Molecular pharmacological differences in the interaction of serotonin with 5-hydroxytryptamine1C and 5-hydroxytryptamine2 receptors.

5-Hydroxytryptamine (5HT)1C and 5HT2 receptors appear to be closely related, from a molecular viewpoint, displaying similar second messenger systems and a high degree of sequence homology. However, there are striking differences in the interactions of 5HT with 5HT1C and 5HT2 receptors; 5HT is generally more potent in stimulating responses mediated through 5HT1C receptors than responses mediated through 5HT2 receptors. Also [3H]5HT labels 5HT1C receptors and not 5HT2 receptors. In order to explore more fully the molecular rationale for these differences, radioligand binding studies were performed in rat, human, and porcine brain and choroid plexus tissues and in mammalian cells transfected with rat 5HT1C or 5HT2 receptors; second messenger studies (inositol phosphate accumulation) were performed in the transfected cells. The second messenger studies confirmed the approximately 10-fold higher potency of 5HT in stimulating intracellular responses through 5HT1C receptors (EC50 = 8.3 nM) than in stimulating intracellular responses through 5HT2 receptors (EC50 = 101 nM). An agonist radioligand selective for the 5HT1C and 5HT2 receptors, 2,5-dimethoxy-(4-[125I]iodo)phenylisopropylamine, was used, as well as [3H]5HT, [3H]mesulergine (antagonist radioligand for 5HT1C receptors), and [3H]ketanserin (antagonist radioligand for 5HT2 receptors). Computer-assisted analyses of the binding data revealed two agonist affinity states for the 5HT1C receptor. The agonist high affinity state of the receptor was modifiable by guanyl nucleotides. The proportion of agonist high affinity states, relative to the total receptor population, was approximately 10% for both receptors. The apparent higher affinity of 5HT for the radiolabeled 5HT1C receptors was due to the higher affinity 5HT displayed for the agonist low affinity state of the 5HT1C receptor, compared with the affinity of 5HT for the agonist low affinity state of the 5HT2 receptor. The correspondence between the higher affinity of 5HT for the agonist low affinity state of the 5HT1C receptor, relative to the 5HT2 receptor, and the higher potency of 5HT in stimulating 5HT1C responses indicates that 5HT interacts with the agonist low affinity state of the 5HT1C and 5HT2 receptors in initiating its biological effects. These observations indicate that guanine nucleotide-binding protein (G protein)-coupled receptors can exhibit high affinity for neurotransmitters in both the free receptor and the G protein-coupled states and that receptors exhibiting this property may represent a novel subfamily of G protein-coupled receptors.     

Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurones in the dorsal raphe of the rat brain.

1. The effect of bath application of sulphated cholecystokinin octapeptide (CCK-8) was studied on neurones in slices containing rat raphe nucleus. 2. Intracellular recordings were made from neurones in the dorsal raphe nucleus. Some of the neurones with the characteristics of 5-hydroxytryptamine (5-HT)-containing cells which were inhibited by 5-HT and excited by noradrenaline were excited by cholecystokinin. The response to cholecystokinin was dose-dependent over the range 10 to 1000 nM. 3. The response to CCK-8 persisted in the presence of tetrodotoxin. Either reduction of extracellular calcium or addition of 25 mM magnesium did not block the CCK response, suggesting it was mediated by receptors located on the membrane of the raphe neurones. 4. The agonist and antagonist specificity of the CCK response was determined. The CCKB selective agonist, pentagastrin, was inactive when applied at concentrations up to 10 microM. the CCKA receptor antagonist L-364,718 (1 to 100 nM) blocked the response to cholecystokinin. Much higher (1-10 microM) concentrations of the CCKB receptor antagonist L-365,260 were required for inhibition of the CCK response. 5. These data support the existence of a CCK receptor, located on raphe neurones in the rat, with a pharmacological profile very similar to that described for the CCKA type.     

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.     

Effect of cholecystokinin tetrapeptide amide on the metabolism of 5-hydroxytryptamine in the rat brain

The effect of cholecystokinin tetrapeptide amide (CCK-4) on the content of 5-hydroxytryptamine (5-HT) and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), was studied in various regions of the brain of rat. After intracerebroventricular administration of CCK-4, the content of 5-HT decreased, while the content of 5-HIAA increased in some areas. These changes were marked 30 min after the injection and relatively small doses (10 and 100 ng) of CCK-4 produced a pronounced effect, but larger doses (1 and 10 micrograms) were less effective. The ratio of 5-HIAA/5-HT increased significantly in most regions of the brain, except in the striatum and the olfactory tubercle. The results indicate that exogenous CCK-4 in small doses stimulates the metabolism of 5-HT in the brain of rat.     

The interaction of some kynurenine pathway metabolites with 5-hydroxytryptophan and 5-hydroxytryptamine

The kynrenine pathway metabolites kynurenine, 3-hydroxykynurenine and xanthurenic acid have been tested against 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan (5-HTP)-induced head twitches in the mouse in a dose-range of 0.5–5.0 mg/kg. Kynurenine and 3-hydroxykynurenine were highly active. Low doses caused marked potentiation of the twitch response to both 5-HT and 5-HTP with increased toxicity of 5-HT. High doses caused antagonism of both responses. Xanthurenic acid was inactive over the same dose range. The effects of kynurenine could not be duplicated in the guinea-pig ileum. The relevance of these results to the involvement of kynurenine pathway metabolites in depressive illness is discussed.     

Attenuated 5-hydroxytryptamine receptor-mediated responses in aortae from streptozotocin-induced diabetic rats.

1. This study was designed to examine further the attenuated contractile responses to 5-hydroxytryptamine (5-HT) previously observed in aortae from diabetic rats. 2. Cumulative concentration-response curves to 5-HT, and the 5-HT receptor agonists, alpha-methyl 5-HT (alpha-Me-5-HT, 5-HT2/1C agonist), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI, 5-HT2/1C agonist) and 5-carboxamidotryptamine (5-CT, 5-HT1A/1B/1D agonist), were examined in endothelium-intact and -denuded aortae from 2-week streptozotocin (STZ)-diabetic and control rats. 3. In endothelium-intact and -denuded aortae from diabetic rats, maximum responses to 5-HT and alpha-Me-5-HT were significantly reduced compared to those of aortae from control rats. Responses to these agonists were inhibited by the 5-HT2/1C receptor antagonist, ketanserin (0.1 microM). 4. The attenuated responses to 5-HT of aortae from diabetic rats were normalized by chronic insulin treatment of the rats (5 units day-1, s.c.), but not by altering the glucose concentration of the bathing fluid. 5. The nitric oxide synthase inhibitor N-nitro-L-arginine (NOLA, 0.1 mM) significantly potentiated responses to both 5-HT and alpha-Me-5-HT in endothelium-intact aortae. However, the difference between maximum responses of aortae from diabetic and control rats was still evident in the presence of NOLA. 6. Endothelium-intact rings, in the presence of ketanserin (0.1 microM) and preconstricted with the thromboxane A2-mimetic, U46619 (0.1-0.3 microM), from control and diabetic rats, did not relax to cumulative additions of 5-HT (1 nM-30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hindlimb

The vascular response to bolus injections of 5-hydroxytryptamine (5HT) over the range 0.01-10.0 microgram was measured in isolated perfused rat hindlimbs. The hindlimb vasculature displayed a vasoconstrictor response to 5HT, which was potentiated by the addition of 0.01 microgram/ml noradrenaline (NA) to the perfusate at all doses tested. The response to 5HT was also enhanced by the monoamine oxidase (MAO) inhibitor, tranylcypromine (0.05 mg/ml), suggesting that MAO located in the vascular wall is involved in the termination of the response to 5HT in the peripheral circulation. MAO inhibition abolishes the potentiation of 5HT by NA, indicating that this potentiation results from competition for MAO by both amines, leaving more amine intact to activate membrane receptors. Corticosterone (0.01 mg/ml), catecholamine extraneuronal uptake inhibitor, did not alter the response to 5HT, nor the increase of this response by NA, it thus appears that 5HT and NA have different membrane transport systems in the peripheral vasculature.     

Changes in brain monoamine levels of rats during cholecystokinin octapeptide-induced suppression of feeding

Cholecystokinin octapeptide (CCK-8) in doses of 5 or 10 micrograms/kg was injected intraperitoneally to 24 hr food-deprived rats before a 30 min feeding period, and the dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT) contents of the hypothalamus, mesencephalon, amygdala, hippocampus and striatum were measured thereafter. The experimental procedure (deprivation + food intake) alone could induce changes in the brain monoamine contents of saline-treated animals as compared to the nondeprived control group. The most striking effect was observed in the hypothalamus, in which the contents of all three monoamines decreased. In the deprived control group there was a significant positive correlation calculated by linear regression analysis between the amount of food eaten and the DA contents of the amygdala. Injection of CCK-8 before food intake testing decreased the DA contents of the hypothalamus. In the CCK-8-treated animals the correlation between food intake and amygdaloid DA contents disappeared. The CCK-8 treatment specifically gave rise to a significant positive correlation between the amount of food eaten and the NE content of the hypothalamus; such a relation could not be observed in the saline-treated group. The hypothalamic NE contents altered in parallel with the effectiveness of both doses of CCK-8 in inhibiting food intake. The results indicate the importance of the hypothalamic NE system in the food intake-suppressing effect of CCK-8.     

Evidence for the non-specificity of the interaction between 5-hydroxytryptamine and botulinum toxin

A comparison of the effects of the drugs 5-hydroxytryptamine and pargyline on intoxication by botulinum toxin and nereistoxin has been made. Results indicate that the former can be a non-specific antagonizer of poisoning; the latter, a non-specific augmenter of poisoning. Histamine is shown to be without effect on intoxication by either poison. The effect of 5-hydroxytryptamine appears to be a function of the interval between its injection and injection of a toxin. If given before a toxin, 5-hydroxytryptamine will antagonize it; if given at the same time as a toxin, 5-HT will augment it. 5-HT is impotent when administered after a toxin. The actions of both 5-HT and pargyline are suggested as being mediated through their effects on the circulatory system.     

Fenfluramine and 5-hydroxytryptamine. Part 2: Involvement of brain 5-hydroxytryptamine in the anorectic activity of fenfluramine.

As it is well-known, fenfluramine produces anorexia and decrease in brain 5-hydroxytryptamine (5-HT). As it has been suggested that the anorectic effect of fenfluramine may be due to a release of brain 5-HT, we have examined the influence of several drugs active on 5-HT mechanisms and metabolism, on the anorexigenic activity of fenfluramine. These studies were made in relationship with the depletion of 5-HT levels and the concentration of brain fenfluramine or m-trifluoromethyl-isopropylamine. The results have confirmed the involvement of a tryptaminergic mechanism in fenfluramine anorexia and suggest the hypothesis that fenfluramine itself can interfere with the serotoninergic system in the brain (stimulation of tryptaminergic neurons directly).     

The interaction of 5-hydroxytryptamine and related hydroxyindoles with horseradish and mammalian peroxidase systems.

5-Hydroxytryptamine (5-HT) was found to be a competitive inhibitor with respect to guaiacol, and a non-competitive inhibitor with respect to H₂O₂ in a guaiacol-H₂O₂-horseradish peroxidase system. Other indolic compounds possessing a hydroxyl group in the C5 position behaved similarly to 5-HT, but those devoid of a nuclear substituted hydroxyl group were poor inhibitors. 5-HT altered the spectral characteristics of peroxidase, a shift from 417 to 399 nm occurring when an equimolar ratio of 5-HT and H₂O₂ is present. During such interaction 5HT fluorescence is lost and a yellow-brown product is formed. Attempts to characterize this product were only partially successful but it is suggested that it may possibly possess a dimeric structure. The formation of this product from ¹⁴C 5-HT could be demonstrated in disrupted mammalian spleen preparations but not in spleen slices or brain synaptosomes.     

Further evidence of anticonvulsant role for 5-hydroxytryptamine in genetically epilepsy-prone rats.

1. This study was designed to evaluate further the role of 5-hydroxytryptamine (5-HT) in regulating susceptibility and/or intensity of audiogenic seizures in genetically epilepsy-prone rats. 2. The effects of sertraline, a highly selective and potent inhibitor of 5-HT uptake, on both the intensity of the audiogenic seizures and the extracellular concentrations of 5-HT in the thalamus were evaluated in severe seizure genetically epilepsy-prone rats. 3. Sertraline (7.5, 15 and 30 mg kg-1, i.p.) produced a dose-dependent reduction in the intensity of the audiogenic seizures. 4. Brain microdialysis studies showed that the same doses of sertraline also caused dose-dependent increases in the extracellular 5-HT concentration in the thalamus of the freely moving rats. 5. The peak anticonvulsant effect correlated temporally with the peak increases in the extracellular 5-HT concentration for this drug. 6. It is concluded that enhancement of 5-hydroxytryptaminergic transmission may contribute to the anticonvulsant effect of sertraline in severe seizure genetically epilepsy-prone rats. 7. The present results coupled with earlier investigations support the hypothesis that 5-HT plays an anticonvulsant role in genetically epilepsy-prone rats.     

The inhibitory action of 5-hydroxytryptamine on gastric secretory function in rats.

The inhibitory action of 5-hydroxytryptamine (5-HT) on gastric function was studied in vagotomized rats. 5-HT (0.6, 1 or 5 mgkg-1, s.c.) dose-dependently reduced gastric acid secretion evoked by histamine, pentagastrin or methacholine. Pepsin secretion induced by pentagastrin or methacholine was also depressed by 5-HT. Basal secretion of both acid and pepsin was not significantly affected by any of the three 5-HT doses. Indomethacin pretreatment, which significantly decreased gastric mucosal prostaglandin E2 content, did not modify the inhibitory effects of 5-HT on histamine-induced acid secretion, nor did phentolamine or propranolol. This study suggests that 5-HT inhibits gastric secretory function through mechanisms other than by sympathetic influence or increased prostaglandin synthesis. The inhibitory action appears not to be vagus-dependent. Other mechanisms of action are discussed.     

Noradrenergic and purinergic involvement in spinal antinociception by 5-hydroxytryptamine and 2-methyl-5-hydroxytryptamine.

The adrenergic involvement in spinal antinociception by 5-hydroxytryptamine (5-HT) and 2-methyl-5-hydroxytryptamine (2-Me-5-HT) was examined using the alpha-adrenoceptor antagonists phentolamine, yohimbine and prazosin, and the neurotoxin 6-hydroxydopamine. Intrathecal pretreatment with phentolamine and yohimbine (7.5-30 micrograms), but not prazosin (30 micrograms), reduced the action of 5-HT and 2-Me-5-HT in both the tail flick and hot plate tests. Pretreatment with 6-hydroxydopamine (100 micrograms) reduced (5-HT) or increased (2-Me-5-HT) antinociception in the hot plate test, while tail flick responses were largely unaffected. In other experiments, 8-phenyltheophylline, an adenosine receptor antagonist, reduced the action of 5-HT, but not 2-Me-5-HT, in both tests. These results indicate that (a) antinociception by both 5-HT and 2-Me-5-HT involves some form of interaction with spinal alpha 2-adrenoceptors, but the nature of the interaction for these two agents is different because only 5-HT is dependent on endogenous noradrenaline, (b) release of adenosine from the spinal cord contributes to spinal antinociception by 5-HT but not by 2-Me-5-HT.