The existence of functionally distinct intracellular Ca²⁺ stores has been proposed in some types of smooth muscle. Ca²⁺ release from these stores can induce both contraction and relaxation or regulate non-contractile events such as gene expression. To date, characterization of intracellular Ca²⁺ stores in the gallbladder smooth muscle is lacking. In this study, we sought to examine Ca²⁺ stores in the gallbladder by measuring [Ca²⁺]_i in fura-2 loaded isolated myocytes, and by recording membrane potential and isometric contractions in whole mounted preparations.

Tissue was obtained from adult guinea pigs killed by rapid cervical dislocation. Application of caffeine (10 mM) to single myocytes induced a rapid, spike-like elevation in [Ca²⁺]_i that reached a peak within 6-9 s after the onset of elevation and had a mean amplitude of 0.60 ± 0.09 (expressed as a ratio of the fluorescence at 380 and 340 nm, F₈₀/F₃₄₀, n = 10). When cholecystokinin (CCK, 10 nM) was used to induce IP₃-dependent Ca²⁺ release, the mean amplitude of the peak was 0.22 ± 0.03 (n = 14). These data support the idea that ryanodine receptors (RyRs) could release a substantial amount of Ca²⁺ in these cells. When caffeine was applied in Ca²⁺-free solution, the increases in [Ca²⁺]_i were smaller, indicating a possible leakage of Ca²⁺ in these stores. The refilling of caffeine-sensitive stores involved SERCA activation, as it was inhibited in the presence of 1 µM thapsigargin. Similarly, IP₃-sensitive stores had a leak of Ca²⁺ and were depleted by thapsigargin. The moderate Ca²⁺ elevation caused by CCK induced a contraction of 13.1 ± 1.3 mN (n = 8), but caffeine-induced Ca²⁺ elevation was unable to induce gallbladder contraction. Thus, addition of caffeine (100 µM-10 mM) to the organ bath did not alter resting tone. Similarly, no changes were recorded in response to ryanodine application. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips precontracted with 10 nM CCK or 60 mM KCl, which could be related to the hyperpolarization (6.6 ± 0.9 mV, mean ± S.E.M., n = 5) that this drug caused in gallbladder smooth muscle or to the inhibition of IP₃ receptors. The inability of caffeine to trigger contraction could be related to accumulation of c-AMP as the result of phosphodiesterase (PDE) inhibition. However, in the presence of IBMX, a general inhibitor of PDE, the relaxing effects of caffeine persisted.

All together our results suggest that there is no evidence for multiple, pharmacologically distinct Ca²⁺ pools but the location of IP₃ and ryanodine receptors must be different, with the latter facing the subsarcolemmal space.

This work was supported by SAF-2001-0295.