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Using several 5-hydroxytryptamine (5-HT) agonists and antagonists, we attempted to characterize the receptor subtypes involved in the contractile response to 5-HT in the in situ autoperfused rat kidney. An intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1 microg/kg) increased renal perfusion pressure in a dose-dependent way but did not affect the systemic blood pressure. The selective 5-HT2 receptor agonist alpha-methyl-5-HT (alpha-methyl-5-hydroxytryptamine) and the non-selective 5-HT2C receptor agonist (1-(3-chlorophenyl)piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney, whereas BW723C86, a selective 5-HT2B receptor agonist, the 5-HT1 receptor agonist 5-carboxamidotryptamine, 5-CT, and the selective 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)-biguanide) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by alpha-methyl-5-HT and m-CPP was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), SB 206553 (3,5-Dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a selective 5-HT2B/2C receptor antagonist and enalapril, but was not modified by pretreatment with spiperone (a 5-HT2A receptor antagonist). The results of protein expression analyses allow us to postulate that 5HT-SRC (a 5-HT2C receptor protein) is expressed in renal tissue and differentially expressed in renal artery. Our data suggest also that the serotonergic vasoconstrictor response induced in the in situ autoperfused rat kidney would be mediated by local 5-HT2C receptor activation.

This study evaluated the possible involvement of 5-HT 2B receptors in long-lasting hypotension to 5-hydroxytryptamine (5-HT), which is predominantly mediated by 5-HT 7 receptors, in anaesthetised vagosympathectomized rats. Intravenous injections of 5-HT and 5-carboxamidotryptamine (5-CT) elicited a dose-dependent hypotension that was dose-dependently antagonised by ( R )-1-{(3-hydroxyphenyl)sulfonyl}-2-{2-(4-methyl-1-piperidinyl) ethyl} pyrrolidine (SB-269970; a selective 5-HT 7 receptor antagonist), but not by saline. Interestingly, α-methyl-5-(2-thienylmethoxy)-1 H -indole-3-ethanamine (BW723C86; a 5-HT 2B receptor agonist) produced vasopressor responses without affecting hypotension to 5-HT. These results suggest that hypotension to 5-HT and 5-CT is mainly mediated by 5-HT 7 receptors, whilst the role of 5-HT 2B receptors seems unlikely.

Serotonin 5-HT2B receptor antagonists have been proposed as migraine prophylactic drugs, but previously available 5-HT2B receptor antagonists displayed multiple monoaminergic side effects and had to be withdrawn from the market. Here, we set out to identify a novel antagonist with high affinity and selectivity towards 5-HT2B receptors. To test the affinity of new compounds towards various receptors, we generated a broad series of cells functionally coupling human monoaminergic receptors to luciferase. Using the cell lines we revealed pimethixene (1-methyl-4-(9H-thioxanthen-9-ylidene)piperidine) as highly potent, albeit non-selective 5-HT2B receptor antagonist and optimized its chemical structure to create highly potent and selective 5-HT2B receptor antagonists. We selected the methoxythioxanthene BF-1 for further analysis. In comparison to pimethixene, it lacked high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2 as well as muscarinic M1 and M2 receptors. BF-1 was tested as potential migraine prophylactic drug by blocking meta-chlorophenylpiperazine, (mCPP) or BW723C86 (5-((thiophen-2-yl)methoxy)-α-methyltryptamine) induced neurogenic dural plasma protein extravasation in a guinea pig model that may resemble a migraine attack. BF-1 was significantly more potent in this assay compared to the well know non-selective 5-HT2B antagonists, methysergide ((6aR,9R)-N-[(2S)-1-Hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide) or pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-thiophene). Therefore, we propose BF-1 as a new compound that may be developed for prophylactic migraine treatment without the typical monoaminergic side effects.

1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-1(2H)-pyridinyl]ethyl]-3-isopropyl-6-(methylsulphonyl)-3,4-dihydro-1H-2,1,3-benzothiadiazine-2,2-dioxide (LY393558) is a potent inhibitor of [3H]5-hydroxytryptamine ([3H]5-HT) uptake into rat cortical synaptosomes (pIC(50)=8.48+/-0.12). It produces a dextral shift of the 5-HT dose-response curves for the binding of GTPgamma[35S] to human 5-HT(1B) (pK(b)=9.05+/-0.14) and 5-HT(1D) (pK(b)=8.98+/-0.07) receptors and inhibits the contractile response of the rabbit saphenous vein to the 5-HT(1B/D) receptor agonist, sumatriptan (pK(b)=8.4+/-0.2). In addition, it is an antagonist at the 5-HT(2A) (pK(i)=7.29+/-0.19) and 5-HT(2B) (pK(i)=7.35+/-0.11) receptors. Presynaptic autoreceptor antagonist activity was demonstrated by its ability to potentiate the K(+)-induced outflow of [3H]5-HT from guinea pig cortical slices (pEC(50)=7.74+/-0.05 nM) in which the 5-HT transporter had been inhibited by a maximally effective concentration of paroxetine. It is concluded that LY393558 should be an effective antidepressant with the potential to produce an earlier onset of efficacy than selective serotonin uptake inhibitors.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Using a number of agonist and antagonist compounds, we attempted to characterize the responses and receptors involved in the effects of 5-hydroxytryptamine (5-HT) in the in situ blood perfused rat mesentery. An intra-arterial (i.a.) bolus injection of 5-HT increased mesenteric perfusion pressure in a dose-dependent way but did not change the systemic blood pressure. The selective 5-HT(2) receptor agonists alpha-methyl-5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (m-CPP) and (+/-)-1-(4-iodo-2, 5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI), caused a local vasoconstrictor effect in the autoperfused vascular mesenteric bed. Intra-arterial injection of 5-carboxamidotryptamine (5-CT) and 1-(m-chlorophenyl)-biguanide (m-CPBG) did not modify the mesenteric perfusion pressure. The vasoconstrictor effect elicited by 5-HT and alpha-methyl-5-HT was significantly decreased by ritanserin and by a selective 5-HT(2B/2C) receptor antagonist, N-3-pyridinyl-3, 5-dihydro-5-methyl-benzo[1,2-b:4,5-b']dipyrrole-1(2H)-carboxamide hydrochloride (SB 206553), but was not modified by prazosin, propranolol, indomethacin or enalapril pretreatment. Our data suggest that the vasoconstrictor serotonergic response induced in the in situ autoperfused rat mesenteric vascular bed is mainly mediated by activation of 5-HT(2B) and/or 5-HT(2C) receptors.

The effects of a serotonin (5-HT) releasing drug, p -chloroamphetamine, on plasma glucose levels were investigated in rats. p -Chloroamphetamine elicited a significant hyperglycemia. The hyperglycemic effects of p -chloroamphetamine were completely prevented by the 5-HT synthesis inhibitor, p -chlorophenylalanine. Prior adrenodemedullation abolished the hyperglycemia elicited by p -chloroamphetamine. p -Chloroamphetamine-induced hyperglycemia was prevented by methysergide, which blocks the 5-HT 1 and 5-HT 2 receptor, the 5-HT 1A/1B/2C receptor antagonist, (−)-propranolol, the selective 5-HT 1A receptor antagonist, 4-(2′-methoxyphenyl-1-[2′- n -2″pyridinyl)- p -iodobenzamido]-ethyl-piperazine ( p -MPPI), the 5-HT 2A/2B/2C receptor antagonists, ritanserin and 4-isopropyl-7-methyl-9-(2-hydroxy-1-methyl-propoxycarbonyl)-4,6A,7,8,9,10,10A-octahydro-indolo[4,3-FG]quinolone maleate(LY 53857). However, the 5-HT 3 and 5-HT 4 receptor antagonist, tropisetron, the 5-HT 4 receptor antagonist, 2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester (SDZ 205-557), and the 5-HT 2A receptor antagonist, ketanserin, did not affect the p -chloroamphetamine-induced hyperglycemia. These results suggest that p -chloroamphetamine-induced hyperglycemia is elicited by an enhanced 5-HT release and facilitated adrenaline release. Moreover, our results indicate that p -chloroamphetamine-induced hyperglycemia is mediated by 5-HT 1A and 5-HT 2B/2C receptors.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by sustained elevation of pulmonary arterial pressure that leads to right ventricle failure and death. Pulmonary resistance arterioles in PAH undergo progressive narrowing and/or occlusion. Currently approved therapies for PAH are directed primarily at relief of symptoms by interfering with vasoconstrictive signals, but do not halt the microvascular cytoproliferative process. In this study we show that C-122 (2-amino-N-(2-{4-[3-(2-trifluoromethyl-phenothiazin-10-yl)-propyl]-piperazin-1-yl}-ethyl)-acetamide trihydrochloride, a novel antagonist of serotonin receptor 5-HT(2B) (Ki=5.2 nM, IC(50)=6.9 nM), when administered to rats for three weeks in daily oral 10mg/kg doses, prevents not only monocrotaline (MCT)-induced elevations in pressure in the pulmonary arterial circuit (19 ± 0.9 mmHg vs. 28 ± 2 mmHg in MCT-vehicle group, P

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

Ghrelin is an orexigenic peptide present in the stomach with gastroprokinetic properties. Previous in vivo studies have shown that the ghrelin receptor antagonist, D-Lys 3 -GHRP-6, reduced food intake and delayed gastric emptying in rodents but these effects are at variance with the normal phenotype of the ghrelin knockout mice. To verify the specificity of the effects observed with D-Lys 3 -GHRP-6 this study aimed to investigate the pharmacology of D-Lys 3 -GHRP-6 in vitro. Rat fundic strips were suspended in a tissue bath and the contraction of strips to 10 − 5 M of ghrelin, GHRP-6 or D-Lys 3 -GHRP-6 was measured isometrically in the absence and presence of blockers. Neither ghrelin, nor GHRP-6, induced significant contractions in the absence of electrical field stimulation thereby excluding the presence of ghrelin receptors on smooth muscle cells. In contrast D-Lys 3 -GHRP-6, induced a pronounced biphasic contraction of 13.9 ± 1.8% and 40.5 ± 3.2% relative to the response to 60 mM KCl. The contraction was blocked by the 5-HT 1,2 receptor antagonist methysergide and was markedly reduced by the 5-HT 2B receptor antagonist, yohimbine, which also profoundly affected 5-HT induced contractions in fundic strips. The existence of 5-HT 2B receptors in the fundus was confirmed by use of the 5-HT 2B receptor agonist, BW 723C86. In contrast to ghrelin and GHRP-6, the ghrelin receptor antagonist, D-Lys 3 -GHRP-6, induced pronounced smooth muscle contractions in the rat fundus by interacting with 5-HT 2B receptors. This may question the role of endogenous ghrelin in the effects observed with D-Lys 3 -GHRP-6 on food intake and gastric emptying in vivo.

We report the cloning, molecular characterization, and pharmacological characterization of the canine 5-HT2A and 5-HT2B receptors. The canine and human 5-HT2A receptors share 93% amino acid homology. The canine and human 5-HT2B receptors are also highly conserved (87% homology) with the exception of the carboxyl termini where the canine protein is 62 amino acids shorter. Both the canine 5-HT2A and 5-HT2B receptors have high affinity for [3H]5-HT (KD=4.50+/-0.89 nM and 3.10+/-0.82 nM, respectively) and, in general, the pharmacology of these two receptors matches closely the pharmacology of their human homologs for the 19 serotonergic ligands tested. However, the functional response (Ca2+ mobilization) of the canine 5-HT2B receptor to several agonists was weaker compared to the human 5-HT2B receptor. Using quantitative reverse transcriptase polymerase chain reaction, a high expression level of canine 5-HT2A receptor mRNA was detected in the brain and lower levels in peripheral tissues, whereas the highest levels of canine 5-HT2B receptor mRNA were observed in lungs and smooth muscles. A significant level of canine 5-HT2B receptor mRNA was detected in brain tissue. The availability of the full sequence and pharmacology of the canine 5-HT2A and canine 5-HT2B receptors provides useful information for the interpretation of previous and future pharmacological studies of 5-HT2A/2B ligands in dog.

Effects of the 5-HT(2C/2B) receptor agonist m-chlorophenylpiperazine (mCPP) on plasma glucagon levels were investigated in rats. mCPP dose dependently increased plasma glucagon levels. Hyperglucagonemia elicited by mCPP was prevented by the 5-HT(2A/2B/2C) receptor antagonist, ritanserin, while the 5-HT(2A) receptor antagonist, ketanserin, did not show any effect. Increases in glucagon levels induced by mCPP were inhibited by prior adrenodemedullation. These results indicate that increases in plasma glucagon levels induced by mCPP are mediated by the 5-HT(2C/2B) receptor which in turn facilitates adrenaline release.

Acute administration of the 5-HT2C/2B receptor agonist 1-(3-chlorophenyl)piperazine (mCPP, 5-10 mg/kg i.p.) induced hyperglycemia in rats. These changes were diminished in a dose-dependent manner by the 5-HT1/5-HT2 receptor antagonist methysergide and the 5-HT2A/2B/2C receptor antagonist ritanserin. In addition, mCPP-induced hyperglycemia was dose dependently diminished by the ganglionic blocker hexamethonium and was prevented by prior adrenodemedullation. Neither the 5-HT2A receptor antagonist ketanserin nor the 5-HT3/5-HT4 receptor antagonist (3-alpha-tropanyl)-1 H-indole-3-carboxylic acid ester (ICS 205-930) proved effective against mCPP-induced hyperglycemia. Lastly, administration of the 5-HT2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane (DOI) increased plasma glucose levels through ketanserin- and ritanserin-sensitive processes. Our results suggest that hyperglycemia elicited by mCPP is mediated by 5-HT2C and/or 2B receptors, and in turn adrenomedullary catecholamine release, whereas that elicited by DOI involves 5-HT2A receptors.