The purpose of this project is to continue work carried out by our group over the past 13 years on the metabolic properties of the myelin sheath. Present emphasis is on the role of receptors which produce second messengers within the myelin complex, and the enzymatic reactions involved in the formation and inactivation of such messengers. Following our observations on receptor-mediated activation of phosphoinositide phosphodiesterase and adenylyl cyclase, we plan to follow up preliminary observations myelin also contains guanylyl cyclase activity leading to the synthesis of two products: 3',5'-cGMP and 2',3'-cGMP. The latter is of special interest because it is potentially the in situ substrate for myelin-associated 2',3'-cyclic nucleotide phosphodiesterase (CNP); the function of the latter enzyme has been one of the long-standing mysteries in the myelin field. We plan to prove unequivocally that this is the product formed, and to characterize as fully as possible the kinetic and related properties of the two guanylyl cyclases. In the case of the cyclase catalyzing formation of the 3',5' isomer, which we hypothesize to be a second messenger, we shall attempt to find an agonist that activates the enzyme; this will involve incubation of myelin with [3H]GTP and various agonists (e.g.. natriuretic peptides, prostaglandin, angiotensin) that have been shown to activate a similar enzyme in other systems. It is also possible that the 2',3' isomer is formed as a second messenger, but that will not be pursued during the coming period. An additional signal transduction mechanism which we have discovered to occur in myelin is phosopholipase D-catalyzed cleavage of phosphatidylcholine, leading to phosphatidic acid. We shall investigate the enzymology of that reaction, explore possible agonists that trigger it, and determine whether the final product is phosphatidic acid or diacylglycerol; the latter is a possibility to be considered since it is known that myelin contains phosphatidate phosphohydrolase. Finally, we shall investigate two enzymes involved in the phosphoinositide cycle to determine whether this cycle occurs in a manner requiring concerted action of both axon and myelin.
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