Characterization of intracellular Ca2+ stores in gallbladder smooth muscle

Abstract

The existence of functionally distinct intracellular Ca2+ stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca2+ stores in the gallbladder by measuring intracellular Ca2+ concentration ([Ca2+]i) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca2+]i that persisted in Ca2+-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca2+]i that was insensitive to 2-APB but was abolished by pretreatment with 10 μM ryanodine. These data support the idea that both inositol trisphosphate (IP3) receptors (IP3R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca2+-free solution, the [Ca2+]i transients decreased as the interval between Ca2+ removal and caffeine application was increased, indicating a possible leakage of Ca2+ in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca2+-ATPase activation, similar to IP3-sensitive stores. The moderate Ca2+ elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 μM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca2+ pools do not exist, but IP3R and RyR must be spatially separated because Ca2+ release via these pathways leads to opposite responses.