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Radioligand binding to muscarinic receptors of bovine aortic endothelial cells
Author(s) -
Brunner Friedrich,
Kukovetz Walter R.
Publication year - 1991
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1991.tb12181.x
Subject(s) - muscarinic acetylcholine receptor , radioligand , pirenzepine , chemistry , acetylcholine , binding site , stereochemistry , receptor , radioligand assay , biophysics , biochemistry , biology , endocrinology
1 Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (−)‐[ 3 H]‐N‐methyl quinuclidinyl benzilate ([ 3 H]‐NMeQNB) was used as radioligand. 2 Binding of [ 3 H]‐NMeQNB to crude membranes of freshly isolated endothelial cells was atropine‐displaceable and of high affinity ( K D = 0.48 n m ) 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 [ 3 H]‐NMeQNB binding was inhibited by dexetimide in the nanomolar range ( K I = 0.63 n m ) and by levetimide, its stereoisomer in the micromolar range ( K I = 3.2 μ m ) (selectivity factor: ∼5000). 3 Drug competition curves indicated a single class of binding sites for antagonists and the following apparant affinities ( K I , n m ): 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 [ 3 H]‐NMeQNB was best described by two affinity sites (or states) ( K H = 0.82 μ m , K L = 1.6 μ m ). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 μ m ), acetylcholine affinity (IC 50 ) was slightly, but significantly reduced (factor ∼4). 4 Binding of [ 3 H]‐NMeQNB to freshly harvested intact cells was also atropine‐displaceable, stereospecific (selectivity factor: ∼3500) and of high affinity ( K D = 0.35 n m ). 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 M 1 subtype and is of comparatively very low density, and (3) cultured endothelial cells lose their receptors during isolation or culture procedures.