5-HT3 receptor antagonism by anpirtoline, a mixed 5-HT1 receptor agonist/5-HT3 receptor antagonist.

1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.     

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT_1A (pK _i = 7.72) and 5-HT_2A (pK _i = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT_2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC₅₀ = 6.09) and in the hippocampus (pEC₅₀ = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pK _i = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT_1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT_1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT_1A receptors and antagonism at 5-HT_2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).     

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist,directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT_1A receptor agonist/5-HT_2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1–10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1–1000 g/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1–10 g/kg and 0.1–3 g/kg i.v. respectively, and reduced it at higher doses, 30–300 g/kg and 10–30 g/kg i.v., respectively.The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT_1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 g/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OHDPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.These findings suggest that BIMT 17 directly inhibits the electrical activity of medial prefronto-cortical neurons through its dual mode of receptor interaction.     

Restraint accentuates the effects of 5-HT2 receptor antagonists and a 5-HT1A receptor agonist on lordosis behavior

The effect of restraint on lordosis behavior was examined in proestrous and ovariectomized, hormone-primed rats. Restraint durations from 5 to 60 min had no effect on lordosis behavior of proestrous rats. There was also no effect of 5 min restraint on lordosis behavior of ovariectomized rats hormonally primed with 10 microg estradiol benzoate and 500 microg progesterone. However, after intraperitoneal treatment with 1.0 mg/kg ketanserin tartrate (ketanserin), 5 min of restraint significantly reduced lordosis behavior of both groups of rats. The 5-min restraint combined with 0.50 or 0.75 mg/kg ketanserin reduced lordosis to mount (L/M) ratios of ovariectomized rats, while L/M ratios of proestrous rats were inhibited only by the 1.0 mg/kg dose. Increasing the restraint duration (10 or 15 min) reduced the dose of ketanserin necessary to reduce the L/M ratios of proestrous rats. Treatment with the selective serotonin (5-HT)(2C) receptor antagonist, SB206553 (2.5 or 5.0 mg/kg), in combination with 5 min of restraint, also reduced L/M ratios of hormonally primed, ovariectomized rats. The neural sites responsible for ketanserin's additivity with restraint are unknown, but infusion of the drug into the ventromedial nucleus of the hypothalamus (VMN) did not mimic the systemic treatment. However, 5 min of restraint did enhance the effects of VMN infusion with the 5-HT(1A) receptor agonist, 8-OH-DPAT. In contrast, 8-OH-DPAT's systemic potency was not enhanced by restraint. These findings support the hypothesis that a mild stressor increases the lordosis-inhibiting effects of 5-HT(1A) receptor agonists and that 5-HT(2) receptors may protect against such disruption of lordosis behavior.     

Interactive effects of the mGlu5 receptor antagonist MPEP and the mGlu2/3 receptor antagonist LY341495 on nicotine self-administration and reward deficits associated with nicotine withdrawal in rats

Stimulatory actions of nicotine on mesocorticolimbic dopamine transmission are partly mediated by nicotine-induced glutamate release acting on ionotropic and metabotropic glutamate (mGlu) receptors. Because both presynaptic inhibitory mGlu2/3 and postsynaptic excitatory mGlu5 receptors provide potential targets for treatment of aspects of nicotine dependence, we examined interacting effects of mGlu5 (2-methyl-6-(phenylethynyl)-pyridine, MPEP) and mGlu2/3 (LY341495) receptor antagonists on nicotine self-administration and brain reward threshold elevations associated with spontaneous nicotine withdrawal in rats. We hypothesized that increasing glutamate transmission by blocking presynaptic inhibitory mGlu2/3 autoreceptors would antagonize MPEP-induced decreases in nicotine self-administration. We also hypothesized that blocking postsynaptic actions of glutamate on mGlu5 receptors would exacerbate nicotine withdrawal-induced reward deficits, and that this effect would be attenuated by co-administration of the mGlu2/3 receptor antagonist LY341495. MPEP selectively decreased nicotine, but not food, self-administration in rats. LY341495 slightly decreased both nicotine and food self-administration. Co-administration of LY341495 with MPEP attenuated the effectiveness of MPEP in decreasing nicotine intake, although MPEP was still effective. Spontaneous nicotine withdrawal induced somatic signs of withdrawal and reward threshold elevations indicating reward deficits. MPEP increased somatic signs and reward deficits in both nicotine- and saline-withdrawing rats. Thus, while mGlu5 receptor antagonists may be therapeutically useful in decreasing tobacco smoking, they worsen nicotine withdrawal. Co-administration of LY341495 reduced MPEP-induced reward deficits in both nicotine- and saline-withdrawing rats. Thus, increasing glutamate transmission via mGlu2/3 autoreceptor blockade reduces the effects of mGlu5 receptor blockade on nicotine self-administration and MPEP-induced exacerbation of brain reward deficits associated with nicotine withdrawal.     

Inverse agonist activity of sarpogrelate, a selective 5-HT2A-receptor antagonist, at the constitutively active human 5-HT2A receptor

Mutations producing constitutively active G-protein coupled receptors have been found in the pathophysiology of several diseases, implying that inverse agonists at the constitutively active receptors may have preferred therapeutic applications. Because of the involvement of 5-HT(2A) receptors in mediating many cardiovascular diseases, constitutively active mutants of the 5-HT(2A) receptor may be responsible for the disease states. Thus, the purpose of the present study was to investigate the inverse agonist activity of sarpogrelate, a selective 5-HT(2A)-receptor antagonist, and its active metabolite, M-1; and we compared their activities with those of other 5-HT(2A)-receptor antagonists such as ritanserin, ketanserin, and cyproheptadine. Using a constitutively active mutant (C322K) of the human 5-HT(2A) receptor, we demonstrated that like other 5-HT(2A)-receptor antagonists, sarpogrelate acts as a potent inverse agonist by significantly reducing basal inositol phosphate levels. However, there were no significant differences between sarpogrelate and other 5-HT(2A)-receptor antagonists for their inverse agonist activity. Compared with the wild type receptor, mutant receptor displayed significantly higher affinity for 5-HT and lower affinity for sarpogrelate. These results indicate that stabilization of the inactive conformation of the 5-HT(2A) receptor may be a key component of the mechanism of action of sarpogrelate.     

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.     

Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement

The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT(2A) and 5-HT(2C) receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT(2C) receptor agonist Ro60-0175 and the 5-HT(2A) receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT(2C) receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT(2C) and 5-HT(2A) receptors may be potential targets for therapies to treat some aspects of nicotine dependence.     

ACP-103, a 5-HT2A receptor inverse agonist

ACADIA Pharmaceuticals is developing ACP-103, lead compound in a series of 5-HT2A inverse agonists, as a potential antipsychotic agent and for the potential treatment of insomnia. Phase II clinical trials in treatment-induced psychosis in Parkinson's disease (PD) patients and in schizophrenic patients are ongoing, as are phase II trials evaluating the effects of the drug on PD symptoms and dyskinesias.     

The 5-HT2 receptor antagonist, MDL 28,133A, disrupts the serotonergic-dopaminergic interaction mediating the neurochemical effects of 3,4-methylenedioxymethamphetamine.

The selective 5-HT₂ receptor antagonist MDL 28,133A dose dependently blocked the long-term deficits in rat brain 5-HT concentrations produced by the substituted amphetamine analogue 3,4-methylenedioxymethamphctamine (MDMA). This protective effect of MDL 28,133A could be abolished by coadministration of the dopamine precursor, L-dihydroxyphenylalanine (L-DOPA). Electrophysiological experiments demonstrated that the ability of MDL 28,133A to block the MDMA-induced slowing of A⁹ dopaminergic neurons was also sensitive to L-DOPA administration. Both sets of experiments suggest an interaction of MDL 28,133A at the level of dopamine synthesis. Consistent with this explanation, MDL 28,133A antagonized the MDMA-induced stimulation of dopamine synthesis in vivo. MDMA-induced 5-HT release did not reduce the firing rate of dopaminergic neurons as assessed by dopamine depletion following synthesis inhibition with -mcthyl-p-tyrosinc (-MPT). This indicates that the effect of 5-HT₂ receptor antagonists on MDMA-induced dopamine synthesis is not due simply to the removal of an inhibitory serotonergic input followed by an increase in dopamine cell firing and autoreceptor activation. MDL 28,133A was also shown to be without effect on the sensitivity of terminal dopamine autoreceptors. The results are consistent with the hypothesis that 5-HT₂ receptors are permissive for the stimulation of dopamine synthesis necessary to support MDMA-induced transmitter efflux.     

R-zacopride, a 5-HT3 antagonist/5-HT4 agonist, reduces sleep apneas in rats.

The effects of R-zacopride, a benzamide with potent 5-HT3 receptor antagonist and 5-HT4 receptor agonist properties, on spontaneous apneas were studied in 10 Sprague-Dawley rats by monitoring respiration and sleep for 6 h. R-zacopride (0.5, 1.0 and 10.0 mg/kg) suppressed spontaneous central apneas during non-rapid-eye-movement (NREM) sleep by 50% (P=.05 for 0.5 mg/kg, P=.02 for 1.0 mg/kg and P=.001 for 10.0 mg/kg dose vs. control), and during rapid-eye-movement (REM) sleep by 80% by all doses tested (P<.0007) for at least 2 h after intraperitoneal injection. We conclude that R-zacopride, over a 20-fold dose range, significantly reduces central apnea expression during NREM and REM sleep in the rat. The efficacy of this compound to suppress central apneas most probably arises from its antagonist actions at 5-HT3 receptors or from its mixed agonist/antagonist profile at 5-HT4/5-HT3 receptors.     

Anti-thrombotic and vascular effects of AR246686, a novel 5-HT2A receptor antagonist

We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.     

Temperature,food intake,and locomotor activity effects of a 5-HT3 receptor agonist and two 5-HT3 receptor antagonists in rats

This study investigated three physiologic functions known to be modulated by serotonin — temperature, food intake and locomotor activity — using the 5-HT₃ receptor agonist,m-chlorophenylbiguanide (m-CPBG), and two 5-HT₃ antagonists, MDL-72222 and ondansetron. m-CPBG produced dose-dependent elevations in rectal temperature. MDL-72222, which had no effects on temperature when given alone, significantly attenuated m-CPBG-induced hyperthermia. Food intake in food-deprived rats was reduced during the first hour by the highest dose of m-CPBG. Food intake was also dose-dependently reduced by MDL-72222; m-CPBG plus MDL-72222 led to greater reductions in food intake. Food intake in freely fed rats was unaffected by m-CPBG or MDL-72222. Locomotor activity was unaffected by m-CPBG, but was dose-dependently reduced by MDL-72222, an effect which may have contributed to its hypophagic effects. Ondansetron, used in ten-fold lower doses than MDL-72222, was inactive in all of these paradigms. These data: (1) provide some evidence for 5-HT₃ receptor-mediated changes in temperature; (2) are in agreement with two prior studies which reported locomotor activity reductions following 5-HT₃ antagonists; but (3) do not support an important role for 5-HT₃ receptors in the regulation of food intake in rats.     

Effect of sustained administration of the 5-HT1A receptor agonist flesinoxan on rat 5-HT neurotransmission.

A short-term treatment with flesinoxan (2.5 and 5 mg/kg/day x 2 days, s.c., delivered using osmotic minipumps) decreased significantly the spontaneous firing activity of dorsal raphe serotonin (5-HT) neurons of male Sprague-Dawley rats. This firing was still decreased following 1 week of treatment with flesinoxan (5 mg/kg/day) but was back to normal after a treatment of 2 weeks. This recovery of firing was associated with a 3-fold shift to the right of the dose-response curve of the effect of the 5-HT autoreceptor agonist lysergic acid diethylamide on the firing activity of 5-HT neurons, indicating a desensitization of somatodendritic 5-HT1A autoreceptors. At the postsynaptic level, long-term treatment with flesinoxan (5 mg/kg/day x 14 days) did not modify the responsiveness of dorsal hippocampus CA3 pyramidal neurons to microiontophoretic applications of 5-HT and flesinoxan nor to endogenous 5-HT released by the electrical stimulation of the ascending 5-HT pathway, indicating an unchanged sensitivity of postsynaptic 5-HT1A receptors. Finally, in rats treated with flesinoxan for 2 weeks, the administration of the selective 5-HT1A receptor antagonist (N-{2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohe xanecarboxamide trihydroxychloride (WAY 100635, 100 and 500 microg/kg, i.v.) did not increase the firing activity of dorsal hippocampus CA3 pyramidal neurons, thus failing to reveal an enhanced tonic activation of postsynaptic 5-HT1A receptors as for other antidepressant drugs, including the 5-HT1A receptor agonist gepirone. The marked potency and the long dissociation constant of flesinoxan for the 5-HT1A receptors may account for the latter discrepancy. In conclusion, as for selective 5-HT re-uptake inhibitors, monoamine oxidase inhibitors and 5-HT1A receptor agonists, flesinoxan produced most of the adaptive changes exerted by these antidepressant drugs on the 5-HT system.     

Full substitution of the discriminative cue of a 5-HT(1A/1B/2C) agonist with the combined administration of a 5-HT(1B/2C) and a 5-HT(1A) agonist

The present study examined whether animals attend to the individual components of the cue produced by a drug that stimulates different 5-hydroxytryptamine (5-HT) receptor populations, using a drug discrimination task based on the conditioned taste aversion (CTA) procedure. The training drug was indorenate (5-methoxytryptamine beta-methylcarboxylate) (INDO) that has been described as a 5-HT(1A/2C/1B) agonist able to exert discriminative control in both operant and CTA procedures. The principal objective was to examine generalization with the combined administration of agonists for the different receptor sites that may mimic the mechanism of action of the training drug. Male Wistar rats, deprived of water, were trained to discriminate INDO from saline; during the drug trials, the administration of INDO preceded saccharin-LiCl pairings, while, during the saline trials, the administration of saline preceded the saccharin-saline pairings. In generalization tests, INDO, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, a 5-HT(1A) agonist), 1-(3-trifluoromethylphenyl)piperazine (TFMPP, a 5-HT(1B) agonist), alpha-methyl-5-HT (a 5-HT(2C) agonist) or 2-methyl-5-HT (a 5-HT(3) agonist), were administered alone or in combination. The results showed that 8-OH-DPAT, TFMPP and alpha-methyl-5-HT produced dose-dependent generalization, up to 88% in the case of 8-OH-DPAT. The combined administration of the following pairs of drugs, 8-OH-DPAT+TFMPP or 8-OH-DPAT+ alpha-methyl-5-HT, at doses that produced only 15-55% generalization when administered alone, produced greater than 80% generalization to INDO. However, the single administration of 2-methyl-5-HT produced only saline-like responding and its combined administration with 8-OH-DPAT did not modify the generalization produced by the single administration of 8-OH-DPAT. These results suggest that animals attend to the individual components of the drug cue; in the case of INDO, which has three elements, each mediated by a different receptor subpopulation (5-HT(1A), 5-HT(1B) and 5-HT(2C) ), the separate stimulation of at least two receptor subpopulations was 'interpreted' by the subject as the presence of the training drug.     

Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor

Background and purpose:

5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT₁ receptor of an insect model for neurobiology, physiology and pharmacology.

Experimental approach:

A cDNA encoding for the Periplaneta americana 5-HT₁ receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum.

Key results:

The P. americana 5-HT₁ receptor (Pea5-HT₁) shares pronounced sequence and functional similarity with mammalian 5-HT₁ receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT₁ was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands.

Conclusions and implications:

This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT₁ receptor. The results presented here should facilitate further analyses of 5-HT₁ receptors in mediating central and peripheral effects of 5-HT in insects.     

Interaction between a selective 5-HT1A receptor antagonist and an SSRI in vivo: effects on 5-HT cell firing and extracellular 5-HT.

1. The acute inhibitory effect of selective 5-hydroxytryptamine (serotonin) reuptake inhibitors (SSRIs) on 5-HT neuronal activity may offset their ability to increase synaptic 5-HT in the forebrain. 2. Here, we determined the effects of the SSRI, paroxetine, and a novel selective 5-HT1A receptor antagonist, WAY 100635, on 5-HT cell firing in the dorsal raphé nucleus (DRN), and on extracellular 5-HT in both the DRN and the frontal cortex (FCx). Extracellular electrophysiological recording and brain microdialysis were used in parallel experiments, in anaesthetized rats. 3. Paroxetine dose-dependently inhibited the firing of 5-HT neurones in the DRN, with a maximally effective dose of approximately 0.8 mg kg-1, i.v. WAY 100635 (0.1 mg kg-1, i.v.) both reversed the inhibitory effect of paroxetine and, when used as a pretreatment, caused a pronounced shift to the right of the paroxetine dose-response curve. 4. Paroxetine (0.8 mg kg-1, i.v.), doubled extracellular 5-HT in the DRN, but did not alter extracellular 5-HT in the FCx. A higher dose of paroxetine (2.4 mg kg-1, i.v.) did increase extracellular 5-HT in the FCx, but to a lesser extent than in the DRN. Whereas 0.8 mg kg-1, i.v. paroxetine alone had no effect on extracellular 5-HT in the FCx, in rats pretreated with WAY 100635 (0.1 mg kg-1), paroxetine (0.8 mg kg-1, i.v.) markedly increased extracellular 5-HT in the FCx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular effects of SL65.0472, a 5-HT receptor antagonist

In this study, we describe the cardiovascular effects of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c] pyridin-4-yl) piperazin-l-yl] ethyl]-1, 2-dihydroquinoline-1-acetamide), a novel 5-hydroxytryptamine (5-HT) receptor antagonist developed for the treatment of cardiovascular disease, in several in vivo models. The haemodynamic profile of SL65.0472 was evaluated in anaesthetised dogs. Following i.v. bolus doses of 0.03 mg/kg i.v. and 0.3 mg/kg, no significant changes in cardiac output, contractility or rate, systemic and pulmonary pressures, regional blood flows and vascular resistances or electrocardiogram were noted. After 1 mg/kg i.v. SL65.0472 significantly reduced arterial blood pressure. In conscious spontaneously hypertensive rats administration of SL65.0472 0.5 mg/kg p.o. had no effect on mean arterial blood pressure or heart rate. Vasoconstriction produced by 5-HT results primarily from the stimulation of two receptor subtypes, 5-HT(1B) and 5-HT(2A) receptors. In anaesthetised dogs SL65.0472 antagonised sumatriptan-induced decreases in saphenous vein diameter (5-HT(1B)-receptor mediated) with an ID(50) of 10.1 microg/kg i.v. (95% c.l. 8.3-12.4). In anaesthetised pithed rats SL65.0472 inhibited 5-HT pressor responses (5HT(2A)-receptor mediated) with ID(50) values of 1.38 microg/kg i.v. (95% c.l. 1.15-1.64) and 31.1 microg/kg p.o. (95% c.l. 22.6-42.6). The duration of the 5-HT(2A)-receptor antagonist effect of SL65.0472 following oral administration was evaluated in conscious rats. SL65.0472 (0.1 mg/kg p.o.) markedly inhibited 5-HT pressor responses 1 and 6 h after administration. Therefore, in vivo, SL65.0472 potently antagonises vasoconstriction mediated by 5-HT(1B) and 5-HT(2A) receptors but has minimal direct haemodynamic effects.