Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding.
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.
Johnson
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DE
Miles Inc., Miles Research Center, West Haven, CT 06516.
Heald
S L
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Dally
R D
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Janis
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RA
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
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; C038806:1,4-dihydropyridine; C078814:anandamide