Effects of Methiothepin and Lysergic Acid Diethylamide on Serotonin Release In Vitro and Serotonin Synthesis In Vivo: Possible Relation to Serotonin Autoreceptor Function

Abstract: An in vitro system characterizing the presyn- aptic serotonin (5-HT) autoreceptor which controls the release of 5-HT from rat brain slices is described. Using this system, methiothepin (1–10 μ M) demonstrated 5-HT autoreceptor antagonist activity -by enhancing 5-HT release, while several recognized postsynaptic 5-HT receptor antagonists were inactive: mianserin, cinanserin, cyproheptadine, methysergide. The activity of methiothepin was highest in hypothalamic slices and lowest in striatal slices and was inhibited by the autoreceptor agonists lysergic acid diethylamide (LSD) and 5-methoxy- tryptamine (5-MT). The reversal of the methiothepin-enhanced 5-HT release from hypothalamic slices by LSD was not influenced by 0.3 μ M tetrodotoxin. The peripheral administration of LSD to rats has been shown to reduce 5-HT synthesis and release by a mechanism thought to involve, in part, an autoreceptor-mediated reduction in impulse flow of 5-HT neurons. In the present experiments, intraperitoneal injection of methiothepin antagonized the LSD-induced reduction in hypothalamic 5-HT synthesis (5-hydroxytryptophan accumulation) while exerting no influence by itself. Conversely, compounds which were not active as 5-HT autoreceptor antagonists in vitro (i.e., cyproheptadine, methysergide, cinanserin) did not influence the effect of LSD on 5-HT synthesis. Further, the reduction in 5-hydroxytryptophan (5-HTP) accumulation by LSD showed regional differences in inhibition by methiothepin (hypothalamus > cortex > striatum) which paralleled the autoreceptor antagonist activity of methiothepin in vitro. These data suggest that similar autoreceptor mechanisms control 5-HT release and synthesis in terminal 5-HT projection areas and that the reduction in 5-HT accumulation by LSD and the antagonism by methiothepin may represent a useful biochemical measure of 5-HT autoreceptor activity in vivo.     

Histochemical demonstration of transmitter release from noradrenaline,dopamine and 5-hydroxytryptamine nerve terminals in field stimulated rat brain slices

Summary The effect of electrical field stimulation on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) nerve terminals in rat brain slicesin vitro was investigated. Slices prepared from the cerebral cortex or the neostriatum were incubated in physiologic buffer for 30 min and then superfused by buffer and stimulated by an electrical field (biphasic pulses, 10 Hz, 12 mA, 2 ms) for various time periods. The effect of the stimulation was studied at the cellular level with the histochemical fluorescence technique of Falck and Hillarp. The transmitter overflow into the superfusing buffer caused by the stimulation was studied with isotope technique. Cerebral Cortex NA Nerve Terminals. Stimulation caused release of NA from cortical NA nerve terminals. Already after 2 min stimulation a slight decrease of the fluorescence intensity of the nerve terminals could be found. Stimulation for 15 to 30 min greatly reduced the fluorescence intensity. In slices preincubated with³H-NA the stimulation-induced overflow of tritium during 2 min stimulation was about 15% (i.e. 15% of the tissue tritium content was overflowing into the superfusing buffer in response to stimulation for 2 min). During prolonged stimulation there was a considerable decline of the tritium efflux. Cerebral Cortex 5-HT Nerve Terminals. The 5-HT-analogue 6-hydroxytryptamine (6-HT) which is readily taken up into 5-HT nerve terminals was used to permit good visualization of these nerve terminals. Uptake of 6-HT into cortical NA nerve terminals was prevented by preincubation with 6-hydroxydopamine (6-OH-DA) or protriptyline. Stimulation for 15 to 30 min reduced the fluorescence intensity of the 5-HT nerve terminals. In slices preincubated with³H-5-HT the stimulation-induced overflow of tritium during 2 min stimulation was about 5%. The tritium efflux slowly decreased during continuous stimulation. Neostriatal DA Nerve Terminals. In slices frozen directly after preparation an intense diffuse fluorescence could be seen. After incubation in drug-free buffer at 37° C the fluorescence was localized in the varicosities of the DA nerve terminals. The central parts of the slices almost completely lacked specific fluorescence, while the outer zones were brightly fluorescent. No clear reduction of the fluorescence intensity of the DA nerve terminals in the outer zone could be observed after stimulation for 30 min. In slices preincubated with³H-DA the stimulation-induced overflow of tritium during 2 min stimulation was about 2%. The tritium efflux slowly decreased during continuous stimulation.It is suggested that the differences in release between the various nerve terminal systems foundin vitro reflect differences in transmitter release occurringin vivo. The comparably high release of NA per impulse from the cortical NA nerve terminals implicate that the discharge rate of these neuronsin vivo is very low.This investigation has been supported by grants from the Swedish Medical Research Council (B72-14X-2330-05A) and Magnus Bergwall's Foundation.The author is greatly indebted to Mrs. Annika Hamberger for her skillful technical assistance. For generous supplies of drugs thanks are due to Hässle, Göteborg, Sweden, through Dr. H. Corrodi (6-HT, 6-OH-DA and H44/68).     

Serotoninergic control of somatostatin and gastrin release from the isolated rat stomach

The influence of exogenous serotonin on the secretion of gastric somatostatin and gastrin was investigated under in vitro conditions using an isolated, vascularly perfused rat stomach preparation. Serotonin stimulated gastrin release, maximal effects were observed at 10(-6) M which increased gastrin levels by 78%; on the contrary, somatostatin secretion was inhibited (maximal inhibition of 56% at 10(-6) M). Changes in hormone secretion in response to serotonin were reversed by combined blockade of 5-HT1 and 5-HT2 receptors by methysergide and blockade of 5-HT2 receptors by ketanserin (10(-5) and 10(-6) M, respectively), and of cholinoreceptors by atropine (10(-5) M). It is concluded that in rats in vitro serotonin inhibits release of gastric somatostatin and stimulates gastrin secretion via specific serotonin receptors but muscarinic cholinergic receptors are also involved.     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Effect of vasoactive intestinal peptide on serotonin metabolism in the suprachiasmatic area of the rat: mechanism of action

Vasoactive intestinal peptide (VIP) inhibits serotonin (5-HT) uptake in the suprachiasmatic area (SCA) of the rat. The present study investigates the possibility of a functional relationship between 5-HT uptake mechanisms and 5-HT autoreceptor activity in this effect of VIP in the SCA. The hypothesis of a linkage between these two mechanisms of 5-HT regulation has been recently proposed. We investigated the possibility of the presence of 5-HT autoreceptors in the SCA. Using superfusion system, exogenous 5-HT (500 and 50 nM) increased the release of newly synthesized 3H-5-HT. In contrast, 5 nM of exogenous 5-HT inhibited this release. This latter effect was antagonized by methiothepin (10(-7) M). In contrast, the concentration of methiothepin required to inhibit the VIP effect was 10(-6) or 10(-5) M, the same molarity found to decrease the 5-HT uptake. On the other hand, the increase of the 3H-5-HT in the synaptic cleft, induced by VIP, did not modify the inhibition of 3H-5-HT release induced by 5 nM of exogenous 5-HT. We conclude that the effect of VIP on 5-HT metabolism in the SCA is linked to the 5-HT uptake mechanism but not to the activity of 5-HT presynaptic autoreceptors. In our experimental conditions, the activity of 5-HT autoreceptors is independent of the 5-HT uptake processes.     

Behavioral and biochemical actions of p-chlorophenylethylamine (p-CPEA) in mice.

p-chlorophenylethylamine (p-CPEA), a metabolite of p-chlorophenylalanine (p-CPA) induces the “serotonin syndrome” which consists of lateral head weaving, Straub tail, hindlimb abduction, tremor, hyperactivity, reciprocal fore-paw treading, salivation and piloerection. These p-CPEA-induced behavioral signs were partially prevented by pretreatment with serotonin (5-HT) uptake blockers (fluoxetine, chlorimipramine, Org 6582) and 5-HT receptor blockers (methiothepin, methysergide, cinnanserin) but not by two depletors of brain 5-HT (p-CPA, reserpine). p-CPEA (50 mg/kg) produces an initial decrease in 5-HT associated with a concurrent increase in 5-hydroxyindoleacetic acid with a maximum change at 30 minutes after injection; these early biochemical changes are prevented by pretreatment with fluoxetine (10 mg/kg). p-CPEA also competes with (³H)-5-HT for 5-HT receptors. The reported paradoxical effects of p-CPA on several behavioral paradigms could be due to its decarboxylation to p-CPEA which may both stimulate 5-HT receptors and enhance 5-HT release.     

Modulation of spontaneous and stimulation-evoked transmitter release from rat sympathetic neurons by the cognition enhancer linopirdine: insights into its mechanisms of action

The mechanisms by which the cognition enhancer linopirdine may affect transmitter release were investigated in cultures of rat superior cervical ganglion neurons. Overflow of previously incorporated [3H]noradrenaline evoked by 10 microM UTP or 0.1 microM bradykinin was enhanced by linopirdine at > or =3 microM, overflow evoked by 25 mM K(-), 100 microM nicotine, or 300 microM ATP was enhanced by linopirdine at > or =10 microM, and overflow due to 40 mM K+ or electrical field stimulation was not altered by linopirdine. Ba2+ (0.3 mM) augmented the same types of stimulation-evoked overflow to a similar extent as linopirdine. K+ (25 mM), nicotine (100 microM), and ATP (300 microM) triggered transmitter release in a partially tetrodotoxin-resistant manner, and the release-enhancing action of linopirdine was lost in the presence of tetrodotoxin (1 microM). Linopirdine (10 microM) raised spontaneous tritium outflow and reduced currents through muscarinic K+ (K(M)) channels with a similar time course. The secretagogue action of linopirdine was concentration- and Ca2(+)-dependent and abolished by tetrodotoxin (1 microM) or Cd2+ (100 microM). Linopirdine (10 microM) added to the partial inhibition of K(M) channels by 1 or 3 mM Ba2(+) but not to the complete inhibition by 10 mM Ba2(+). Likewise, the secretagogue action of 1 and 3 mM, but not that of 10 mM, Ba2+ was enhanced by linopirdine. These results indicate that linopirdine facilitates and triggers transmitter release via blockade of K(M) channels and suggest that these K+ channels are located at neuronal somata rather than at presynaptic sites.     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of Inhibitory 5-Hydroxytryptamine Receptors That Modulate Dopamine Release in the Striatum

Abstract: The effect of a series of indoleamines on the potassium-evoked tritium release of previously accumulated [³H]dopamine from rat striatal slices has been investigated. The indoleamines 5-hydroxytryptamine, 5-methoxy-tryptamine, 5-methoxy- N, N' -dimethyltryptamine and tryptamine (10⁻⁷ to 10⁻³ M) all reduced potassium-evoked release of tritium, to a maximum of 50%. The uptake of [³H]dopamine was unaffected by these compounds. A series of 5-hydroxytryptamine antagonists were examined for their ability to reduce the inhibition of potassium-evoked tritium release induced by 5-methoxytryptamine. The relative order of antagonist potency obtained was methysergide > metergoline > methiothepin > cinanserin > cyproheptadine > mianserin, and was consistent with an action on 5-hydroxytryptamine receptors. It is concluded that there are inhibitory 5-hydroxytryptamine receptors located on the terminals of dopaminergic neurones in the striatum.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Serotonergic influences on pituitary gland and hypothalamic induction of prolactin and luteinizing hormone release in the young turkey (Meleagris gallopavo)

Primary anterior pituitary cell cultures were utilized to study the influence of serotonin (5-HT) directly on the pituitary. Cells incubated with 10(-5) and 10(-4) M 5-HT exhibited a significant prolactin (Prl) release, whereas cells incubated with 10(-10) to 10(-6) M 5-HT did not. Cells incubated with 10(-10) to 10(-4) M quipazine (5-HT agonist) or methysergide (MES; 5-HT antagonist) did not release Prl in amounts greater/less (P greater than 0.01) than spontaneous release. Luteinizing hormone (LH) release from cells incubated in the presence of 5-HT, quipazine, or MES was similar to spontaneous release. The hypothalamic extract-induced Prl and LH release from cells was not influenced by quipazine, but Prl release was diminished in a dose-related fashion by MES. The influence of 5-HT on hypothalamic induction of Prl and LH release was investigated utilizing in vitro culture of hypothalamic fragments (HF). Media samples from HF incubated with 10(-6) and 10(-4) M 5-HT induced a release of Prl. Media samples from HF incubated with 10(-4) M MES induced less Prl release than media samples from control fragments. When HF were incubated with both 10(-4) M 5-HT and 10(-4) M MES, the expected 5-HT-mediated Prl release was not evident. These culturing situations had no influence on LH release. In vitro Prl release from pituitary cells of the young turkey was stimulated through 5-HT activity at the hypothalamus, but not by direct 5-HT action on the pituitary cells.     

The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5-Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes

The effect of the serotonergic receptor agonist 1-(m-trifluoromethylphenyl)piperazine (TFMPP) was studied on the K(+)-evoked [3H]acetylcholine [( 3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5-1,000 microM) inhibited the evoked ACh release in a dose-dependent manner (IC50 = 81.8 microM). The inhibitory effect of TFMPP was mimicked by CGS-12066B (10, 30, and 100 microM), a 5-hydroxytryptamine1B (5-HT1B)/5-HT1D receptor agonist; 1-(m-chlorophenyl)piperazine (100 microM), a 5-HT1C/5-HT1B receptor agonist; and 5-carboxamidotryptamine (10 microM), a nonselective 5-HT1 receptor agonist. 8-Hydroxy-2-(di-n-propylamino)tetralin (10 and 100 microM), a 5-HT1A receptor agonist, and quipazine (10 and 100 microM), a 5-HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 microM), metergoline (0.1 microM), methysergide (0.5 and 1 microM), or yohimbine (1 and 10 microM), blocked the TFMPP effect dose-dependently. In contrast, methiotepine (0.3 and 1 microM), propranolol (1 microM), ketanserin (0.1 microM), mesulergine (0.1 microM), ICS 205930 (0.1 and 1 microM), and spiroperidol (1 and 7 microM) did not affect the TFMPP-induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K(+)-evoked ACh release is modulated by a 5-HT1 receptor distinct from the 5-HT1A, 5-HT1B, and 5-HT1C subtypes.     

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction with alpha 1-adrenoceptors; (c) GABA inhibits melatonin production in vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.     

Pharmacologic evidence for direct dopaminergic regulation of striatal acetylcholine release

This study was done to provide pharmacologic evidence for the location of those striatal dopamine D-1 and D-2 receptors that participate in the regulation of local acetylcholine (ACh) release. Striatal tissue slices from adult male Sprague-Dawley rats were preloaded with [3H]choline and superfused in separate experiments with buffer containing either: a D-2-specific agonist (LY141865 or LY171555), a D-2 specific antagonist (L-sulpiride), a D-1 specific agonist (SKF38393), or a D-1 antagonist (SCH23390), in the presence or absence of tetrodotoxin (TTX), used to block interneuronal activity. With either D-2 agonist there was a dose-dependent decrease in K+-stimulated [3H]ACh release, maximally at 5 X 10(-7)-10(-6) M [agonist] and to the same extent with each drug. Both SKF38393 and SCH23390 increased [3H]ACh release at tested concentrations of these agents. Results were unchanged when any of the drugs used was superfused in the presence of TTX, 5 X 10(-7) M. These data are consistent with the hypothesis that populations of striatal D-1 and D-2 receptors exist on local cholinergic neurons, where they regulate ACh release. Alternative interpretations are discussed.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

The in vitro release of immunoreactive dynorphin and alpha-neoendorphin from the perfused rat duodenum

The release of immunoreactive (ir) dynorphin (DYN) and alpha-neoendorphin (ir-ANEO) from the isolated perfused rat duodenum was demonstrated using specific radioimmunoassays (RIAs). Depolarization of the tissue by increasing the potassium (K+) concentration up to 108 mM enhanced the release of ir-DYN and ir-ANEO in Ca2+-dependent manner. Administration of the serotonin-releasing agent fenfluramine (10(-6) M) and the serotonin receptor agonist m-chlorophenylpiperazine (m-CPP, 10(-6) M) stimulated the release of ir-DYN and ir-ANEO from the duodenum. A subsequent study revealed that serotonin (5-HT, 10(-6)-10(-4) M) induced a dose-dependent increase in the release of ir-DYN and ir-ANEO from the duodenum. The effect of 5-HT on the release of ir-DYN and ir-ANEO from the duodenum was antagonized by 5-HT antagonist cyproheptadine (10(-6) M). The presence of dynorphin and the related peptides in the gastrointestinal tract (GIT) and their release from the duodenum in vitro indicate that these peptides may act as transmitters involved in some GIT functions. Furthermore, our results suggest that at least part of 5-HT effects on the GIT may be mediated by the release of dynorphin and the related peptides.