oleylamide and Neuroblastoma

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.

Topics: Amides; Animals; Cells, Cultured; Choroid Plexus; Ethanolamine; Ethanolamines; Fatty Acids; Fatty Acids, Monounsaturated; Linoleic Acids; Mice; Neuroblastoma; Oleic Acids; Palmitic Acids; Sheep; Sheep, Domestic; Tumor Cells, Cultured

Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Culture Media; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Glycine; Mice; Mixed Function Oxygenases; Multienzyme Complexes; Neuroblastoma; Oleic Acid; Oleic Acids; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity

cis-9,10-Octadecenoamide (cOA) accumulates in cerebrospinal fluid during sleep deprivation and induces sleep in animals, but its cellular actions are poorly characterized. In earlier studies, like a variety of anesthetics, cOA modulated gamma-aminobutyric acidA receptors and inhibited transmitter release/burst firing in cultured neurones or synaptoneurosomes.. Here, radioligand binding ([3H]batrachotoxinin A 20-alpha-benzoate and mouse central nervous system synaptoneurosomes) and voltage clamp (whole cell recording from cultured NIE115 murine neuroblastoma) confirmed an interaction with neuronal voltage-gated sodium channels (VGSC).. cOA stereoselectively inhibited specific binding of toxin to VGSC (inhibitor concentration that displaces 50% of specifically bound radioligand, 39.5 microm). cOA increased (4x) the Kd of toxin binding without affecting its binding maximum. Rate of dissociation of radioligand was increased without altering association kinetics, suggesting an allosteric effect (indirect competition at site 2 on VGSC). cOA blocked tetrodotoxin-sensitive sodium currents (maximal effect and affinity were significantly greater at depolarized potentials; P < 0.01). Between 3.2 and 64 microm, the block was concentration-dependent and saturable, but cOA did not alter the V50 for activation curves or the measured reversal potential (P > 0.05). Inactivation curves were significantly shifted in the hyperpolarizing direction by cOA (maximum, -15.4 +/- 0.9 mV at 32 microm). cOA (10 microm) slowed recovery from inactivation, with tau increasing from 3.7 +/- 0.4 ms to 6.4 +/- 0.5 ms (P < 0.001). cOA did not produce frequency-dependent facilitation of block (up to 10 Hz).. These effects (and the capacity of oleamide to modulate gamma-aminobutyric acidA receptors in earlier studies) are strikingly similar to those of a variety of anesthetics. Oleamide may represent an endogenous ligand for depressant drug sites in mammalian brain.

Topics: Animals; Batrachotoxins; Brain; Cells, Cultured; Hypnotics and Sedatives; Male; Mice; Neuroblastoma; Oleic Acids; Patch-Clamp Techniques; Receptors, GABA; Sodium Channels

The fatty-acid primary amide, oleamide, is a novel signaling molecule whose mechanism of biosynthesis is unknown. Recently, the N(18)TG(2) cell line was shown to synthesize oleamide from oleic acid, thereby demonstrating that these cells contain the necessary catalytic activities for generating the fatty-acid primary amide. The ability of peptide alpha-amidating enzyme, peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3), to catalyze the formation of oleamide from oleoylglycine in vitro suggests this as a function for the enzyme in vivo. This investigation shows that N(18)TG(2) cells, in fact, express PAM and that cellular differentiation dramatically increases this expression. PAM expression was confirmed by the detection of PAM mRNA, PAM protein, and enzymatic activity that exhibits the functional characteristics of PAM isolated from mammalian neuroendocrine tissues. The regulated expression of PAM in N(18)TG(2) cells is consistent with the proposed role of PAM in the biosynthesis of fatty-acid primary amides and further establishes this cell line as a model for studying the pathway.

Topics: Animals; Base Sequence; Cell Differentiation; DNA Primers; Enzyme Induction; Mice; Mixed Function Oxygenases; Models, Biological; Multienzyme Complexes; Neuroblastoma; Oleic Acids; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Cis-9,10-octadecenoamide (oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The enzyme catalyzing the hydrolysis of the amide bond of oleamide as well as of anandamide, the putative endogenous ligand of cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named fatty acid amide hydrolase (FAAH). The enzymatic synthesis of oleamide from oleic acid and ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of oleamide, necessary in order to postulate for this compound a role as a neuromodulator. Here we show for the first time that oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of oleic acid and ammonia. A lipid metabolite was extracted and purified from mouse neuroblastoma N18TG2 cells through a sequence of chromatographic steps and characterized as oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic oleamide, was 55.0+/-09.5 pmols/10(7) cells, compared to less than 0.7 pmol/10(7) cells for anandamide in the same cells. When N18TG2 cells were prelabeled with [14C]oleic acid and the lipids extracted and purified, a radioactive component with the same chromatographic behavior as oleamide was found whose levels: (1) were not significantly influenced by stimulation with ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF); (3) appeared to correlate with [14C]oleic acid incorporation into phospholipids but not with free [14C]oleic acid levels. N18TG2 cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of oleamide from oleic acid and ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and methylarachidonoylfluorophosphonate, as well as by an excess of anandamide, and by PMSF at the same concentration which increased oleamide formation in intact cells. These data suggest that a FAAH-like enzyme working "in reverse" may be responsible for the formation of oleamide in cell-free preparations but not in whole cells.

Topics: Animals; Arachidonic Acids; Cannabinoids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Endocannabinoids; Gas Chromatography-Mass Spectrometry; Mice; Neuroblastoma; Oleic Acids; Polyunsaturated Alkamides; Sleep; Tumor Cells, Cultured