Myo-inositol does not modulate PI turnover in MDCK cells under hyperosmolar conditions

Abstract

The relationship between free cellular myo-inositol concentration and phosphatidylinositol turnover was evaluated in Madin-Darby canine kidney (MDCK) cells under isosmolar and hyperosmolar conditions. MDCK cells exposed to high extracellular sodium chloride were documented to increase their free myo-inositol content as measured by gas-liquid chromatography in both a time- and concentration-dependent manner. Measurement of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate mass failed to reveal changes under conditions where the myo-inositol concentration was more than threefold higher compared with control conditions. CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase activity, measured in plasma membranes from MDCK cells grown under control and hyperosmolar conditions, was kinetically similar with comparable observed Michaelis constant (Km) and maximal rate of enzyme reaction. Moreover, the apparent Km was significantly below the estimated intracellular myo-inositol concentration consistent with exposure of the transferase to saturating concentrations of myo-inositol under both conditions. Finally, bradykinin-stimulated myo-inositol trisphosphate mass was measured by use of a competitive binding assay. Both basal and hormone-stimulated myo-inositol 1,4,5-trisphosphate levels were not significantly different under control and hyperosmolar conditions. These data indicate that bulk free myo-inositol content is unlikely to regulate phosphatidylinositol turnover and myo-inositol trisphosphate formation under hyperosmolar conditions.