Presynaptic serotonin receptors regulate [3H]serotonin release from rat spinal cord synaptosomes

Superfused rat spinal cord synaptosomes were studied to determine if inhibitory serotonin (5-HT) receptors (autoreceptors) exist on spinal serotonergic nerve terminals. Exogenous 5-HT (1-50 nM) produced a concentration-dependent inhibition of K+-induced [3H]5-HT release but did not affect basal [3H]5-HT release. A 32-44% inhibition was produced by 30 nM 5-HT. The inhibitory effect of 30 nM 5-HT was effectively antagonized by 100 nM metitepine, a 5-HT autoreceptor antagonist. The results provide evidence for the existence of 5-HT autoreceptors in rat spinal cord tissue.     

Differential effects of triethyllead on synaptosomal [3H]dopamine vs. [3H]acetylcholine and [3H]gamma-aminobutyric acid release.

In vitro exposure to tetraethyllead (Et4Pb, 10 microM) did not alter the release of [3H] dopamine (DA), [3H]acetylcholine (ACh), or [3H]gamma-aminobutyric acid (GABA) from superfused synaptosomes isolated from rat brain striatum, hippocampus, and cortex, respectively. On the other hand, a concentration-dependent increase in the spontaneous release of these transmitters was observed following exposure to triethyllead (Et3Pb, 0.1-10 microM). The magnitude of 1 microM Et3Pb-induced [3H]DA release was 5-fold greater than that observed for [3H]ACh or [3H]GABA release. Removal of [Ca2+]e did not alter the Et3Pb-induced increase in the release of these three transmitter substances, nor did Et3Pb alter synaptosomal 45Ca efflux. EtePb-induced [3H]ACh and [3H]GABA release, but not [3H]DA release, was blocked by lowering [Na+]e from 140 to 50 mM. Similarly, the release of [3H]ACh and [3H]GABA, but not [3H]DA, induced by either Na,K-ATPase inhibition or veratridine (a Na(+)-ionophore), was attenuated by lowering [Na+]e from 140 to 50 mM. However, Et3Pb did not inhibit isolated synaptic membrane Na,K-ATPase, nor did the magnitude or temporal patterns of Et3Pb-induced transmitter release resemble transmitter release induced by Na,K-ATPase inhibition. Et3Pb and veratridine, but not Na,K-ATPase inhibition, produced an increase in synaptosomal [3H] deoxyglucose phosphate (dGluP) efflux, suggesting that both compounds increase membrane permeability. A Et3Pb-induced increase in membrane permeability is further supported by electrophysiological studies using the frog neuromuscular junction in which Et3Pb was found to reduce both the input resistance and membrane potential of muscle cells. As with [3H]ACh and [3H]GABA release, the Et3Pb-induced increase in synaptosomal [3H]dGluP efflux was attenuated by lowering [Na+]e.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct stimulatory effect of calcitonin on [3H]5-hydroxytryptamine release from the rat spinal cord

The in vitro effects of porcine, salmon and human calcitonin on the K+-evoked overflow of [Met5]enkephalin, substance P and [3H]5-HT (previously taken up) were investigated in superfusion experiments with spinal cord slices. Porcine and salmon calcitonin did not affect the release of [Met5]enkephalin and substance P but enhanced that of [3H]5-HT. In contrast, human calcitonin was inactive. The stimulatory effect of porcine and salmon calcitonin on K+-evoked [3H]5-HT overflow was found with slices from the dorsal or the ventral half of the lumbar enlargement but not with hippocampal or hypothalamic slices. The calcitonin effect on [3H]5-HT outflow persisted in the absence of extracellular Ca2+ but was totally suppressed by 5-HT uptake inhibitors such as citalopram and chlorimipramine and by the 5-HT-releasing agent, p-chloroamphetamine. Direct investigation of the possible action of porcine calcitonin on [3H]5-HT uptake and release demonstrated that the enhanced [3H]5-HT overflow resulted from a p-chloramphetamine-like 5-HT-releasing effect of the hormone at the spinal level. This action might be involved in the potent analgesic effect of intrathecal calcitonin.     

Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H(+) ions may prevent desensitization of some AMPA receptor subtypes.     

5-HT3 receptors are not involved in the modulation of the K(+)-evoked release of [3H]5-HT from spinal cord synaptosomes of rat.

The ability of 5-HT3 receptor agonists to modulate the resting efflux or K(+)-evoked release of [3H]5-HT from superfused synaptosomes from the spinal cord of the rat was investigated. Phenylbiguanide did not alter the resting efflux of [3H]5-HIAA or [3H]5-HT or modify the K(+)-evoked release of [3H]5-HT. 2-Methyl-5-HT (10 microM) caused an increase in resting efflux of [3H]5-HIAA, an effect that was blocked by the inhibitor of the uptake of 5-HT fluoxetine. No effect on K(+)-evoked release of tritium was observed. Bufotenine (100-1000 nM) increased the resting efflux of [3H]5-HT and [3H]5-HIAA. These effects were not antagonized by the 5-HT3 antagonist ICS 205-930 but were antagonized by fluoxetine. The drug ICS 205-930 (1 microM) did not alter resting efflux or block the ability of serotonin (30 and 100 nM) to decrease K(+)-evoked release of tritium. Quipazine, a potent antagonist of peripheral 5-HT3 receptors (subnanomolar concentrations), was also unable to alter resting or K(+)-evoked release of [3H]5-HT. It did, however, attenuate the inhibitory effect 5-HT on K(+)-evoked release. The concentrations required were in the micromolar range, consistent with the ability of the drug to antagonize the 5-HT1B autoreceptor. These results support the idea that 5-HT3 receptors do not act as nerve terminal autoreceptors in the spinal cord of the rat.     

Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels.

The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.     

Involvement of protein kinase C in the presynaptic nicotinic modulation of [(3)H]-dopamine release from rat striatal synaptosomes.

1. Presynaptic nicotinic ACh receptors modulate transmitter release in the brain. Here we report their interactions with protein kinase C (PKC) with respect to [(3)H]-dopamine release from rat striatal synaptosomes, monitored by superfusion. 2. Two specific PKC inhibitors, Ro 31-8220 (1 microM) and D-erythro-sphingosine (10 microM) significantly reduced (by 51 and 26% respectively) [(3)H]-dopamine release stimulated by anatoxin-a (AnTx), a potent and selective agonist of nicotinic ACh receptors. The inactive structural analogue of Ro 31-8220, bisindolylmaleimide V (1 microM) had no effect. 3. Two phorbol esters, PDBu (1 microM) and PMA (1 microM) potentiated AnTx-evoked [(3)H]-dopamine release by 50 - 80%. This was Ca(2+)-dependent and prevented by PKC inhibitors. In the absence of nicotinic agonist, phorbol esters enhanced basal release through a PKC-independent mechanism. 4. A (86)Rb(+) efflux assay of nicotinic ACh receptor function confirmed that Ro 31-8220 has no nonspecific effect on presynaptic nicotinic ACh receptors. 5. These results suggest that PKC is activated by nicotinic ACh receptor stimulation and mediates a component of AnTx-evoked [(3)H]-dopamine release. In addition, independent activation of PKC can further amplify the response, offering a potential mechanism for receptor crosstalk.     

Effect of dotarizine on K+-stimulated [3H]-serotonin and [3H]-acetylcholine release from rat hippocampus

• 1.1. The effect of the diphenylmethyl-piperazine derivative dotarizine on K⁺-stimulated release of [³H]serotonin ([³H]5-HT) and [³H]acethylcholine ([³H]Ach) in rat hippocampal slices was studied.
• 2.2. Dotarizine at a concentration of 10⁻⁶ M significantly decreased the basal [³H]5-HT release and, at a concentration of 10⁻⁵ M, it significantly decreased the K⁺-stimulated [³H]5-HT release compared to vehicle controls.
• 3.3. Dotarizine, at a concentration of 5 × 10⁻⁷ M, significantly increased both basal and K⁺-stimulated [³H]Ach release. At higher concentrations (10⁻⁶ and 2 × 10⁻⁶ M), dotarizine did not change the basal release but significantly increased the K⁺-stimulated [³H]Ach release. The effect of dotarizine on K⁺-stimulated [³H]Ach release decreased with increasing dotarizine concentrations.
• 4.4. It is speculated that, in addition to its Ca²⁺ antagonistic activity, dotarizine exerts an antagonistic effect on the presynaptic 5-HT autoreceptors, which could account for the facilitation of [³H]Ach release.

     

Protein kinase C: regulation of serotonin release from rat brain cortical slices

The effects of phorbol esters on serotonin release were examined in an attempt to investigate the role of protein kinase C in the regulation of serotonin release. Rat brain parietal cortical slices were incubated with [3H]5-HT in the presence of pargyline in order to label the serotonin stores. Potassium stimulated (30 s) release and spontaneous [3H]5-HT efflux were examined in slices during superfusion with Krebs-Ringer solution containing chlorimipramine. Repeated K+ stimulations elicited reproducible responses with release ratios of approximately 1.0. Introduction of phorbol 12-myristate, 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu) 20 min prior to S2, or S3 resulted in dose-related increases in [3H]5-HT or [3H]NE release. PMA was slightly more potent (93% increase) than PDBu in potentiating K+-stimulated [3H]5-HT release. Phorbol and 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) which do not activate protein kinase C did not alter serotonin release. In contrast, basal [3H]5-HT and [3H]NE release were altered to a far lesser extent which was not always dose related. The response to the phorbol esters was reversible, Ca2+-dependent and reached maximal effect after 20 min of superfusion. The putative protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited K+-induced [3H]5-HT release significantly (11%) but did not alter basal efflux. The PMA facilitation of serotonin release was, however, markedly prevented by the enzyme inhibitor. The effect of PMA on release was found not to be directly mediated through the prejunctional serotonin autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of verapamil on [3H]acetylcholine release in the striatum of spontaneously hypertensive rats.

In the present study, we describe the effects of Ca2+ channel antagonist (verapamil) on [3H]acetylcholine (ACh) release in the central nervous system of spontaneously hypertensive rats (SHR). The electrically stimulated release of [3H]ACh from striatal slices was not different between SHR and normotensive Wistar Kyoto (WKY) rats. Verapamil inhibited electrically stimulated [3H]ACh release in a dose-related fashion. The inhibitory effect of verapamil was significantly greater in SHR than in WKY rats. These results suggest that the Ca2+ sensitivity of central cholinergic neurons might be enhanced in SHR, which could attribute, at least partially, to the pathogenesis of hypertension.     

Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas

The effects of acetylcholine (ACh) on the release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in synaptosomes prepared from rat cerebral cortex, hypothalamus and hippocampus and depolarized with 15 mM KCl under superfusion conditions. ACh inhibited the release of [3H]5-HT in all three brain areas. This effect was not modified by hexamethonium but was antagonized by atropine and by the non-classical antagonists pirenzepine and dicyclomine.     

Serotonin-2 receptor-mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat

5-Hydroxytryptamine (5-HT) inhibited the K+-induced release of [3H]acetylcholine [( 3H]ACh) from slices of the hippocampus of the rat, dose-dependently. Minaprine (3-(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine, Fig. 1) had no effect on the release of [3H]ACh. However, it inhibited the (formula; see text) Fig. 1. Chemical structure of minaprine dihydrochloride. attenuation of the release of [3H]ACh by 5-HT dose-dependently. The 5-HT2 receptor antagonists, mianserine, methysergide and spiperone, prevented the inhibitory effect of the 5-HT, as well as did minaprine. The attenuating effect of 5-HT was not mimicked by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and was not prevented by a 5-HT1A and 5-HT1B mixed receptor antagonist, propranolol, or by the 5-HT3 receptor antagonists, cocaine and metoclopramide. Minaprine inhibited the bindings of [3H]5-HT, [3H]8-OH-DPAT and [3H]ketanserin in the hippocampus. The inhibitory effect of minaprine on the binding of [3H]ketanserin was more marked than on the binding of [3H]5-HT and [3H]8-OH-DPAT, and was non-competitive. The Ki value of minaprine for the binding of [3H]ketanserin was 2.9 microM. The inhibitory effect of 5-HT on the release of [3H]ACh was observed in the presence of tetrodotoxin. By electrolytic lesioning of the medial septum, the K+-induced release of [3H]ACh from the slices of hippocampus was significantly reduced and the release was no longer inhibited by 5-HT. The lesioning significantly decreased the binding of [3H]ketanserin in the hippocampus, with hardly any reduction in the binding of [3H]5-HT and [3H]8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotine-evoked [3H]5-hydroxytryptamine release from rat striatal synaptosomes

The aim of this study was to characterize the pharmacology of presynaptic nicotinic cholinoceptors (nAChRs) that modulate release of 5-hydroxytryptamine (5-HT) from superfused rat brain synaptosomes preloaded with [3H]5-HT. Nicotine increased 5-HT release from striatal synaptosomes (maximally by 15-30%) but not from cerebral cortex or hippocampal synaptosomes. Release of striatal 5-HT was increased in a concentration-dependent manner by nicotine, epibatidine, cytisine, and ACh (with added esterase inhibitor and muscarinic antagonist). Respective EC50 values were: 0.5, 0.003, 0.1 and 0.7 microM. The maximal effect of each agonist was virtually completely blocked by a high concentration of the insurmountable nicotinic antagonist mecamylamine; at a higher concentration of epibatidine (3 microM), a mecamylamine-insensitive effect was revealed. Nicotine, ACh and epibatidine appeared equally efficacious, whereas cytisine was of lower efficacy (60-70% of ACh). Release evoked by a half-maximal concentration of nicotine was inhibited by the nicotinic antagonists dihydro-beta-erythroidine (IC50 0.04 microM) and methyllycaconitine (IC50 0.06 microM). Nicotine-evoked 5-HT release was not reduced by tetrodotoxin given in a concentration that blocked veratridine-evoked release. These findings provide functional evidence for a direct action of nicotine on 5-HT neurons in the brain. The presynaptic nAChRs that modulate striatal 5-HT release appear to possess a novel pharmacological profile.     

Raised [3H]-5-HT release and 45Ca2+ uptake in diazepam withdrawal: inhibition by baclofen.

The effect of diazepam withdrawal (2 mg/kg/day) on release of [3H]-5-hydroxytryptamine(5-HT) and [14C]-GABA from rat cortical and hippocampal slices was studied. No changes in [14C]-GABA release (basal, K(+)-evoked, uptake) from slices of either region were observed. Similarly, all parameters of [3H]-5-HT release were unchanged in cortical slices. However, during diazepam withdrawal, depolarised [3H]-5-HT release from hippocampal slices was raised with no changes in basal release or uptake into the slices being found. This increase could be prevented by in vivo administration of 1 mg/kg baclofen--this dose having no significant effect on [3H]-5-HT release from hippocampal slices of control rats. To further investigate this effect, 45Ca2+ uptake into hippocampal synaptosomes was examined and found to be increased during withdrawal. This was blocked by in vitro addition of 10 microM (-)baclofen, which had no effect on 45Ca2+ uptake in controls. Inhibition of 45Ca2+ uptake by (-)baclofen was also enhanced in nonwithdrawn diazepam-treated rats, but not in rats treated acutely with diazepam. The results from both studies indicate that chronic diazepam treatment increases neuronal sensitivity to baclofen. These results are discussed with reference to the anxiogenic state during diazepam withdrawal and a recent report of reversal of this behaviour by baclofen.     

Increased release of [3H]acetylcholine in vitro from the mouse hippocampus by a convulsant barbiturate

The effects of the convulsant barbiturate, 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB), on the spontaneous release of [³H]acetylcholine (ACh) from mouse hippocampal slices in an in vitro superfusion system have been evaluated. The pattern of the release of [³H]ACh by a single treatment with CHEB or an elevated potassium concentration was similar, with the peak release occurring in the same fraction. A maximally effective concentration of CHEB (500 μM) caused a 177% stimulation of spontaneous release of [³H]ACh, while 50 mM KCl increased the release by 2100% above the baseline. The stimulation of the spontaneous release of [³H]ACh by CHEB was concentration-dependent with an EC₅₀ of 180 μM. The pattern of release of [³H]ACh induced by multiple treatment with CHEB and elevated potassium appeared to differ, suggesting that different pools of [³H]ACh in the cholinergic neurons might be affected by these treatments. The action of CHEB on the sponaneous release of [³H]ACh was unique among some other convulsant drugs that were studied. Another convulsant barbiturate, S(+)-1-methyl-5-phenyl-5-propyl barbituric acid [S(+)-MPPB], pentylenetetrazol and a convulsant benzodiazepine 1,3-dihydro-5-methyl-2H-1,4-benzodiazepine-2-one (Ro-5-3663) did not affect the spontaneous release of [³H]ACh. The relationship between the stimulation of release of ACh and the convulsant action of the barbiturates is discussed.     

Halothane enhances exocytosis of [3H]-acetylcholine without increasing calcium influx in rat brain cortical slices.

1. The effect of halothane on the release of [3H]-acetylcholine ([3H]-ACh) in rat brain cortical slices was investigated. 2. Halothane (0.018 mM) did not significantly affect the basal and the electrical field stimulation induced release of [3H]-ACh. However, halothane (0.063 mM) significantly increased the basal release of [3H]-ACh and this effect was additive with the electrical field stimulation induced release of [3H]-ACh. 3. The release of [3H]-ACh induced by 0.063 mM halothane was independent of the extracellular sodium and calcium ion concentration and was decreased by tetracaine, an inhibitor of Ca(2+)-release from intracellular stores or dantrolene, an inhibitor of Ca(2+)-release from ryanodine-sensitive stores 4. Using 2-(4-phenylpiperidino)-cyclohexanol (vesamicol), a drug that blocks the storage of ACh in synaptic vesicles, we investigated whether exocytosis of this neurotransmitter is involved in the effect of halothane. Vesamicol significantly decreased the release of [3H]-ACh evoked by halothane. 5. It is suggested that halothane may cause a Ca2+ release from intracellular stores that increases [3H]-ACh exocytosis in rat brain cortical slices.     

The mechanism by which monoamine oxidase inhibitors give rise to a non-calcium-dependent component in the depolarization-induced release of 5-HT from rat brain synaptosomes.

1. The effects of the monoamine oxidase inhibitors pargyline and nialamide on the Ca2+-dependency of [3H]-5-hydroxytryptamine release from superfused rat brain synaptosomes has been studied in order to evaluate the discrepancies that have occasionally been observed in studying transmitter release by in vivo and in vitro techniques. 2. The application of K+ pulses of low concentration (12.5-20 mM) caused an essentially Ca2+-dependent release of [3H]-5-HT. However, at K+ concentrations above 30 mM, a small non-Ca2+-dependent component appeared. 3. At high concentrations of K+ (30-55 mM), nialamide (18 microM) or pargyline (7 microM) increased the amount of [3H]-5-HT released which could be accounted for by an increase in the non-Ca2+-dependent component of release. 4. The elevation of the non-Ca2+-dependent component of release caused by the monoamine oxidase inhibitors was totally abolished by the inhibitors of the plasma membrane 5-HT carrier, chlomipramine (500 nM), citalopram (50 nM) and fluoxetine (1 microM). 5. The results suggest that the non-Ca2+-dependent component of release seen with high depolarizing concentrations of K+, particularly in the presence of monoamine oxidase inhibitors, is caused by the efflux of [3H]-5-HT through the plasma membrane carrier which seems to be activated during depolarization. 6. The significance of these findings to the physiological in vivo situation, and to the use of in vitro preparations in the study of transmitter release is discussed.     

Serotonin modulation of cell excitability and of [3H]GABA and [3H]D-aspartate efflux in primary cultures of rat cortical neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal excitability, evaluated as depolarization-induced firing rate, and on amino acid release, measured as electrically-evoked [(3)H]GABA and [(3)H]d-aspartate efflux, were investigated in rat primary cortical neuronal cultures. 5-HT displayed a concentration-dependent, bimodal effect on neuronal excitability: at 3-10microM it increased excitability through 5-HT(2A) receptors, and was blocked by the selective 5-HT(2A) antagonist MDL 100907, whereas at 30-100microM it reduced excitability through 5-HT(1A) receptors, and was, in turn, blocked by the selective 5-HT(1A) antagonist WAY 100135. The electrically-evoked [(3)H]GABA efflux was concentration-dependently inhibited by 5-HT (pEC(50)=4.74) and such inhibition was prevented by WAY 100135, but not by GR 55562, a selective 5-HT(1D/B) receptor antagonist. Conversely, 5-HT concentration-dependently increased stimulus-evoked [(3)H]d-aspartate efflux (pEC(50)=4.71). The increase was facilitated by methiothepin and was reversed into inhibition by ICS 205930, a selective 5-HT(3) receptor antagonist. In the presence of ICS 205930, the inhibition induced by 5-HT was prevented by the selective 5-HT(1D/B) receptor antagonist GR 55562, but not by WAY 100135. These findings suggest that 5-HT inhibits GABA release through 5-HT(1A) receptors and exerts a dual modulation on glutamate release, mostly facilitatory (through 5-HT(3) receptors) but also inhibitory (through 5-HT(1D/B) receptors), leading to a prevalently positive modulation of the excitatory signal by amino acid neurotransmitter containing neurons.     

Effect of (+/-)-methyl 3-ethyl-2,3,3a,4-tetrahydro-1H-indolo-[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (OM-853), a new vincamine analogue, on the metabolism and function of cerebral serotonergic neurons

Effect of (+/-)-methyl 3-ethyl-2,3,3a,4-tetrahydro-1H-indolo[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (OM-853), a new vincamine analogue, on the metabolism and function of cerebral 5-hydroxytryptamine (5-HT) neurons was investigated using male Wistar rats. The single administration of OM-853 (200 mg/kg, p.o.) induced the facilitation of metabolic turnover of 5-HT in various brain areas except the cerebral cortex, pons-medulla and cerebellum. In vitro addition of OM-853 inhibited the uptake of [14C]5-HT in striatal slices only at a high concentration (10(-4) M). On the other hand, a low concentration of OM-853 (10(-8)-10(-6) M) induced the increase of the spontaneous and high K+ (30 mM)-evoked releases of [14C]5-HT from striatal slices. OM-853 had more potent inhibitory effect on the binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-hydroxy DPAT) to 5-HT1A receptors and/or 5-HT autoreceptors than that of [3H]-ketanserin to 5-HT2 receptors. The stimulatory effect of OM-853 (10(-7) M) on [14C]5-HT release was antagonized by 10(-7) M 8-hydroxy DPAT, which is known to act at presynaptic 5-HT autoreceptors as an agonist. These results suggest that OM-853 may induce facilitation of 5-HT turnover by enhancing 5-HT release, probably via the inhibition of presynaptic 5-HT autoreceptor.     

Modulation of [3H]serotonin release by dihydropyridines in spinal cord synaptosomes.

The release of [3H]monoamines from preloaded synaptosomes from spinal cord is K(+)-dependent and can be modulated by L-type Ca2+ channel agonists such as the 1,4-dihydropyridine (1,4-DHP), Bay K 8644. Whereas the basal release of [3H]monoamines was not altered by Bay K 8644, K(+)-stimulated release of [3H]norepinephrine was enhanced 35% and [3H]serotonin 50%. Modulation of release by Bay K 8644 was dependent on the K+ concentration in the medium, being present only at submaximal depolarization with 15 mM K+. Enhanced release in the presence of Bay K 8644 was concentration-dependent and Ca2(+)-dependent. Ca2(+)-independent release induced by fenfluramine was not enhanced by Bay K 8644. Both nimodipine and nitrendipine, 1,4-DHP antagonists, produced a concentration-dependent block of the Bay K 8644-induced monoamine release and had no independent effect on basal or K(+)-stimulated release. omega-Conotoxin GVIA (omega-CgTx) produced a concentration dependent decrease of K(+)-stimulated serotonin release, which antagonized the stimulatory effect of low concentrations of Bay K 8644. However, omega-CgTx did not alter the enhancement of K(+)-stimulated release at higher concentrations of Bay K 8644. The data from the present work establish the conditions for modulation of K(+)-evoked monoamine release in spinal cord by 1,4-DHP agonists and suggest a role for the L-type voltage dependent Ca2+ channel in this process.