Interaction of calmodulin- and PKC-dependent contractile pathways in cat lower esophageal sphincter (LES).

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 doses of acetylcholine (ACh) or Inositol Triphosphate (IP3) depends on the release of Ca2+ from intracellular stores and activation of a Ca2+-calmodulin (CaM)-dependent pathway. On the contrary, 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 (TG) 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(-9) 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 TG (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(-9) M) 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.