Characterization of 5-hydroxytryptamine1B receptors in rat spinal cord via [125I]iodocyanopindolol binding and inhibition of [3H]-5-hydroxytryptamine release.
The aim of the present study in rat spinal cord synaptosomes was to compare the pharmacological characteristics of the serotonin (5-HT)1B receptor defined by [125I]iodocyanopindolol [( 125I] ICYP) binding and the 5-HT autoreceptor defined by inhibition of [3H]-5-HT release. In Percoll gradient Fractions 3 and 4 of spinal cord synaptosomes, a single saturable binding site for [125I]ICYP with a maximum binding of 70 and 134 fmol/mg, respectively, was demonstrated in the presence of 30 microM isoproterenol. The Kd of 0.16 nM did not vary between fractions. Competition for [125I]ICYP binding by various 5-HT agonists and antagonists also indicated a single site model based on a Hill coefficient of approximately 1.0. The most potent compounds at displacing [125I]ICYP binding were RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole), 5-carboxyamidotryptamine HCl, 5-methoxytryptamine, 5-HT and CGS 12066B (7-trifluoromethyl-4(4 methyl-1-pyrolo[1,2-a]-quinoxaline malate). [125I]ICYP binding was not altered by compounds with activity at 5-HT1A, 5-HT1C, 5-HT2, 5-HT3 or alpha-2 receptor sites. Similar to the pharmacological characteristics of the 5HT1B site defined by [125I]ICYP, compounds most active at inhibiting 15 mM K(+)-stimulated release of [3H]-5-HT were RU24969 = 5-carboxyamidotryptamine HCl = CGS 12066B greater than 5-methoxytryptamine greater than 5-HT. Compounds with activity at 5-HT1A, 5-HT1C, 5-HT2 or 5-HT3 sites were inactive. A correlation analysis of selective 5-HT1B compounds comparing the pKD for displacement of [125I]ICYP vs. the IC50 for inhibition of [3H]-5-HT release demonstrated the pharmacological similarity of the presynaptic inhibitory 5-HT autoreceptor and the 5-HT receptor site defined by [125I]ICYP binding in spinal cord synaptosomes (r = 0.791, P = .0193). Although [125I]ICYP binding was unaltered, alpha-2 agonists such as clonidine, norepinephrine and UK 14304 [5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) as well as the alpha-2 antagonists rauwolscine and yohimbine also decreased the K(+)-stimulated release of [3H]-5-HT and phentolamine, an alpha-2 antagonist increased release. The action of these alpha-2 compounds to alter [3H]-5-HT release suggests the presence of heteroreceptors localized on 5-HT terminals in the spinal cord. These results point out that [125I]ICYP identifies the 5-HT1B receptor, and affinity of compounds for this site predicts action at the 5-HT1B autoreceptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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5-HT [125I]ICYP [125I]iodocyanopindolol [3H]-5-HT 5-HT 5-HT [125I]ICYP [( 125I] ICYP) [125I]ICYP clonidine [125I]ICYP norepinephrine UK 14304 [5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline rauwolscine yohimbine K(+) [3H]-5-HT phentolamine 5-HT [3H]-5-HT serotonin 5-HT [125I]ICYP [3H]-5-HT [125I]ICYP isoproterenol [125I]ICYP 5-HT [125I]ICYP RU 24969 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole K(+) 5-carboxyamidotryptamine HCl 5-methoxytryptamine 5-HT CGS 12066B 7-trifluoromethyl-4(4 methyl-1-pyrolo[1,2-a]-quinoxaline malate [125I]ICYP [3H]-5-hydroxytryptamine [125I]iodocyanopindolol 5-HT1A 5-HT1C [3H]-5-HT 5-HT2 5-HT3 5HT1B serotonin (5-HT)1B receptor 5-HT1A 5-HT1C 5-HT2 5-HT3 5-HT1B 5-HT autoreceptor RU24969 5-HT receptor 5-HT autoreceptor 5-HT1B 5-HT1B 5-hydroxytryptamine1B 5-carboxyamidotryptamine HCl CGS 12066B 5-methoxytryptamine