anandamide and 1-4-dihydropyridine

This article represents a timely opportunity to express my affection, admiration and gratitude to Professor David Triggle. David was my Ph.D. advisor as well as a key consultant in the 1980s and early 1990s for research programs at Miles Institute for Preclinical Pharmacology in West Haven, CT, the U.S. research operation of Bayer AG, in the areas of Ca(2+) and K(+) channel ligands. The binding methodology developed in his laboratory was used to search for an endogenous ligand for L-type Ca(2+) channels. We did not find the substance that we were searching for, a genetically-determined, competitive inhibitor for the 1,4-dihydropyridine binding site, but instead isolated the endogenous ligand for the brain's own marijuana, anandamide. Devane, Mechoulam and coworkers first discovered that this compound was the endogenous ligand for delta-9-tetrahydrocannabinol, the active substance in cannabis. The endogenous endocannabinoid system is now the target of many exciting new approaches to drug discovery.

Topics: Academies and Institutes; Animals; Arachidonic Acids; Brain; Calcium Channels; Cooperative Behavior; Dihydropyridines; Drug Discovery; Endocannabinoids; History, 20th Century; History, 21st Century; Humans; Ligands; Polyunsaturated Alkamides; Potassium Channels; Research; United States

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [3H]PN-200. AEA competed for [3H]PN-200 binding with a K(I) of 40 +/- 4 microM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K(I) of AEA (48 +/- 6 microM). Arachidonic acid had a slightly higher K(I) (120 +/- 11 microM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-gamma-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [3H]PN-200 binding at concentrations as high as 10 microM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Binding Sites; Binding, Competitive; Brain; Cell Membrane; Dihydropyridines; Endocannabinoids; Ethanolamines; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Tosyl Compounds

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.

Topics: Animals; Arachidonic Acids; Calcium Channel Blockers; Cattle; Cerebral Cortex; Chemical Fractionation; Chromatography, High Pressure Liquid; Dihydropyridines; Dose-Response Relationship, Drug; Endocannabinoids; Heart Ventricles; Magnetic Resonance Spectroscopy; Mass Spectrometry; Polyunsaturated Alkamides; Rats