Stoichiometry of contraction and Ca²⁺ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP₃). The contractile response to InsP₃ was prompt (peak < 5 s), concentration-dependent (EC₅₀-0.3 μM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP₃ or CCK-8 was accompanied by a concentration-dependent increase in free Ca²⁺ that was directly correlated with the magnitude of contraction. Both InsP₃ and CCK-8 caused rapid net efflux of Ca²⁺ from cells preloaded with ⁴⁵Ca²⁺. Contraction, increase in free Ca²⁺ concentration, and net ⁴⁵Ca²⁺ efflux elicited by a combination of maximal concentrations of InsP₃ and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP₃ or CCK-8 in Ca²⁺-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca²⁺, implying equal access of InsP₃ and receptor-linked agonists to the same intracellular Ca²⁺ store. The results demonstrate that InsP₃ mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca²⁺ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP₃ as membrane-derived messenger responsible for mobilization of intracellular Ca²⁺ in smooth muscle cells.