The involvement of phosphoinositides (PI, PI-P and PI-P2) in receptor-mediated stimulus-response coupling has been well-demonstrated in brain. Recent studies have provided evidence that this response is initiated by interaction of the stimuli with a phosphodiesterase specific for hydrolysis of PI-P2. The main objective of the investigation is to examine the biochemical properties of PI-P2 phosphodiesterase and the products formed after stimulated hydrolysis of the polyphosphoinositides (poly-PI). Inositol phosphates formed will be analyzed after separation by Dowex-1 AG x 4 column chromatography, and Li+ will be used to block the hydrolysis of inositol phosphates. (C14)-arachidonoyl-diacylglycerol (DG) will be generated in plasma membranes by converting labeled PI and poly-PI to DG using an in vitro assay system. The DG-labeled membranes will be used to study the DG lipase and DG kinase activities and any factors which may regulate the metabolism. Studies of the cytosolic and membrane-bound phosphoinositide phosphodiesterase will be carried out using a plasma membrane fraction containing phosphoinositides labeled with (P32), (C14)-arachidonate, and (H3)-inositol. Cytosolic and solubilized enzyme(s) will be studied using heat-treated prelabeled membrane as substrate. Specific experiments are designed to examine the substrate specificity, response to detergent and amphiphiles, Ca++ dependency, and to assess whether the membrane and cytosolic reactions are mediated by the same or different enzymes. In addition, the response of isolated plasma membranes to muscarinic cholinergic agonists in mediating poly-PI breakdown will be assessed and compared to the results obtained with brain slices. Conventional chromatographic methods will be used to purify both cytosolic and membrane-bound enzyme(s) from bovine brain and to compare their properties and substrate specificity. The effects of carbamylcholine stimulation and atropine blocking on metabolism of phosphoinositides in vivo will be examined in young rats after prelabeling brain with Pi32 or H3-inositol. Results obtained from this project are expected to provide new information regarding phosphodiesterase involvement in the stimulus-mediated response. Furthermore, new insights will be obtained regarding the physiological significance of the cyclic event involving phosphoinositides, phosphatidic acids, and DG in the central nervous system.
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