Abstract
Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.
Highlights
Amount for N18TG2 Cells pmoles per107 cells
Palmitic acid, 6-thioguanine, and fatty acid-free BSA (BSA) were from Sigma; elaidic acid and palmitoleic acid were from Fisher; penicillin/streptomycin, EMEM, and DMEM were supplied by Mediatech Cellgro; fetal bovine serum (FBS) was from Atlanta Biologicals; BSTFA and silica were from Suppelco; [2H33]heptadecanoic acid (margaric acid, C2H3-(C2H2)15-COOH) was from C/D/N isotopes; goat ant-fatty acid amide hydrolase (FAAH) (V-17) antibody and its blocking peptide were from Santa Cruz Biotech; donkey antigoat IgG fused to horse radish peroxidase (HRP) was from ICN Biomedical; SuperSignal chemiluminescent HRP substrate was from Bio-Rad; and MagicMark XP molecular weight markers were from Invitrogen
Our study is the first report of the conversion of free fatty acids to their respective Primary fatty acid amides (PFAM) in N18TG2 cells, and it is the first finding of the other endogenous PFAMs in N18TG2 cells
Summary
Amount for N18TG2 Cells pmoles per107 cells. Amount for SCP Cells pmoles per107 cells 410 ± 450To facilitate comparison of these data to the endogenous PFAM levels in the N18TG2 and SCP cells and to the cellular production data, the moles of amide found in these blanks was divided by an average number of viable cells (1.24 × 107 for SCP or 7.9 × 107 for N18TG2). Amount for N18TG2 Cells pmoles per107 cells. Background levels of elaidamide (trans-9-octadecenic acid) are most likely ف0 [3]. Incubation of the N18TG2 cells for 0, 12, 24, or 48 h in the presence of exogenously added fatty acids led to continued production of the corresponding PFAM (Table 4 and Fig. 2A, C). Tridecanamide was produced by the N18TG2 cells from two precursors added to the cell culture medium, tridecanoate and N-tridecanoyethanolamine (Table 4 and Fig. 3A, C). Palmitamide was the most abundant PFAM after incubation of the N18TG2 cells with palmitic acid. Palmitoleamide was the most abundant PFAM, followed by elaidamide, oleamide, linoleamide, and tridecanamide after 48 h incubation with the corresponding free fatty acid
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