Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

Abstract

1 Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (−)-[³H]-N-methyl quinuclidinyl benzilate ([³H]-NMeQNB) was used as radioligand. 2 Binding of [³H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (K_D = 0.48 nm) to a single class of sites (maximum binding capacity: 14 ± 3 fmol mg⁻¹ protein). Stereospecificity of the binding sites was demonstrated in experiments in which [³H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (K_I = 0.63 nm) and by levetimide, its stereoisomer in the micromolar range (K_I = 3.2 μm) (selectivity factor: ∼5000). 3 Drug competition curves indicated a single class of binding sites for antagonists and the following apparant affinities (K_I, nm): methyl atropine: 1.1; 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-(diethylamino-methyl)-1-piperidinyl-acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116): 2.500. Competition of acetylcholine with [³H]-NMeQNB was best described by two affinity sites (or states) (K_H = 0.82 μm, K_L = 1.6 μm). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 μm), acetylcholine affinity (IC₅₀) was slightly, but significantly reduced (factor ∼4). 4 Binding of [³H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: ∼3500) and of high affinity (K_D = 0.35 nm). The maximum binding capacity (9 ± 2 fmol mg⁻¹ total cell protein) was comparable to that of membranes and corresponded to ∼900 binding sites per endothelial cell. Binding to enzymatically harvested and cultured endothelial cells, or membranes derived therefrom, showed no atropine-displaceable binding. 5 The results suggest that (1) bovine aortic endothelial cells contain muscarinic binding sites with all necessary criteria of functional muscarinic receptors; (2) the receptor most closely corresponds to the M₁ subtype and is of comparatively very low density, and (3) cultured endothelial cells lose their receptors during isolation or culture procedures.