The primary fatty-acid amide, oleamide, is representative of a newly discovered, highly novel class of intercellular messengers whose actions are mediated by specific receptor mechanisms. Oleamide was originally discovered as an agent that accumulates in the cerebral spinal fluid of cats during sleep deprivation. Subsequently, it was shown to induce sleep/sedation following either central or peripheral administration. Currently, intense investigations focus on the mechanisms by which oleamide induces sleep. Findings reported to date indicate that oleamide regulates sleep/wake cycles by potentiating the serotonergic neurotransmission. This effect is mediated through the direct action of oleamide on the serotonin receptor protein. Oleamide is also reported to alter cellular communication by potentiating information transfer across gap junctions. The broader importance of primary fatty-acid amide messengers is illustrated by the clinical effectiveness of several primary fatty-acid amides as anxiolytics and antipsychotics. While the precise mechanism(s) by which oleamide acts is being elucidated, its pathway of biosynthesis remains unknown. The goal of this project is to define the physiologic mechanisms for oleamide production. This research is predicated upon the principal investigator's published report that alpha-amidating enzyme [a-AE: peptidylglycine alpha-amidating monooxygenase (PAM)], is capable of generating the formation of the primary fatty-acid amide, myristamide, from myristoylglycine. Importantly, the reaction occurs in a fashion analogous to the generation of peptide amides by a-AE. This seminal observation has been extended by Dr. Merkler's preliminary findings showing that a-AE can also generate oleamide and several other fatty-acid amides from their respective fatty acylglycine precursors. Prior to these findings, a-AE was only recognized for its ability to catalyze the essential and rate limiting step in the bioactivation of peptide messengers. The finding that a-AE is able to catalyze the formation of fatty-acid and well as peptide amides raises the possibility that the same enzyme regulates the formation of two distinct classes of bioactive messengers that coordinate vital body functions including sleep and virtually all neuroendocrine function. Proving that a-AE mediates the biosynthesis of primary fatty-acid amides in vivo would have far reaching implications for understanding the regulation and functions of all amidated cellular messengers. The research proposed here is designed to determine the physiologic importance of a-AE in mediating the amidation oleamide. The experimental approach is focused upon the murine N(18)TG(2) neuroblastoma cell line which synthesizes oleamide from oleic acid, produces alpha-amidated peptides and, as shown in supplemental materials submitted here, to express the a-AE gene.
Three specific aims are proposed to determine if a-AE protein, indeed, mediates the formation of oleamide in N(18)TG(2) cells. The first specific aim will determine the extent to which N(18)TG(2) a-AE is capable of catalyzing the formation of fatty-acid amides from their respective N-acylglycine derivatives. To accomplish this goal, Dr. Merkler will synthesize radiolabeled N-oleoylglycine, N- isovaleroylglysine and N-lauroglycine substrates using enzymatic procedures he has established. He will then evaluate the ability of a-AE prepared from N(18)TG(2) cells to catalyze their conversion to primary fatty-acid amides. The kinetic properties of N(18)TG(2) a- AE well be determined for each substrate and compared to those obtained with purified recombinant a-AE catalyzing the same reactions. The second Specific Aim will determine if pharmacologic inhibition of a-AE in N(18)TG(2) cell leads to a decrease in oleamide production and a corresponding accumulation of N- oleoylglycine precursor. This demonstration will indicate the existence of a precursor-product relationship between oleylglycine and oleamide and reveal a-AE as mediating the intervening step. The third specific aim will establish a mechanism by which the oleamide substrate, N-oleylgycine, is synthesized in N(18)TG(2) cell. Reverse transcriptase-polymerase chain reactions (RT-PCR) will be used to demonstrate the co-existence of a long chain, acyl- CoA:glycine N-acyltransferase (ACGNAT) together with a-AE in N(18)TG(2) cells. Accomplishing this goal will establish the role of a-AE and oleoylglycine in the biosynthesis oleamide in the mammalian cells of neural origin.
