Neuroeffector Ca2+ transients are caused by P2X1 receptors

Richard D Wassall1, Keith L Brain1, Richard J Evans2, Thomas C Cunnane1
1University of Oxford, Department of Pharmacology, Oxford, United Kingdom, 2University of Leicester, Department of Cell Physiology & Pharmacology, Leicester, United Kingdom

ATP is the major fast-acting excitatory neurotransmitter in the mouse vas deferens. Targeting P2X1 receptors (P2X1Rs) has been hailed as an innovative treatment for male infertility, or conversely, as a novel contraceptive (Mulryan et al., 2000). However, selective drugs to identify P2X receptor subtypes and characterize their functions have been difficult to develop. High-resolution techniques to study neurotransmission at the level of individual neuroeffector junctions have been used to investigate focal purinergic neuroeffector Ca2+ transients (NCTs), which reflect the release of packets of ATP from sympathetic varicosities (Brain et al., 2002). The hypothesis that P2X1R activation results in NCTs was explored using P2X1R knockout (KO) mice (see Mulryan et al., 2000), and the specific P2X1R antagonist, NF449 (10 microM), with confocal microscope Ca2+-imaging.

Vasa deferentia were removed from 8-12 week-old MF-1 and P2X1R KO mice (129Ola-MF-1), which had been killed humanely by cervical fracture in accordance with the UK Animals Act 1986 and European Communities Council Directive 86/09/EEC, and the tissues were loaded with the Ca2+ indicator, Oregon Green 488 BAPTA-1,AM.

Evoked NCTs (i.e. following 1 Hz nerve stimulation) were absent in KO mice (number of preparations (n) = 6; number of areas investigated (ni) = 26; P < 0.001), and abolished upon application of NF449 in wild-type (MF-1) mice (n = 8; ni = 8; P < 0.001). During high frequency nerve stimulation, occasional focal Ca2+ activity could be observed. Ca2+ waves mediated by alpha1-adrenoceptors could not be found on higher frequency stimulation (MF-1: n = 4, ni = 19, P = NS; KO: n = 4, ni = 19, P = NS), but were observed when exogenous noradrenaline (10 or 100 microM) was applied.

This work demonstrates that P2X1R activation is required for the generation of NCTs, and validates the action of NF449 on a whole organ preparation. The differences between nerve-evoked and exogenously applied noradrenaline have also been investigated.

Brain KL et al. (2002). J Physiol 541, 849-862
Mulryan K et al. (2000) Nature 403, 86-89