RESUMO
This study utilised the selective 5-ht(5A) receptor antagonist, SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), to investigate 5-ht5A receptor function in guinea pig brain. SB-699551-A competitively antagonised 5-HT-stimulated [35S]GTPgammaS binding to membranes from human embryonic kidney (HEK293) cells transiently expressing the guinea pig 5-ht5A receptor (pA2 8.1+/-0.1) and displayed 100-fold selectivity versus the serotonin transporter and those 5-HT receptor subtypes (5-HT(1A/B/D), 5-HT2A/C and 5-HT7) reported to modulate central 5-HT neurotransmission in the guinea pig. In guinea pig dorsal raphe slices, SB-699551-A (1 microM) did not alter neuronal firing per se but attenuated the 5-CT-induced depression in serotonergic neuronal firing in a subpopulation of cells insensitive to the 5-HT1A receptor-selective antagonist WAY-100635 (100 nM). In contrast, SB-699551-A (100 or 300 nM) failed to affect both electrically-evoked 5-HT release and 5-CT-induced inhibition of evoked release measured using fast cyclic voltammetry in vitro. SB-699551-A (0.3, 1 and 3 mg/kg s.c.) did not modulate extracellular levels of 5-HT in the guinea pig frontal cortex in vivo. However, when administered in combination with WAY-100635 (0.3 mg/kg s.c.), SB-699551-A (0.3, 1 or 3 mg/kg s.c.) produced a significant increase in extracellular 5-HT levels. These studies provide evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain.
Assuntos
Neurônios/efeitos dos fármacos , Receptores de Serotonina/fisiologia , Antagonistas da Serotonina/farmacologia , Serotonina/metabolismo , Análise de Variância , Animais , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Compostos de Bifenilo/farmacologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Linhagem Celular , Citalopram/farmacocinética , Relação Dose-Resposta a Droga , Eletroquímica/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Cobaias , Humanos , Isótopos/farmacocinética , Dietilamida do Ácido Lisérgico/farmacocinética , Masculino , Microdiálise/métodos , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Ensaio Radioligante/métodos , Antagonistas da Serotonina/química , Antagonistas da Serotonina/farmacocinética , Agonistas do Receptor de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacocinéticaRESUMO
The guinea pig 5-hydroxytryptamine(5A) (gp5-ht(5A)) receptor was cloned from guinea pig brain using degenerate polymerase chain reaction (PCR) and shows 88%, 85% and 84% amino acid sequence identity versus the human, rat and mouse 5-ht(5A) receptors, respectively. The receptor was transiently expressed in human embryonic kidney (HEK) 293 cells. [(3)H]-Lysergic acid diethylamide (LSD) bound saturably to gp5-ht(5A)/HEK293 membranes with a K(d) of 2.3+/-0.1 nM and B(max) of 15.7+/-3.4 pmol/mg protein. The receptor binding profile, determined by competition with [(3)H]LSD, correlated well with that for the human 5-ht(5A) receptor. 5-HT stimulated [(35)S]GTPgammaS binding to gp5-ht(5A)/HEK293 membranes (pEC(50) 8.1+/-0.2), and the response was surmountably antagonised by methiothepin and ritanserin, giving apparent pK(B) values of 8.0 and 7.2, respectively. The 5-HT response was absent using membranes prepared from gp5-ht(5A)/HEK293 cells pretreated with pertussis toxin (PTX). These data suggest that the gp5-ht(5A) receptor couples to G(i)-proteins in this expression system and shows a similar pharmacological profile to that for the human 5-ht(5A) receptor.