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-t ype calcium channels. The screening for active fractions was done by m easuring inhibition [H-3]1,4-dihydropyridine (DHP) binding to rat card iac and cortex membranes. An inhibitory fraction, termed lyophilized b rain hexane-extractable inhibitor (LBHI), was isolated from hexane ext racts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. C-13 nuclear magnetic resonance (NM R), H-1 coherence spectroscopy (COSY) NMR and fast atom bombardment (F AB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hyd roxyethylamide. Synthesis of this substance and subsequent high perfor mance liquid chromatography (HPLC) and NMR analysis confirmed this str ucture. Synthetic LBHI (SLBHI) inhibited [H-3]DHP binding to rat corte x membranes with an IC50 value of congruent-to 15 muM and a Hill coeff icient of congruent-to 2. Saturation analysis in the presence of SLBHI showed a change in K(D) (equilibrium dissociation constant), but not maximal binding capacity (B(max)). SLBHI produced an increased dissoci ation rate, which, along with the Hill slope of > 1, suggested a non-c ompetitive interaction with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the D HP calcium antagonist binding site.