[3H]pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes.
Studies show [3H]PZ identified selectively a subpopulation of muscarinic binding sites compared to classical antagonists like (-)-[3H]QNB in many central and peripheral tissues. We characterized the binding and regulation of selected antagonists to high-affinity [3H]PZ (putative M1) and low-affinity PZ (putative M2) sites in rat cerebral cortex (predominantly M1) and heart (predominantly M2). Saturation isotherms of [3H]PZ and (-)-[3H]QNB were performed under various conditions. Guanyl-5'-yl-imidodiphosphate (30 microM) showed little effect on Kd (dissociation constant) or total binding capacity (total receptor density) values. Higher ionic strength buffers yielded lower affinity values for [3H]PZ and (-)-[3H]QNB. Kinetic studies confirmed high affinity Kd values seen in steady-state assays. We conducted inhibition studies of selected muscarinic antagonists including the reportedly cardioselective (putative M2) drug, AF-DX 116 (11-[(2-(diethylamino)methyl-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the reportedly M1 selective compound, PZ, and the classical antagonist (-)QNB, using [3H]PZ and (-)-[3H]QNB-labeled cerebral cortical and cardiac homogenates. Assays were done with and without guanyl-5'-yl-imidophosphate at 25 degrees C in 10 mM Na-K-phosphate, 50 mM Na-K-phosphate and modified Krebs-phosphate buffer. Studies showed antagonists generally had higher affinity in 10 mM Na-K-phosphate buffer, were insensitive to guanyl-5'-yl imidodiphosphate and had Hill values (nH) nearly equal to one. Cardiac PZ/[3H]QNB curves were steep.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001570:Benzodiazepinones / Q000378:metabolism*; D002021:Buffers; D002540:Cerebral Cortex / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010276:Parasympatholytics / Q000378:metabolism*; D010890:Pirenzepine; D011812:Quinuclidines / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000032:analysis / Q000378:metabolism*; D014316:Tritium
D001570:Benzodiazepinones; D002021:Buffers; D010276:Parasympatholytics; D011812:Quinuclidines; D011976:Receptors, Muscarinic; D014316:Tritium; D006165:Guanylyl Imidodiphosphate; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D005033:Ethylmaleimide