[3H] GR67330, a very high affinity ligand for 5-HT3 receptors

Summary GR67330 potently inhibited 5-hydroxytryptamine (5-HT)-induced depolarizations of the rat isolated vagus nerve. At the higher concentrations used (0.3 nmol/l–1 nmol/l) this was accompanied by a marked reduction in the maximum response to 5-HT. The calculated pK_B value was 10.2.The binding of the tritiated derivative of GR67330 to homogenates of rat entorhinal cortex was examined. Kinetic analysis revealed that specific ³H] GR67330 (0.1 nmol/l) binding was rapid and reversible. Association and dissociation rate constants were 1.48 ± 0.36 × 10⁸ mol/l^–1 s^–1 and 7.85 ± 0.41 × 10^–3 s^–1 respectively. Equilibrium saturation analysis revealed specific binding was to a single site (Bmax 22.6±0.21 fmol/mg protein) of high affinity (Kd 0.038±0.003 nmol/l). At low ligand concentrations, specific binding was up to 90% of total binding. If unlabelled GR67330 was used to define non-specific binding two sites were evident (Kd₁ 0.066 ± 0.007 nmol/l, Kd₂ 20.1 ± 9.7 nmol/l; Bmax₂ 31.5 ± 3.2 fmol/mg protein, Bmax₂ 1110 ± 420 fmol/mg protein). ³H] GR67330 binding was inhibited potently by 5-HT₃ antagonists and agonists. Ligands for other 5-HT receptors and other neurotransmitter receptors were either only weakly active or inactive at inhibiting binding. Hill numbers for antagonist inhibition of binding were close to unity, except for quipazine which was significantly greater than one. In common with other 5-HT₃ binding studies, all 5-HT₃ agonist tested had Hill numbers greater than one (1.51–1.71). GR38032 and GR65630 inhibited a greater proportion of binding than other 5-HT₃ antagonists, this additional binding was interpreted as inhibition from a second saturable site unrelated to the 5-HT₃ receptor.Homogenates of five areas of rat brain were examined for specific ³H]-GR67330 binding (entorhinal cortex, cingulate cortex, parietal cortex, hippocampus and nucleus accumbens/olfactory tubercle). In each brain area a site of very high affinity was labelled. Drug inhibition profiles were also very similar in each brain area. It is concluded that, because of its high affinity, ³H] GR67330 will be a useful ligand to label 5-HT₃ receptors especially in tissues with low receptor densities and to map 5-HT₃ receptors autoradiographically.Abbreviations 5-HT 5-Hydroxytryptamine - 8-OH-DPAT 8-hydroxy-2-di-N-propylaminotetralin - 5-CT 5-carboxyamidotryptamine - GR38032 (±) 1,2,3,9-tetrahydro-9-methyl-3(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one - GR65630 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl) -1-propanone - GR67330 (±)1,2,3,9 - tetrahydro-9-methyl-3-(5-methyl-1H-imidazol-4-yl)methyl]-4H-carbazol-4-one - MDL72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - ICS 205–930 (3-tropanyl)-1H-indole-3-carboxylic acid ester - BRL24924 endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo3,3,1]non-4-yl)benzamide - BRL43694 endo-N-(9-methyl-9-azabicyclo3,3,1]non-3-yl)-1-methyl-indazole-3-carboxamide - SDZ 206-830 (3-homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid ester - mCPP meta-chlorophenylpiperazine Send offprint requests to G. J. Kilpatrick at the above address