Local SR Ca²⁺ release events, sparks, have been shown to influence excitability in tonic vascular smooth muscle (Herrera & Nelson, 2002 ), but little is known of their role in phasic muscles. In the present study we have therefore investigated the role of Ca²⁺ sparks in the control of excitability in guinea-pig ureter.

Guinea-pigs were humanely killed with CO₂. Fluo-4-loaded multicellular preparations or freshly isolated single cells, and wide-field fast confocal imaging were used. Results are expressed as mean values ± S.E.M.; n is the number of preparations and significance was tested using Student’s paired t test.

In cells in situ localised, short-lasting Ca²⁺ sparks, potentiated by caffeine (1-2 mM) and inhibited by ryanodine (50 µM) and CPA (20 µM) were seen in 30-40 % of isolated cells (122 cells observed from 20 animals) and in 87 % of intact preparations (47 observed from 20 animals). The Ca²⁺ sparks originated at the cell membrane close to the middle of the cell, from one to four frequent discharging sites (FDSs). Following global Ca²⁺ transients evoked by electrical field stimulation (7 V, 200 ms), an increase in the number of sparking cells (5-6 times) and FDSs per cell (3-5 times) and the frequency (from 0.1-0.4 to 2.5 Hz) of Ca²⁺ sparks of active FDSs were seen. Stimulation of the ureter during this period of elevated Ca²⁺ spark activity (30-40 s) failed to induce a propagating global rise of Ca²⁺, suggesting that the ureteric muscle was refractory to stimulation during this time. In whole cell voltage-clamp experiments, with K⁺ in the pipette solution, voltage step from -60 mV to 0 mV produced initial inward current followed by small sustained outward current with spontaneous transient outward currents (STOCs) superimposed on it. STOCs appeared after about 100 msec delay following inward current. The temporal characteristics of the STOCs were close to those of Ca²⁺ sparks. The STOCs were inhibited by 1 mM TEA, 50 µM ryanodine or 20 µM CPA and potentiated by 1 mM caffeine. TEA significantly (paired t test n = 7) decreased the refractory period from 60 s to 10 s. Similar effects on the refractory period were also seen with ryanodine (n = 4) and CPA (n = 5). In contrast, caffeine, which enhanced Ca²⁺ spark frequency and STOCs, blocked electrical field stimulated propagating global Ca²⁺ transients (n = 11). The inhibitory action of caffeine on the global Ca²⁺ rise was completely reversed by TEA, ryanodine and CPA.

These data suggest that Ca²⁺ uptake by the SR during Ca²⁺ entry on the action potential plays a key role in controlling the refractory period, by stimulating Ca²⁺ spark activity, which by targeting BK channels affects the resting membrane potential. This is achieved through an increase in FDSs, the number of firing cells and the frequency of Ca²⁺ sparks.

This work was supported by the Welcome Trust.