Inositol 1,4,5-Trisphosphate-induced Increase in Ca²⁺-ATPase Activity in the Microsomes of Tracheal Epithelial Cells

Inositol 1,4,5-Trisphosphate-induced Increase in Ca²⁺-ATPase Activity in the Microsomes of Tracheal Epithelial Cells

Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was 329±10 nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular Ca²⁺-ATPase, inhibited about 50% of the activity, leaving 178±18 nmol/min .mg protein (n=6), indicating that the Ca²⁺-ATPase is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in <Sup>45</sup>Ca²⁺ uptake experiments. Inositol 1,4,5-trisphosphate InsP₃, 4 μM, an agonist of InsP_{3}-sensitive Ca²⁺ release channel (InsP₃, receptor), and Ca-ionophore A23187 (10 μM) induced <Sup>45</sup>Ca²⁺ releases of 20% and 50% of stored <Sup>45</sup>Ca²⁺, respectively. The addition of (10 μM InsP₃ also increased the microsomal ATPase activity from 282±8 nmol/min mg protein to 334±21 nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ;InsP₃-induced effects were blocked by either thapsigargin or heparin suggesting that: 1) the InsP₃-induced increase in ATPase activity is mediated by microsomal Ca²⁺-ATPase, and 2) dissipation of Ca²⁺ gradient across the microsomal membrane is responsible for the InsP₃-induced effect. In order to test the dependence of the Ca²⁺-ATPase activity on the activity of InsP₃-induced the activity of ATPases was monitored in various concentrations of free Ca²⁺ using EGTA-Ca²⁺ buffers. The Ca²⁺-dependent biphasic change is the well-known character of InsP₃ receptor but not of microsomal Ca²⁺-ATPase in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of 397±36nmol/min .mg protein was obtained in the solution containing 100 nM free Ca²⁺. Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal Ca²⁺-ATPase to the InsP₃ receptors in epithelial microsomes.