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1 Department of Physiology, Nursing School, University of Extremadura, Caceres, Spain
* To whom correspondence should be addressed. E-mail: mjpozo{at}unex.es.
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 [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-APB. Application of caffeine induced a rapid spike-like elevation in [Ca2+]i that was insensitive to 2-APB, but was abolished by pre-treatment with 10 µM ryanodine. These data support the idea that both 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 SERCA 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 pre-contracted 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.
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