-dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC₅₀=25 nM). The partial D₂ receptor agonist, (−)-(3-hydroxyphenyl)-N-n-propylpiperidine [(−)-3-PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC₅₀=91 nM).
2. The AA-releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca²⁺ release, 8-(N N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), by excluding calcium from the medium, by the phospholipase A₂ (PLA₂) inhibitor, quinacrine, and by long-term pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, it was antagonized by the D₂ antagonists, raclopride and (−)-sulpiride–but not by (+)-sulpiride–and absent in sham-transfected CHO cells devoid of D₂ receptors.
3. The results obtained contrast to the previous notion that dopamine and other D₂ receptor agonists require the concomitant administration of calcium-mobilizing agents such as ATP, ionophore A-23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines.

     

Ritonavir and Saquinavir directly stimulate anterior pituitary prolactin secretion,in vitro

An association between human immunodeficiency virus type I (HIV-1) protease inhibitors (PIs) and galactorrhoea/hyperprolactinemia adverse effect has recently been reported in four HIV-1-infected women treated with PIs (indinavir, nelfinavir, ritonavir or saquinavir). This could be explained by a direct effect of ritonavir and saquinavir on anterior pituitary prolactin (PRL) release, and/or an indirect effect of PIs on the secretion of hypothalamic dopamine, which is the main PRL inhibitory factor. Anterior pituitaries were explanted from adult male Wistar rats, the cells were trypsin dispersed, plated into multiwell cultures and incubated for 1 h with either ritonavir or saquinavir (0.01 nM-1μM). PRL release into the incubation medium was evaluated by radioimmunoassay. Hypothalamic neuronal endings (synaptosomes) were prepared by tissue homogenization, incubated with ³H-dopamine, substituting for the endogenous dopamine pool, and perfused with ritonavir or saquinavir, both basally and during depolarization (K⁺ 15 mM)-induced dopamine release. Beta-emission from 2 min perfusate fractions, corresponding to ³H-dopamine release, was detected by liquid scintillation scanning. We found that both ritonavir and saquinavir are able to significantly stimulate PRL secretion, with saquinavir slightly more effective than ritonavir. On the contrary, both protease inhibitors do not modify either basal or depolarization-induced dopamine release. We can speculate that HIV PIs despite a high affinity for the catalytic site of HIV protease, could also bind to and inhibit homologous mammalian proteins in the anterior pituitary that are involved in PRL secretion.     

Effects of morphine on the rat striatal dopamine metabolism.

Concomitant with total suppression of the spontaneous unitary discharges of neurons in the rat corpus striatum, intracarotidly injected morphine (5 mg/kg) was also found to increase the levels of dopamine, homovanillic acid and cyclic AMP by 80, 65 and 46 per cent respectively, measured 5 min after injection. This provides further support to the hypothesis that the nigrostriatal dopaminergic pathway is stimulated by acutely administered morphine. Morphine (10^?5-10^?3 M) did not alter the activity of striatal tyrosine aminotransferase. The drug, added in vitro (10^?6-10^?4 M) or by intracarotid injection (5 mg/kg) did not affect the activity of striatal tyrosine hydroxylase. Moreover, morphine (10^?4 M) did not interfere with the inhibitory effects of dopamine (10^?6-10^?4 M) on striatal tyrosine hydroxylase. However, it significantly potentiated the stimulatory effects of cyclic AMP on this enzyme. Morphine (10^?5-10^?4) was also found to have no effect on the spontaneous or K⁺-stimulated release of dopamine from striatal homogenate and synaptosomes. However, in the presence of 5 × 10^?5 M and 10^?4 M morphine, the uptake of dopamine by striatal homogenate was inhibited by 14 and 33 per cent respectively. With synaptosomal preparations, dopamine uptake was inhibited by 17 per cent in the presence of 10^?4 M morphine—the inhibition being competitive with dopamine with an apparent K_i of 0.41 mM. The inhibition of dopamine uptake caused by 10^?4 M morphine in either preparation was not reversed by the addition of 10^?4 M naloxone. It was concluded that the increase in dopaminergic activity following acute treatment of morphine is probably due to (1) prolongation of the effect of dopamine on the post-synaptic neurons resulting in increased production of cyclic AMP which in turn potentiates dopamine synthesis and (2) decrease in presynaptic cystosol dopamine which is normally a feedback inhibitor of tyrosine hydroxylase thus leading to increased synthesis of dopamine.     

Tityus zulianus venom induces massive catecholamine release from PC12 cells and in a mouse envenomation model

Scorpion envenomation is a public health problem in Venezuela, mainly produced by Tityus discrepans (TD) and Tityus zulianus (TZ). Accidents by these two species differ clinically. Thus, TZ envenomation is associated with high mortality in children due to cardiopulmonary disorders, as a result of, excessive amounts of plasma catecholamines (Epinephrine) release from adrenal medulla, probably via the voltage-gated sodium-channel activated by specific scorpion toxins. This Epi release is, in part responsible, for some of the envenomation clinical consequences, resembling those described for patients presenting catecholamine-releasing tumors (pheochromocytoma). In this work, BALB/c mice and rat pheochromocytoma-derived PC12 cells were used to provide in vivo and in vitro models, respectively, on which the basis for the TZ-mediated catecholamine release mechanism could be elucidated. In mice, TZ venom increased, at 1 h post-injection, the Epi plasma levels in 4000%, which remained elevated for 24 h. A significant rise in plasma levels of the catecholamine catabolite 3-Methoxy-4-Hydroxy-Phenyl-Glycol (MHPG) was also observed. In [³H]dopamine-loaded PC12 cells, TZ venom potentiated the carbamylcholine (CC)-mediated release of [³H]dopamine, as shown by the leftward shift in the CC-dose-response curves. Moreover, TZ venom also displayed the maximal [³H]dopamine releasing activity compared to TD venom, with significant reduction of the EC50 for CC. The nicotinic-acetylcholine receptor (nAChR) blocker hexamethonium induced a significant inhibition of the [³H]dopamine release produced by CC in PC12 cells but the TZ-elicited release of [³H]dopamine was 70% hexamethonium-insensitive, suggesting unidentified TZ toxins affecting other regulatory mechanisms of catecholamine secretion.     

Comparative effects of benztropine and nomifensine on dopamine uptake and release from striatal synaptosomes

The spontaneous release of ³H-dopamine from preloaded superfused rat striatal synaptosomes was markedly increased by benztropine, but to a much lesser degree by nomifensine. Effects of similar magnitude were also observed in static incubations, indicating that superfusion had little inhibitory effect on dopamine re-uptake. However, benztropine and nomifensine were equivalent in their blockade of ³H-dopamine uptake into striatal synaptosomes. Fenfluramine produced a significant increase in the release of ³H-dopamine from superfused striatal synaptosomes. The fenfluramine-induced increase in ³H-dopamine efflux was additive to the benztropine response, but not to the nomifensine effect. The possibility that nomifensine may also act inhibiting the spontaneous release of dopamine by interaction at presynaptic dopamine receptors is discussed.     

Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2-dopamine-autoreceptors

1. Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D₂-dopamine receptors in the modulation of stimulation-induced (S-I) dopamine release from rat striatal slices incubated with [³H]-dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D₂ receptors since phorbol dibutyrate (PDB) and (−)-sulpiride, respectively, elevated S-I dopamine release.
2. The protein kinase C inhibitors polymyxin B (21 μM) and chelerythrine (3 μM) had no effect on stimulation-induced (S-I) dopamine release. However, when presynaptic dopamine D₂ receptors were blocked by sulpiride (1 μM), an inhibitory effect of both PKC inhibitors on S-I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D₂ receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down-regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished.
3. The inhibitory effect of the dopamine D₂/D₃ agonist quinpirole on S-I dopamine release was partially attenuated by PKC down-regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC-dependent as well as a PKC-independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down-regulation, suggesting that it operated exclusively through a PKC-independent mechanism.
4. These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally-released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.

     

Cardiac electrophysiology of four neuroleptics: Melperone,haloperidol, thioridazine and chlorpromazine

Summary Effects of dopamine receptor agonists and antagonists on the release of dopamine were studied in the caudate nucleus of the rabbit. The nucleus contained 6.7 g/g of dopamine, but negligible levels of noradrenaline and dopamine--hydroxylase. No formation of ³H-noradrenaline was detected in caudate slices preincubated with ³H-dopamine, and more than 95% of the tritium content of the tissue consisted of ³H-dopamine.When caudate slices were preincubated with ³H-dopamine and then superfused with amine-free medium, there was a basal outflow of tritium that was not or only slightly changed by tetrodotoxin (10^–7 and 10^–6 M), apomorphine (up to 10^–5 M), bromocriptine (up to 10^–6 M), chlorpromazine (up to 10^–6 M), haloperidol (up to 10^–7 M), or omission of calcium. Electrical stimulation (3 Hz, 24 mA, 2 ms pulse duration, 2-min periods) greatly increased the outflow of tritium. The stimulation-evoked overflow was abolished by tetrodotoxin (10^–7 and 10^–6 M) and in calcium-free medium. Apomorphine (10^–8–10^–5 M) and bromocriptine (10^–8–10^–6 M) reduced, whereas chlorpromazine (10^–7 and 10^–6 M) and haloperidol (10^–8 and 10^–7 M) enhanced the evoked overflow. The inhibitory effect of apomorphine and bromocriptine was antagonized by chlorpromazine and haloperidol, but not by phentolamine.Silicone tubings that had been in contact with ³H-haloperidol retained tritiated material that was slowly eluted during perfusion with water or physiological salt solution. The material was identified as ³H-haloperidol. When silicone tubings pretreated with unlabelled haloperidol were used in subsequent dopamine release experiments, the inhibitory effect of apomorphine was not reproduced.It is concluded that, in the caudate nucleus of the rabbit, apomorphine and bromocriptine depress, whereas chlorpromazine and haloperidol facilitate action potential-evoked release of dopamine. The effects are mediated by specific receptors which may be located on the dopaminergic nerve terminals. The receptors appear to be normally activated by released dopamine itself, which thus inhibits its own further release. Part of the discrepancies in the literature concerning dopaminergic modulation of dopamine release may be due to retention of neuroleptic drugs in superfusion assemblies, followed by slow elution and interference with subsequent experiments.     

Effect of ethanol on dopamine synthesis and release from rat corpus striatum.

The effect of ethanol upon dopamine (DA) synthesis and release from dopaminergic terminals was studied. Slices from rat corpus striatum were incubated in freshly oxygenated Krebs-Ringer phosphate (KRP) media of variable ionic composition containing l-tyrosine-¹⁴C (U) as dopamine precursor and in the presence and absence of ethanol (0.1 to 0.8%, w/v). The addition of ethanol directly to normal KRP media produced no effect on the conversion of ¹⁴C-tyrosine to ¹⁴C-DA. As reported previously, tne absence of Ca²⁺ from the incubation media markedly increased the formation of ¹⁴C-DA. The presence of ethanol in this media was not able either to block or to potentiate the Ca²⁺-free induced formation of ¹⁴C-DA. The presence of K⁺ (55 mM) in the incubation media also increased about 2-fold the formation of ¹⁴C-DA. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K⁺ markedly blocked the K⁺-induced formation of newly synthesized ¹⁴C-DA. The presence of ethanol did not modify the amount of ¹⁴C-tyrosine taken up by striatal slices incubated either in normal KRP or KRP-high K⁺ media. A superfusion system was used to study both spontaneous and K⁺-induced release of labeled DA from striatal slices. The addition of ethanol (0.4 to 0.8%, w/v) to the superfusion system was not able either to block or to potentiate the K⁺-induced release of ³H-DA previously taken up by the slices nor the K⁺-induced release of newly synthesized ³H-DA. The results reported in this work, as well as others recently reported by Murrin et al. (Pharmacologist16, 128, 1974), suggest the existence of another regulatory mechanism of DA synthesis besides the commonly accepted one of feedback inhibition exerted by DA upon the rate-limiting enzyme, tyrosine hydroxylase. The possibility is also raised that the inhibitory effect of ethanol described in this paper might play a role in the intoxicating effect of ethanol in vivo.