Characterization of dopamine autoreceptor and [3H]spiperone binding sites in vitro with classical and novel dopamine receptor agonists

The specific D2 receptor agonist, LY 141865, but not the specific D1-receptor agonist, SK&F 38393, potently inhibited electrically evoked [3H]dopamine release from slices of the cat caudate. Similarly, LY 141865, but not SK&F 38393, inhibited [3H]spiperone binding to membranes of the cat caudate. The inhibition by dopamine receptor agonists of electrically evoked [3H]dopamine release was antagonized by the specific D2-receptor antagonist S-sulpiride. The inhibition of the electrically evoked release of [3H]dopamine by apomorphine was not, however, antagonized by the specific D1-receptor antagonist, bulbocapnine. Similarly, S-sulpiride but not bulbocapnine potently inhibited [3H]spiperone binding to membranes of the cat caudate. These results suggest that the dopamine autoreceptor modulating the depolarization-evoked release of [3H]dopamine, and the binding site of [3H]spiperone, are valid in vitro models for D2-dopamine receptors. Contrary to some previous reports, DPI was inactive in both in vitro dopamine receptor models. The IC50 values of a series of dopamine receptor agonists correlated very well in the two in vitro dopamine receptor models. One exception to this correlation was bromocriptine, which was more potent at [3H]spiperone binding sites than at the dopamine autoreceptor. With the exception of bromocriptine, all dopamine receptor agonists had one-hundred fold higher potency at the dopamine autoreceptor than at [3H]spiperone binding sites. [3H]Spiperone binding sites are localized primarily postsynaptic to dopamine terminals. Possible differences between the pharmacological properties of pre- and postsynaptic dopamine receptors should become apparent in the comparison of the two in vitro dopamine receptor models. However, the order of potency of dopamine receptor agonists with both in vitro models, dopamine autoreceptor and [3H]spiperone binding, was the same: N-n-propylnorapomorphine greater than TL-99 = 7-HAT greater than M-7 greater than Apomorphine greater than LY 141865.     

Stereoisomers of apomorphine differ in affinity and intrinsic activity at presynaptic dopamine receptors modulating [3H]dopamine and [3H]acetylcholine release in slices of cat caudate

We investigated the effects of R(?)-apomorphine and S(+)-apomorphine on dopamine receptors modulating electrically evoked [³H]dopamine and [³H]acetylcholine release from slices of cat caudate nucleus. R(?)-Apomorphine inhibited the release of both [³H]dopamine and [³H]acetylcholine with an IC₅₀ of 20 nM, while S(+)-apomorphine was without inhibitory action on the electrically evoked release of either neurotransmitter at concentrations up to 1 μM. At a concentration of 1 μM, however, S(+)-apomorphine antagonized the inhibition by R(?)-apomorphine, producing a parallel five-fold shift to the right in the concentration-response curve to R(?)-apomorphine. These results indicate that S(+)-apomorphine is devoid of intrinsic activity to stimulate presynaptic dopamine receptors modulating the electrically evoked release of dopamine and acetylcholine. In addition, S(+)-apomorphine has an approximately ten-fold lower affinity for presynaptic dopamine receptors compared to R(?)-apomorphine.     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.     

Inhibitory effect of prostaglandins on dopamine release from the retina

1. Prostaglandins have been shown to modulate transmitter release from both central and peripheral neuroeffector junctions. In the present study, we examined the effect of prostaglandins on [³H]-dopamine release from isolated, superfused rabbit retina.2. Both naturally occurring and synthetic prostaglandins produced concentration-dependent reduction of electrically evoked [³H]-dopamine overflow without affecting basal tracer efflux. The rank order of potencies of the agonists was: sulprostone > 16, 16-dimethyl PGE₂>PGE₂> > 11-deoxy-PGE₁>PGF_2α.3. The PGE₂-mediated inhibition of field stimulated [³H]-dopamine release was not blocked by the selective EP₁-receptor antagonist, AH6809 (5–30 μM).4. The cyclooxygenase inhibitor, flurbiprofen (3 μM) had no effect on basal or evoked [³H]-dopamine overflow nor did it affect the inhibition caused by PGE₂ suggesting that endogenous prostaglandins are not involved in the regulation of dopamine release in the retina.5. The inhibition of [³H]-dopamine release produced by submaximal concentrations of PGE₂, apomorphine and melatonin were not additive indicating that presynaptic PGE₂, D₂- and melatonin receptors coexist at sites for neurotransmitter release and may share a common mechanism for regulation of dopamine release.6. We conclude that prostaglandin-induced inhibition of electrically evoked [³H]-dopamine release from the rabbit retina may be mediated by specific prostaglandin receptors of the EP₃ subtype.     

No evidence for presynaptic opioid receptors on cholinergic,but presence of κ-receptors on dopaminergic neurons in the rabbit caudate nucleus: involvement of endogeneous opioids

Summary The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [³H]dopamine or [³H] choline, superfused with medium (containing in most experiments the D₂ receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [³H]overflow from slices prelabeled with [³H]dopamine (evoked [³H]dopamine release) was significantly reduced by preferential -opioid receptor agonists, like U-50,488 H, but not by µ- or -opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA₂-value of the -selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors.On the other hand, the [³H]overflow from rabbit caudate nucleus slices prelabeled with [³H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D₂ heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential -opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release.From our data we conlude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic -opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids. Correspondence to R. Jackisch at the above address     

Dopamine receptor mediated inhibition by pergolide of electrically-evoked 3H-dopamine release from striatal slices of cat and rat: Slight effect of ascorbate

Summary The dopamine receptor agonist pergolide inhibited the calcium-dependent, electrically evoked overflow of tritium from slices of the striatum of cat or rat prelabelled with ³H-dopamine. This inhibition of tritium overflow by nanomolar concentrations of pergolide was antagonized by the benzamide neuroleptic S-sulpiride (0.1 M). In millimolar concentrations, l- ascorbate had slight or no effects on this dopamine receptor mediated inhibition, in striatal slices of either the cat or the rat. Since these same concentrations of ascorbate have been reported to completely block the specific binding of ³H-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) and of ³H-apomorphine to presumed dopamine receptors, the present results suggest a dissociation between the characteristics of ³H-ADTN and ³H-apomorphine binding and the dopamine autoreceptor. Previous contradictory results concerning the existence of inhibitory dopamine receptors which modulate depolarization-evoked overflow of dopamine from the striatum of the rat are thus apparently not due to a species difference nor to the use of ascorbate, but rather to differences in experimental conditons.     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

Cannabinoid CB1 receptor-mediated inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain

Guinea-pig hippocampal slices preincubated with [³H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [³H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB₁ receptors was determined in rat brain cortex membranes using [³H]SR 141716. In guinea-pig hippocampal slices preincubated with [³H]noradrenaline, tritium overflow stimulated by NMDA 100 μM and 1000 μM and by kainate 1000 μM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB₁ receptor antagonist SR 141716 increased the NMDA (1000 μM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 μM or kainate 1000 μM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 μM)- and kainate (1000 μM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [³H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 μM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [ ³ H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pK _i 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pK _i < 5). In conclusion, activation of CB₁ receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB₁ receptors or that these receptors are activated by endogenous cannabinoids. Received: 25 February 1999 / Accepted: 12 April 1999     

Irreversible blockade of [3H]spiperone- but not [3H]dopamine-labeled dopamine receptors with phenoxybenzamine

Calf caudate homogenates, preincubated with 10 ⁵M phenoxybenzamine followed by thorough washing, exhibited an essentially complete loss of dopamine-associated high affinity [³H]spiperone specific binding. This effect of phenoxybenzamine was inhibited in a potent and concentration-dependent manner by domperidone, a potent and specific dopamine-receptor blocker. No effect of phenoxybenzamine was observed on [³H]dopamine specific binding. These data indicate that the blockade of [³H]spiperone binding occurred at the dopamine-receptor binding site, or sites, and that the [³H]spiperone binding was molecularly distinct from the [³H]dopamine specific binding. The characteristics of dopamine-associated [³H]spiperone specific binding suggest that there may be two sites labeled by [³H]spiperone.     

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     

Stereoselectivity of presynaptic autoreceptors modulating dopamine release

The effects of the (R)- and (S)-enantiomers of sulpiride and butaclamol were studied on the spontaneous and field stimulation-evoked release of total radioactivity from slices of rabbit caudate nucleus prelabelled with [3H]dopamine. (S)-Sulpiride in concentrations ranging from 0.01--1 microM enhanced the electrically evoked release of [3H]dopamine while (R)-sulpiride was 10 times less potent than (S)-sulpiride. Exposure to (S)-butaclamol (0.01--1 microM) but not to (R)-butaclamol (0.1--10 microM) enhanced the field-stimulated release of [3H]dopamine. The facilitatory effects of (S)- and (R)-sulpiride and (S)-butaclamol on the stimulated release of the labelled neurotransmitter were observed under conditions in which these drugs did not modify the spontaneous outflow of radioactivity. Only the active enantiomers of sulpiride and butaclamol antagonized the inhibition by apomorphine (1 microM) of the stimulated release of [3H]dopamine. Our results indicate that the presynaptic inhibitory dopamine autoreceptors modulating the stimulation-evoked release of [3H]dopamine in the caudate nucleus are, like the classical postsynaptic dopamine receptors, chemically stereoselective.     

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding

A 15-mer, all-phosphorothioate-modified antisense oligodeoxynucleotide (ASO) targeted against rat dopamine D3 receptor mRNA (4 μM, 5 days) significantly reduced (28%) the amount of binding sites labelled with [³H]spiperone in monolayer cultured Chinese hamster ovary (CHO) cells transfected with the complementary desoxyribonucleic acid (cDNA) for the rat D3 receptor. In contrast, D3-ASO treatment did not reduce the amount of bound [³H]spiperone in CHO cells transfected with D2 receptor cDNA. Intracerebroventricular infusion of D3-ASO (osmotic minipump, 10 μg/μl/h, 7 days) influenced dopamine receptor density in the limbic forebrain such that the upper part of the dopamine/[³H]spiperone displacement curve – tentatively representing the D3 receptor – was altered significantly. Spontaneous locomotor activity of non-habituated rats was increased significantly in D3-ASO-treated animals; in addition, in vivo microdialysis revealed a moderate increase in dopamine release in the nucleus accumbens in these animals. In all experiments, an oligodeoxynucleotide comprising the same nucleotides as the antisense sequence, but in random order, was used as control. It is concluded that the antisense strategy is useful for investigating the functional role of dopamine D3 receptors and that the dopamine D3 receptor is involved in rat locomotor behaviour. Received: 23 June 1997 / Accepted: 19 May 1998     

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.     

Unrestricted free-choice ethanol self-administration in rats causes long-term neuroadaptations in the nucleus accumbens and caudate putamen

In the present study, the reactivity of striatal dopamine and dopamine-sensitive neurons in superfused striatal slices of ethanol-experienced rats was compared to that of ethanol-naive rats, 3 weeks after oral ethanol self-administration. During the acquisition phase (17 days), rats were offered increasing concentrations of ethanol (from 2 to 10%, 24 h per day) on an alternate-day schedule in a free choice with water. Following 2 weeks of unrestricted 10% ethanol consumption, the highest and lowest drinkers (representing about 25% of the upper and lower extremes of the total population) were selected. Preliminary experiments revealed that both groups of rats displayed a profound increase in ethanol consumption and preference 3 weeks after cessation of ethanol self-administration (deprivation effect). This deprivation effect was associated with an increase in electrically evoked release of [³H]dopamine from superfused nucleus accumbens slices, whereas the evoked [³H]dopamine release from caudate putamen slices remained unchanged. In slices of the caudate putamen, but not in nucleus accumbens slices, postsynaptic dopamine D₁ receptor-stimulated cyclic AMP production was also enhanced. In addition, prior ethanol consumption enhanced the electrically evoked release of [¹⁴C]acetylcholine release in both striatal regions. Interestingly, the magnitude of these long-term neuroadaptations correlated with the amount of daily ethanol consumption, i.e. neuronal hyperresponsiveness in the striatum was more profound in the high than in the low ethanol drinkers. These data show for the first time that unrestricted free-choice ethanol consumption in rats is associated with a long-term increase in dopaminergic and cholinergic neurotransmission in the nucleus accumbens and caudate putamen. These (and other) neuroadaptations may underlie the enhanced motivation to self-administer ethanol and the maintenance of ethanol consumption long after deprivation. Received: 14 April 1998/Final version: 15 June 1998     

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.     

Effect of extracellular dopamine on the release of dopamine in the rabbit caudate nucleus: Evidence for a dopaminergic feedback inhibition

Summary Slices of the head of the rabbit caudate nucleus were preincubated with 10^–7 M ³H-dopamine and then superfused, and the effect of unlabeled dopamine on the outflow of tritium was investigated. In most experiments, nomifensine was added throughout superfusion in order to block uptake of the unlabeled amine. Nomifensine was a potent inhibitor of the uptake of ³H-dopamine into rabbit caudate synaptosomes, with an IC₅₀ of 5·10^–8 M at a ³H-dopamine concentration of 4·10^–8 M.In the absence of nomifensine, unlabeled dopamine (10^–7 M and higher concentrations) accelerated the basal outflow of tritium from preincubated slices. 10^–5 M nomifensine strongly counteracted the acceleration. In the presence of nomifensine, unlabeled dopamine (10^–7 to 10^–6 M) caused a concentrationdependent decrease of the overflow of tritium evoked by electrical stimulation at 0.1 Hz. Chlorpromazine and haloperidol (in the presence of nomifensine) increased the stimulation evoked overflow and antagonized the inhibitory effect of dopamine.It is concluded that extracellular dopamine shares with other dopaminergic agonists the ability to inhibit action potential-evoked release of intraneuronal dopamine. The inhibition is mediated by specific receptors. The results support the hypothesis that previously released dopamine, by an action on these receptors, can inhibit further release of dopamine.     

Terminal serotonin autoreceptor function in the rat hippocampus is not modified by pertussis and cholera toxins

Summary The possibility that the terminal serotonin (5-HT) autoreceptor in the rat hippocampus is coupled to G_i, G_o or G_s regulatory proteins was investigated using the electrically evoked overflow of [³H]5-HT from preloaded slices. Pertussis toxin, which inactivates G_i/o or cholera toxin, which stimulates GS, was injected directly in the hippocampus 3 to 11 days prior to the experiments. Hippocampus slices were prepared, loaded with [³H]5-HT, superfused continuously, and stimulated electrically 72 min (S₁) and 116 min (S₂) after the beginning of superfusion. In the absence of any drug, the evoked overflow of [³H]5-HT in S₁ was not altered by either toxin. The enhancing effect of the 5-HT reuptake blocker paroxetine (1 mol/I) on the evoked [³H]5-HT overflow was also unaltered by these toxins. 5-Carboxyamidotryptamine, a 5-HT autoreceptor agonist, inhibited in a concentration-dependent manner the stimulation-evoked release of [³H]5-HT The concentration-effect curve (0.001–0.1 mol/I) for this drug was not altered by pretreatment with either pertussis or cholera toxin. Similarly, the effect of another 5-HT autoreceptor agonist, 5-methoxytryptamine (0.1 and I mol/I), was not altered in the pretreated rats. In addition, the reduction of [³H]5-HT overflow obtained by increasing the stimulation frequency from 1 Hz to 5 Hz, which is due to an increase in terminal 5-HT autoreceptor activation at the higher frequency, was not altered by either toxin. The enhancing effect of the 5-HT autoreceptor antagonist methiothepin (1 mol/I) on stimulation-evoked [³H]5-HT overflow was not changed by either pretreatment. N-Ethylmaleimide inactivates G_i/o proteins by alkylation. Preincubation with 30 mol/I N-ethylmaleimide for 30 min did not alter the efficacy of the 5-HT reuptake blocker paroxetine to enhance [³H]5-HT overflow nor did it alter the attenuating effect of 5-methoxytryptamine and the differential effectiveness of 1 and 5 Hz stimulations on the overflow of [³H]5-HT In conclusion, the results suggest that the terminal 5-HT autoreceptor in the rat hippocampus may not be coupled to G_i, G_o or G_s proteins.     

Different effects of serotonin (5-HT) uptake blockers in caudate nucleus and hippocampus of the rabbit: Role of monoamine oxidase in dopaminergic terminals

Slices of rabbit hippocampus or caudate nucleus were incubated with [³H]-5-HT (0.1 µM, 60 min) or with [³H]-DA. In hippocampal tissue, the 5-HT uptake blockers chlorimipramine, fluvoxamine, and 6-nitroquipazine (0.1, 1, 10 µM) reduced the percentage content of [³H]-5-HT in a concentration dependent manner. The degree of inhibition of [³H]-5-HT content produced by the 5-HT uptake inhibitors was not affected by the MAO inhibitors pargyline or amezinium (which by themselves enhanced [³H] loading) or the catecholamine uptake inhibitor nomifensine (which by itself did not affect [³H] loading). In caudate nucleus tissue, however, the [³H]-5-HT accumulation was reduced only at the highest concentration of the 5-HT uptake blockers (10 µM). In the additional presence of the MAO inhibitors or nomifensine (which by themselves increased or diminished, respectively, the [³H] labelling) the 5-HT uptake inhibitors became more potent in reducing the percentage [³H]-5-HT accumulation of caudate nucleus slices. These results indicate (1) that a false labelling of [³H]-5-HT into dopaminergic terminals in the caudate nucleus can be prevented by nomifensine, (2) that the 5-HT uptake blockers seem to accumulate within the dopaminergic terminals, where they may display a MAO inhibitory property. The 5-HT uptake blockers were ineffective on the percentage tritium accumulation of caudate nucleus slices incubated with [³H]-DA, regardless of the presence of pargyline or nomifensine. Tritiated DA and deaminated [³H]-metabolites were separated in the superfusate of [³H]-DA-release experiments in caudate nucleus tissue. In the presence of 6-nitroquipazine the percentage efflux of unmetabolized [³H]-DA was significantly enhanced in a concentration and time dependent manner. In comparison to 6-nitroquipazine, fluvoxamine was less potent in that respect. 6-Nitroquipazine inhibited the electrically evoked [³H]-DA and [³H]-ACh release from caudate nucleus slices in a concentration dependent manner. The effects on [³H]-DA release were abolished in the presence of pargyline. The inhibition of [³H]-ACh release was significantly diminished by the D₂-receptor antagonist domperidone. In conclusion, some 5-HT-related drugs may diminish the release of ACh from caudate nucleus slices via an enhanced dopaminergic transmission due to inhibition of MAO within the dopaminergic terminals.     

Neuropeptide Y-induced enhancement of the evoked release of newly synthesized dopamine in rat striatum: mediation by Y2 receptors

The purpose of the present study was to determine whether or not activation of neuropeptide Y (NPY) receptors resulted in an enhancement or attenuation of the KCl (50 mM) evoked release of [3H]dopamine newly synthesized from [3H]tyrosine in superfused striatal slices and, if so to identify the NPY receptor subtype mediating the effect. Rat striatal slices were prepared and placed in microsuperfusion chambers and continuously superfused with physiological buffer containing 50 microCi/ml of l-3-5-[3H]tyrosine. Superfusate effluents were collected and analyzed for [3H]dopamine by liquid scintillation spectrometry following amberlite CG50 and alumina chromatography. NPY agonists (NPY and PYY3-36) were added 6 min prior to the addition of KCl, while the Y1, Y2, and Y5 antagonist BIBO3304, BIIE0246 and CGP71683A, respectively were added 6 min prior to the agonists. Continuous superfusion with [3H]tyrosine resulted in the production of [3H]dopamine which reached a steady state at approximately 48 min. Depolarization with KCl resulted in a 2- to 3-fold increase in [3H]dopamine overflow. NPY and PYY3-36 produced a concentration dependent enhancement in the KCl induced increase in newly synthesized [3H]dopamine overflow. The Y2 antagonist BIIE0246 produced an attenuation of both the NPY and PYY3-36 induced enhancement while the Y1 antagonist BIBO3304 and theY5 antagonist CGP71683A failed to alter the NPY or PYY3-36 induced enhancement. These results are consistent with the NPY-Y2 receptor subtype mediating the facilitatory effect.     

Antagonistic Properties of RU 24969, a Preferential 5-HT1 Receptor Agonist,at Presynaptic α2-Adrenoceptors of Central and Peripheral Neurones

Abstract: The effect of RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) on the electrically evoked ³H overflow was studied in superfused rat brain cortex slices preincubated with ³H-noradrenaline or ³H-serotonin and in superfused segments of the rat vena cava preincubated with ³H-noradrenaline. In cortex slices preincubated with ³H-noradrenaline, RU 24969 facilitated the electrically (3 Hz) evoked ³H overflow. This effect was abolished by phentolamine but was not affected by desipramine or the 5-HT₃ receptor antagonist ICS 205–930. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow (determined in the presence of desipramine) was shifted to the right by RU 24969 32 and 100 μmol/1. In this respect, RU 24969 was about 500 times less potent than phentolamine. In cortex slices preincubated with ³H-serotonin, the inhibitory effect of 3.2 μmol/1 RU 24969 on the electrically evoked ³H overflow was increased by phentolamine. In segments of the vena cava, RU 24969 inhibited the electrically (0.66 Hz) evoked ³H overflow. The concentration-response curve of RU 24969 was U-shaped, since at concentrations higher than 0.1 μmol/1 the extent of inhibition decreased with increasing concentrations of RU 24969. In the presence of phentolamine, the concentration-dependent attenuation of the RU 24969-induced inhibition of overflow was no longer detectable. The present results suggest that RU 24969 is a weak antagonist at presynaptic α₂-adrenoceptors (by more than 2.5 log units less potent than as an agonist at presynaptic 5-HT_1B auto- and heteroreceptors).