Effects of ω-toxins on noradrenergic neurotransmission in beating guinea pig atria

Abstract

The effects of four ω-toxins, known to block various subtypes of neuronal voltage-activated Ca 2+ channels, on the beating guinea pig left atrium have been analyzed. Atria were suspended in oxygenated Krebs-bicarbonate solution at 32°C and driven with electrical pulses delivered by a stimulator at 1 Hz, 1 ms, 4 V. A 10-fold increase of voltage caused a potent and rapid enhancement of the size of contractions (about 3- to 4-fold above basal), which reflects the release of endogenous noradrenaline from sympathetic nerve terminals. ω-Conotoxin MVIIC, ω-conotoxin MVIIA and ω-conotoxin GVIA inhibited the inotropic responses to 10 × V stimulation with IC 50 values of 191, 44 and 20.4 nM, respectively. ω-Agatoxin IVA did not affect the contractile responses. The inotropic responses to exogenous noradrenaline were unaffected by the toxins. The potent blocking effects of ω-conotoxin GVIA were present even in conditions in which the release of noradrenaline was strongly facilitated by presynaptic α 2-adrenoceptor blockade by phenoxybenzamine. These effects were not reversed upon repeated washing of the tissue with toxin-free medium. In contrast, the blockade induced by ω-conotoxin MVIIC and ω-conotoxin MVIIA were fully reversed, with t 1 2 of 13.5 and 31.2 min, respectively. ω-Conotoxin MVIIC (1 μM) protected against the irreversibility of the blockade induced by ω-conotoxin GVIA (100 nM). The results are compatible with the following conclusions: (i) at cardiac sympathetic neuromuscular junctions, the release of noradrenaline and the noradrenergic transmission is maintained and regulated by N-type Ca 2+ channels; (ii) the modulation by presynaptic α 2-adrenoceptors of noradrenaline release is exerted via N-type Ca 2+ channels; (iii) ω-conotoxin MVIIC and ω-conotoxin MVIIA reversibly block N channels and the noradrenergic neurotransmission; (iv) ω-conotoxin GVIA and ω-conotoxin MVIIC seem to recognize the same binding site on the N-type Ca 2+ channels; however, their binding kinetics might considerably differ; (v) P-type Ca 2+ channels are unlikely involved in the regulation of transmitter release at cardiac sympathetic nerve terminals.