Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens

A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA-1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed 'neuroeffector Ca2+ transients' (NCTs). NCTs were abolished by alpha,beta-methylene ATP (1 mum), but not by nifedipine (1 muM) or prazosin (100 nm), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L-type Ca2+ channels or alpha(1)-adrenoceptor-mediated pathways. Action potential-evoked ATP release was highly intermittent (mean probability 0.019 +/- 0.002; range 0.001-0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty-eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the alpha(2)-adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 +/- 21 % during trains of stimuli at 1 Hz. Frequency-dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse-to-impulse basis.