Hormone-regulated phosphoinositide turnover in permeabilized cells and membranes

This chapter discusses the methods for studying hormone-regulated phosphoinositide turnover in permeable cells and in membranes. There are a large number of methods for rendering animal cells permeable to small- or large-molecular-weight probes. However, for the studies of hormone-regulated phospholipid turnover, there is the stringent requirement that membrane-associated receptors, enzymes, and phospholipid substrates remain functionally intact. Hence, methods that employ detergents (e.g., saponin) might be damaging, as it is found for TRH responsive GH 3 cells (unpublished results) and thrombin-responsive platelets. Electric field-induced permeabilization of cells was extensively characterized for studying Ca 2+ -dependent secretion in adrenal chromaffin cells. Electric field-induced permeabilization is based on the fact that a transmembrane potential difference exceeding 0.5–1 V results in the mechanical breakdown of lipid bilayers leading to a state of high permeability. The chapter describes the measurement of regulated phosphoinositide turnover in permeable cells.