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

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 α1A-adrenoceptors in rat vas deferens, α1B-adrenoceptors in rat spleen and α1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[β-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat α1-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 (pA2∼7.2, n=4). This suggests that ρ-TIA is a competitive antagonist of α1A- and α1D-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 α1B-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 (pIC50) 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 (Bmax) in membranes from HEK293 cells expressing the rat α1B-adrenoceptors without affecting affinity (KD). In contrast, in HEK293 cells expressing rat α1A- or α1D-adrenoceptors, ρ-TIA decreased the KD without affecting the Bmax. It is concluded that ρ-TIA will be useful for distinguishing the role of particular α1-adrenoceptor subtypes in native tissues.