FATTY ACID AND PEPTIDE AMIDATION--A SHARED MECHANSIM

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DESCRIPTION: (Adapted from Applicant's Abstract ) Sleep is ubiquitous among mammals and essential for life. More than seventy types of sleep disorders chronically affect millions of Americans in all age groups. Recently, it was shown that the primary fatty-acid amide, oleamide, is a mediator of sleep and may thus contribute to the genesis of sleep disorders. Oleamide accumulates in cerebral spinal fluid during sleep deprivation and induces profound motor quiescence and a sleep-like state upon administration. Primary fatty-acid amides represent a new class of receptor-active signaling molecules whose biogenesis is unknown. Understanding the pathway involved will offer targets for the therapeutic interventions for treating sleep disorders. Recently, we established in vitro that peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is able to catalyze the formation of oleamide. PAM is known for its role as the rate-limiting enzyme in the production of peptide messengers and may thus regulate the production of primary fatty-acid amides as well. The objective of this research is to elucidate the mechanisms that govern oleamide production in brain. Three specific aims are proposed for testing the hypothesis that PAM is the physiologic mediator of oleamide biosynthesis. Aim 1. Establish in a cell-free system that oleamide biosynthesis is dependent upon the actions of PAM. Cell-free models permit the direct investigation of fundamental reaction components. Subcellular fractions of N18TG2 mouse neuroblastoma cells, a line which expresses PAM and synthesizes oleamide, will be used to determine the effects of PAM inhibition on the biosynthesis of oleamide. Aim 2. Establish in cell culture that the induction of PAM during neuronal differentiation mediates an increase in oleamide production. Antisense RNA inhibition of PAM expression will be used to directly establish PAM as an integral component of the oleamide biosynthesis pathway in whole cells. Aim 3. Establish that the production of oleamide in vivo is dependent upon the action of PAM. Experiments conducted in rats will take into account the full biologic complexity of the mammalian organism. Direct measures of oleamide in brain and assessments of tissue activity for oleamide biosynthetic labeling will be used to demonstrate the extent to which inhibition of PAM in vivo impairs oleamide biosynthesis. These studies will define the physiologic role of PAM in mediating the biosynthesis of oleamide. Demonstrating that oleamide biosynthesis proceeds through PAM, or via an alternative pathway, will provide a basis for improving the diagnosis and treatment of sleep disorders.