When a membrane preparation, obtained by freezing and thawing nerve endings labeled by preincubation with 32pi, is incubated in the presence of millimolar Ca2+, there is a rapid and selective loss of label from the polyphosphoinositides and a concomitant increase in labeled inositol di- and triphosphates recovered. When the membranes are not prelabeled and are exposed to [gamma-32P]ATP under similar conditions, phosphatidate labeling is enhanced, indicating increased availability of diacylglycerol. These observations provide evidence for the presence of membrane-bound, Ca2+-stimulated phosphodiesterase activity (phospholipase C) acting on endogenous polyphosphoinositides. The implications of these findings are discussed in respect to the "phosphatidylinositol" cycle.

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.

No abstract

Two methods for the measurement of receptoractivated phosphoinositide turnover were evaluated for their degree of correspondence in slices of rat brain; they involved the Li+-dependent accumulations of either [3H]-inositol-labeled inositol phosphates or [3H]cytidine-labeled CDP-diacylglycerol. In contrast to the expectation that the ratio of these two responses would remain approximately constant, varying degrees of correspondence were obtained. The two extremes are exemplified by carbachol, which elicited large increases in both inositol phosphate and CDP-diacylglycerol labeling, and endothelin, which gave a robust inositol phosphate response with little or no accumulation

A method is described for the preparation of radioactive inositof lipids for studies of their enzymic degradation. Kidney cytosol fractions have been used to produce diesteratic cleavage. High voltage el~~troph~resis at pM 4.3 is used to separate D-myoinositol 1 : 2-cyclic phosphate and ~-myoinosit~l l-phosphate from hydrolysis of pbos~hatidylinositol. Radioactivity ho-rni~ati~~ with myoinositol diph~~phat~ and triphosphat~ is separated by ele~tropho~es~s at pH 1.5 following enzymatic hydrolysis of ph~sphatidylinositol phosphate and phosphatidylinositol diphosphate. Relative activities for hydrolysis of the various inositides suggest the presence of more than one phosphodiesterase.