Phosphoinositidase C Activation Assay I: Cell Labeling, Stimulation, and Recovery of Cellular [3H]Phosphoinositides and [3H]Phosphoinositols

The minor inositol-containing membrane phospholipids, the phosphoinositides, play a central role in cell signal transduction. Activation of a hormone-sensitive phospholipase C (phosphoinositidase C) at the plasma membrane results in the rapid catabolism of the polyphosphoinositides to form the two second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P
3
), a water soluble phosphoinositol that promotes the release of Ca
2+
from intracellular stores, and diacylglycerol (DG), which remains in the plasma membrane and activates protein kinase C (
1
–
3
). The metabolic pathways involved in the synthesis of phosphatidylinositol 4,5-bisphosphate, and the metabolic fate of the DG and Ins(1,4,5)P
3
formed on activation of phosphoinositidase C, are summarized in Fig 1 .Fig. 1.
Metabolic pathways activated as a consequence of phosphoinositidase C action. (
A
) Major metabolic pathways activated by phosphoinositidase C action on PtdIns(4,5)P
2
are shown with solid arrows. Some of the additional pathways that may be activated are shown by broken arrows. Abbreviations: PtdIns, phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P
2
, phosphatidylinositol 4,5-bisphosphate; DG, diacylglycerol; PtdOH, phosphatidic acid; CDP-DG, CDP-diacylglycerol; Ins, Inositol. For phosphoinositols, abbreviations are in the form Ins(
x,y,z
)Pn, where
x, y
, and
z
refer to the positions of the phosphate groups on the myo-inositol ring and
n
refers to the total number of phosphates. (
B
) A simplified outline of the metabolic pathways in (A) also showing alternative abbreviations. PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP
2
, phosphatidylinositol
bis
-phosphate; DG, PtdOH, CDP-DG and Ins as above. Phosphoinositols are referred to as IPn , where
n
refers to the number of phosphates on the inositol ring. In both panels, sites of Li
+
inhibition are also shown.