Differential antagonism by conotoxin ρ-TIA of contractions mediated by distinct α1-adrenoceptor subtypes in rat vas deferens, spleen and aorta

Abstract The ability of the conotoxin ρ-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa , to antagonize contractions induced by noradrenaline through activation of α 1A -adrenoceptors in rat vas deferens, α 1B -adrenoceptors in rat spleen and α 1D -adrenoceptors in rat aorta, and to inhibit the binding of [ 125 I]HEAT (2-[[β-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat α 1 -adrenoceptors was investigated. ρ-TIA (100 nM to 1 μM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (p A 2 ∼7.2, n =4). This suggests that ρ-TIA is a competitive antagonist of α 1A - and α 1D -adrenoceptors with no selectivity between these subtypes. Incubation of ρ-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that ρ-TIA is a non-competitive antagonist at α 1B -adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of ρ-TIA in inhibiting contractions was examined with similar occupancies (∼25%) at each subtype. Its potency (pIC 50 ) was 12 times higher in spleen (8.3±0.1, n =4) than in vas deferens (7.2±0.1, n =4) or aorta (7.2±0.1, n =4). In radioligand binding assays, ρ-TIA decreased the number of binding sites ( B max ) in membranes from HEK293 cells expressing the rat α 1B -adrenoceptors without affecting affinity ( K D ). In contrast, in HEK293 cells expressing rat α 1A - or α 1D -adrenoceptors, ρ-TIA decreased the K D without affecting the B max . It is concluded that ρ-TIA will be useful for distinguishing the role of particular α 1 -adrenoceptor subtypes in native tissues.