Increases in cytosolic Ca²⁺ ([Ca²⁺ ]_c) evoked by depolarisation from -70 to +20 mV), flash photolysis of caged InsP₃ or caffeine (10 mM, applied by pressure ejection) were measured using fluo-3 in single smooth muscle cells voltage-clamped in the whole-cell configuration. Cells were dissociated by an enzymatic process from the colon of guinea-pigs (500 g, humanely killed by stunning then exsanguination). To determine statistical significance Student’s t tests were applied to test and control conditions, where P < 0.05 was considered significant. Ryanodine (50 µM) alone did not decrease the magnitude of either depolarisation or InsP₃-evoked Ca²⁺ increases, nor did it affect the rate of decline of depolarisation-evoked Ca²⁺ transients. Ryanodine decreased caffeine-evoked increases in [Ca²⁺ ]_c) (273 ± 61 nM before ryanodine, 5 ± 2 nM with ryanodine) after which responses to InsP₃ were also reduced (267 ± 45 nM before ryanodine, 14 ± 6 nM with ryanodine, n = 15, P < 0.001 in both cases, results expressed as means ± S.E.M.), indicating that the sarcoplasmic reticulum (SR) Ca²⁺ load had been reduced significantly. After reduction of the SR Ca²⁺ content the amplitude of the depolarisation-evoked Ca²⁺ transient was still not significantly decreased, suggesting that Ca²⁺-induced Ca²⁺ release does not contribute to the depolarisation-evoked rise in [Ca²⁺]_c. However, interestingly, the rate of decline of the depolarisation-evoked Ca²⁺ transient was considerably increased until the transient had fallen to 70 % of the peak [Ca²⁺]_c in 15 out of 21 cells. For example, it took 1.1 ± 0.1 s to reach the 40 % decay point in control compared with 0.7 ± 0.1 s with ryanodine, n = 15, P < 0.001). The increase in the rate of Ca²⁺ removal caused by ryanodine was completely prevented by thapsigargin (1 µM, a selective inhibitor of SERCA activity). After the SR had been depleted (using caffeine and ryanodine) thapsigargin did not alter resting [Ca²⁺]_c values, suggesting that the sarcolemma Ca²⁺ pump was not inhibited by thapsigargin. The rate of decline of [Ca²⁺]_c in thapsigargin alone did not differ significantly from that in ryanodine and thapsigargin, suggesting that the effect of ryanodine on removal occurred by modulating SERCA and not another Ca²⁺ removal mechanism. Therefore, it is proposed that ryanodine caused sufficient depletion of the SR to trigger a rebound increase in SERCA activity. These results may be explained by the rate of Ca²⁺ uptake by the SR exceeding the rate of Ca²⁺ release via open ryanodine receptors.

This work was supported by The Wellcome Trust and the British Heart Foundation. J.McC. is a Caledonian Research Foundation Fellow.