Methods for Studying Phosphoinositide Metabolism in Cultured Cells

Abstract

In the light of the current focus in scientific research on the role of the phosphoinositides in transmembrane signaling, techniques describing the incorporation of tritiated inositol into the phosphoinositides and inositol phosphates of tissue culture cell lines, and their analysis, are described in this chapter. The phosphoinositide pathway is involved in many aspects of cellular life during the life cycle of the cell including cell growth, differentiation and transformation. It has been implicated in gametogenesis, fertilization, neuromodulation and sensory perception (Michell et al. 1989). In basic terms this signaling route involves the phospholipase C (PLC) catalyzed cleavage of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P₂), yielding two second messengers, inositol(1,4,5)-trisphosphate (Ins(1,4,5)P₃), which acts to release Ca²⁺ from internal Ca²⁺ pools, and diacylglycerol (DAG), which activates protein kinase C (PKC) (Fig. 10.1). These second messengers are responsible for mediating the intracellular consequences of first messengers (e.g., hormones, growth factors, neurotransmitters, agonists; i.e., extracellular molecules) binding to the plasma membrane. These two messenger systems are linked by receptor complexes located in the plasma membrane of the target cell.