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

doi:10.1016/j.ejphar.2004.12.011

European Journal of Pharmacology

Volume 508, Issues 1–3, 31 January 2005, Pages 183-192

https://doi.org/10.1016/j.ejphar.2004.12.011 Get rights and content

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 [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 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 (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 (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 α₁-adrenoceptor subtypes in native tissues.

Introduction

The conotoxin TIA (ρ-TIA) is a 19-amino acid peptide isolated from the marine gastropodus Conus tulipa (Sharpe et al., 2001). Most of the toxins from snails of the Conus genus target voltage-gated ion channels (such as Ca²⁺, Na⁺ and K⁺) and ionotropic receptors (such as nicotinic, 5-HT₃ and NMDA glutamate), and are important pharmacological tools in the study of their properties (for review, see Terlau and Olivera, 2004). Interestingly, ρ-TIA interacts with high affinity with α₁-adrenoceptors (Sharpe et al., 2003), which are members of the seven transmembrane domain, G protein-coupled receptor superfamily.

There are three subtypes of α₁-adrenoceptors (α_1A, α_1B and α_1D) (Zhong and Minneman, 1999), and radioligand binding and functional experiments with heterologously expressed human receptors have shown that ρ-TIA has a 10-fold higher affinity for the α_1B-subtype than for the other two receptors (Chen et al., 2004). Also, ρ-TIA interacts differentially at the human α₁-adrenoceptor subtypes, as a non-competitive antagonist at α_1B-adrenoceptor, and as a competitive antagonist at α_1A- and α_1D-adrenoceptors (Chen et al., 2004).

The selective nature and differential modes of inhibition of ρ-TIA suggests that this toxin should prove valuable in elucidating the role of α₁-adrenoceptor subtypes in mediating different functional effects. This is particularly important since to date no highly selective α_1B-adrenoceptor antagonist has become available. However, most previous experiments have been done with recombinant receptors, where a single subtype is expressed in isolation. In addition, the most extensive characterization of the interaction of ρ-TIA with α₁-adrenoceptor subtypes has been done with the human clones (Chen et al., 2004). Since the role of individual α₁-adrenoceptor subtypes in mediating functional responses is usually done in rodent tissues, often expressing more than a single subtype, it is essential to determine whether similar properties are observed in contractile studies of rat tissues. This will allow the use of this compound in distinguishing responses in tissues expressing mixtures of subtypes. Therefore, the ability of ρ-TIA to inhibit contractions of the rat vas deferens, spleen and aorta by noradrenaline, effects predominantly mediated by activation of α_1A-, α_1B- and α_1D-adrenoceptors, respectively, was examined. In addition, the nature of the interactions between ρ-TIA and rat α₁-adrenoceptors was further investigated in radioligand binding assays using [¹²⁵I]HEAT (2-[[β-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) and membranes from human embryonic kidney (HEK) 293 cells expressing each of the recombinant subtypes.

Section snippets

General

The experimental procedures were approved by the Ethics Committee for the Use of Experimental Animals from UNESP-Botucatu. Male Wistar rats (16 to 20 weeks old, 260 to 380 g) were killed by decapitation and selected tissues were carefully excised and prepared for digital recording of isometric contractions as follows: the vas deferens (epididymal portion), spleen (hemi-sections) and thoracic aorta (∼5 mm rings, endothelium denuded) were cleaned of adherent tissues and mounted in organ baths

Effects of ρ-TIA on contractions induced by KCl

ρ-TIA (300 nM) did not affect the maximal contraction induced by 80 mM KCl in vas deferens, spleen or aorta (Fig. 1).

Effects of adrenoceptor antagonists and of ρ-TIA on concentration–response curves to noradrenaline

The adrenoceptor antagonists prazosin, 5-methylurapidil and BMY 7378 antagonized contractions caused by noradrenaline in vas deferens, spleen and aorta. The antagonism was competitive as indicated by the resulting Schild plots (Fig. 2), where the slopes were not different from unity (Table 1). Except for prazosin, which was equipotent in the vas deferens, spleen and aorta with

Discussion

This work compared the actions of ρ-TIA on α₁-adrenoceptor mediated contractions in rat vas deferens, spleen and aorta. Noradrenaline causes contraction of these three tissues predominantly through α_1A-, α_1B- and α_1D-adrenoceptors respectively. This allowed a determination of the ability of the toxin to antagonize functional responses mediated by each of the α₁-adrenoceptor subtypes natively expressed in rat tissues. Also, the potency and selectivity of ρ-TIA with that of other commonly used α₁

Acknowledgement

V. Lima, A. Mueller and Susana Y. Kamikihara contributed equally to the work. The work was supported by FAPESP (grant 02/10315-4 to A.S. Pupo). V. Lima and V. Raymundi are undergraduate students recipients of fellowships from FAPESP. S.Y. Kamikihara and A. Mueller are MSc students recipients of fellowships from FAPESP and CAPES, respectively. The authors wish to thank Ms. Ana Maria Seraphim for technical assistance. TIA was oxidized and purified by Jason Hodonickzy at Xenome.

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

Abstract

Introduction

Section snippets

General

Effects of ρ-TIA on contractions induced by KCl

Effects of adrenoceptor antagonists and of ρ-TIA on concentration–response curves to noradrenaline

Discussion

Acknowledgement

Anal. Biochem.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

Eur. J. Pharmacol.

Some quantitative uses of drug antagonists

Br. J. Pharmacol.

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