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We have previously shown that, in circular muscle cells of the lower esophageal sphincter (LES) isolated by enzymatic digestion, contraction in response to maximally Effective d oses of acet1choline (Ach) or lnositol Triphosphate (IP3) depends on the release of Ca2+ from intracellular stores and activation of a Ca2+-calmodulin (CaM)-dependent pathway. On the contraryf maintenance of LES tone, and response to low doses of Ach or IP3 depend on a protein kinase C (PKC) mediated pathway. In the present Investigation, we have examined requirements for Ca2+ regulation of the interaction between CaM- and PKC- dependent pathways in LES contraction. Thapsigargin (TC) treatment for 30 min dose dependently reduced Ach-induced contraction of permeable LES cells in free Ca2+ medium. Ach-induced contraction following the low level of reduction of Ca2+ stores by a low dose of TG (10-10 M) was blocked by the CaM antagonist, CGS9343B but not by the PKC antagonists chelerythrine or H7, indicating that the contraction is CaM-dependent. After maximal reduction in intracellular Ca2+ from Ca2+ Stores by TC (10-6 M), Ach-induced contraction was blocked by chelerythrine or H7, but not by CGS9343B, indicating that it is PKC-dependent. In normal Ca2+ medium, the contraction by Ach after TG (10-9 M) treatment was also CaM-dependent, whereas the contraction by Ach after TG (10-9M) treatment was PKC-dependent. We examined whether PKC activation was inhibited by activated CaM. CGS 9343B inhibited the CaM-induced contraction, but did not inhibit the DAG-induced contraction. CaM inhibited the DAG-induced contraction in the presence of CGS 9343B. This inhibition by CaM was Ca2+ dependent. These data are consistent with the view that the switch from a PKC-dependent pathway to a CaM dependent pathway can occur and can be regulated by cytosolic Ca2+ in the LES.
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