title: Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding.
abstract: This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [3H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. 13C nuclear magnetic resonance (NMR), 1H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [3H]DHP binding to rat cortex membranes with an IC50 value of congruent to 15 microM and a Hill coefficient of congruent to 2. Saturation analysis in the presence of SLBHI showed a change in KD (equilibrium dissociation constant), but not maximal binding capacity (Bmax). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interaction with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.
authors.affiliation: Miles Inc., Miles Research Center, West Haven, CT 06516.
authors.forename: D E
authors.initials: DE
authors.lastname: Johnson
authors.forename: S L
authors.initials: SL
authors.lastname: Heald
authors.forename: R D
authors.initials: RD
authors.lastname: Dally
authors.forename: R A
authors.initials: RA
authors.lastname: Janis
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000138:chemical synthesis / Q000302:isolation & purification* / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000037:antagonists & inhibitors*; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D004095:Dihydropyridines / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D006352:Heart Ventricles / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D053284:Polyunsaturated Alkamides; D051381:Rats
chemical_list: D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; C038806:1,4-dihydropyridine; C078814:anandamide