We have recently demonstrated that field (nerve) stimulation of the mouse vas deferens evokes focal, highly intermittent, α,β-methylene ATP-sensitive [Ca²⁺] transients in smooth muscle. These focal [Ca²⁺] transients arise adjacent to nerve terminal varicosities and can be used to detect simultaneously the highly intermittent release of packets of ATP from each varicosity along a nerve terminal branch. These focal transients have been termed neuroeffector Ca²⁺ transients (NCTs; Brain et al. 2002). In the present study, agents that modify Ca²⁺-induced Ca²⁺ release (CICR) or the sequestration of Ca²⁺ by intracellular stores, have been used to investigate mechanisms that might influence the amplitude or time course of NCTs. Mice (Balb/C) were humanely killed according to UK legislation. The Ca²⁺ indicator Oregon Green 488 BAPTA-1 10 kDa dextran was applied to the cut end of the isolated vas deferens for 8Ð10 h to load nerve terminals orthogradely (Brain & Bennett, 1997) and to fill a population of smooth muscle cells (Brain et al. 2002). Smooth muscle cell labelling could also be achieved by exposing the vas deferens to 10 mM Oregon Green 488 BAPTA-1 AM for 2 h at 36 °C. Preparations were examined with a Leica inverted confocal microscope; images were recorded at a frequency of 4 Hz when nerves were field stimulated with trains of stimuli at 2 Hz (0.6 ms pulse width; 10 V amplitude). The amplitude and time course of recovery of NCTs were measured from junctions that demonstrated a relatively high probability of evoking NCTs per field stimulus (0.05Ð0.1).

The amplitude of NCTs was not affected (P > 0.05, Student’s paired two-tailed t test) by either the endoplasmic reticulum Ca²⁺ ATPase blocker cyclopiazonic acid (CPA; 10 mM; 3 ± 12% number of junctions, n_j = 11; number of preparations, n_p = 3; mean ± S.E.M.) or thapsigargin (1 mM; -20 ± 10% n_j = 9, n_p = 4). However, CPA significantly slowed the return of Ca²⁺ to its resting concentration: control time constant of 200 ± 13 ms; time constant in CPA of 600 ± 100 ms (P < 0.05). Thapsigargin slowed the time constant of recovery only slightly (by 12 ± 6% P < 0.05). Caffeine (3 mM) greatly increased the frequency of spontaneous focal [Ca²⁺] transients within the smooth muscle cell (by more than 15-fold; n_p = 4). These caffeine-induced Ca²⁺ transients did not preferentially occur at the same locations as evoked NCTs and may be due to the release of Ca²⁺ from intracellular stores. Hence there is no detectable contribution of CICR to NCTs, but CPA- and thapsigargin-sensitive Ca²⁺ ATPase, presumably located on the endoplasmic reticulum, are responsible for sequestering at least some of the Ca²⁺ that enters through P2X receptors. This sequestration buffers the focal Ca²⁺ transients and presumably contributes to the filling of intracellular Ca²⁺ stores.

All procedures accord with current UK legislation.