Phosphoinositide metbolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentialy recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but signifiant proportion of PtdInsP₂-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP₂ (diacylglycerol kinase and PtdInsP kinase) also were present. CTP: phosphatidate cytidylytransferase and CMP-phosphatidate: inositol phosphatidyltransferase were located exclusively in a microsoma fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol form agonist-stimulated cleavage of PtdInsP₂, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP₂ completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP₂ phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PydInsP₂ in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP₂ in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.