Calcium requirements for acetylcholine-induced contraction of cat esophageal circular muscle cells

Esophageal circular muscle cells isolated by enzymatic digestion contracted in response to acetylcholine (ACh) and in response to the protein kinase C (PKC) agonist 1,2-dioctanoylglycerol (1,2 DAG). Both responses were blocked by PKC antagonists but not by calmodulin antagonists. Furthermore, specific PKC activity, measured in the particulate fraction of the muscle, increased in response to cholinergic stimulation, suggesting that ACh-induced contraction is mediated by a PKC-dependent pathway. ACh-induced contraction decreased with decreasing extracellular Ca2+ and was blocked in Ca(2+)-free physiological salt solution (PSS). Similarly, contraction by the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) was blocked by removal of Ca2+ from the PSS. Diacylglycerol production in response to ACh was reduced when extracellular Ca2+ was reduced from 2 to 0.5 mM and was abolished in Ca(2+)-free PSS. The response to 1,2-DAG, however, did not significantly change as extracellular Ca2+ or cytosolic Ca2+ was reduced to zero. Heparin (10 micrograms/ml), thapsigargin (3 microM), or the Ca2+ ionophore A-23187 (3 microM) had no effect on 1,2-DAG or ACh-induced contraction in permeable cells. The data suggest that contraction in response to ACh is mediated by influx of extracellular Ca2+ and a PKC-dependent pathway. Ca2+ may be required mainly to activate the phospholipases responsible for production of diacylglycerol, since contraction of esophageal muscle cells in response to 1,2-DAG is Ca2+ independent.