Ca2+-dependent elevation of inositol 1,4,5-trisphosphate level induced by freezing or homogenization of tissues and cells

Various cells and tissues contain high basal levels of mositol 1,4,5-trisphosphate, raising questions about the functional significance of inositol 1,4,5-trisphosphate in some tissues such as the heart. We used intact tissue and isolated cells from heart and liver of adult rats to examine if different fixation procedures might artificially elevate the level of inositol 1,4,5-trisphosphate. The basal level of mositol 1,4,5-trisphosphate in intact, freeze-clamped cardiac tissue from adult rats was 10 times higher than in isolated, non-frozen cardiomyocytes, while freeze-clamped liver contained approximately 4 times higher mositol 1,4,5-trisphosphate levels than isolated, non-frozen hepatocytes. Stimulation with norepinephrine induced a significant increase in the inositol 1,4,5-trisphosphate level in isolated cardiomyocytes, whereas no significant increase was observed in freeze-clamped cardiac tissue. Freezing of isolated cardiomyocytes or hepatocytes before extraction increased basal inositol 1,4,5-trisphosphate levels 3 times. In cellular homogenates prepared in the presence of EGTA and stored at 4degrees, readdition of calcium resulted in a time-dependent increase in inositol 1,4,5-trisphosphate mass and a decrease in the mass of phosphatidylinositol 4,5-bisphosphate (PIP2). The reaction was essentially complete within 30 sec. in homogenates from cardiomyocytes, while PIP2 hydrolysis was slower in hepatocyte homogenates. Perfusion of intact rat hearts with EGTA present during the last 2 min. of perfusion, followed by freeze-clamping, resulted in basal mositol 1,4,5-trisphosphate levels comparable to those in isolated cardiomyocytes, and norepinephrine stimulation increased inositol 1,4,5-trisphosphate mass by approximately 80%. The presence of EGTA did not significantly affect PIP2 levels in perfused hearts. The results suggest that freezing or homogenization of intact tissue and isolated cells may result in Ca2+-dependent activation of phospholipase C, leading to high basal mositol 1,4,5-trisphosphate levels that may mask agonist-induced changes.