PHARMACOLOGICAL CHARACTERIZATION OF MUSCARINIC RECEPTORS IN NEONATAL RAT CARDIOMYOCYTES

-[N-methyl-H-3]scopolamine methylchloride ([H-3]NMS) was used to chara cterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes . The specific binding of [H-3]NMS was proportional to cell concentrat ion, saturable with respect to [H-3]NMS concentration, and time depend ent. Scatchard analysis of binding isotherms showed that [H-3]NMS boun d to the freshly isolated and cultured cardiomyocytes with dissociatio n constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum recepto r densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respe ctively. Heterogeneity of mAChRs was demonstrated by competitive bindi ng experiments against [H-3]NMS with M2 and M3 antagonists. These rece ptors (80%) exhibited high affinities for amino)methyl]-1-piperidinyl] -acetyl}-5,-11-dihydro - 6H -pyrido [2,3 - b] [ 1, 4] benzodiazepine-6 -one (AF-DX- 1 16) and methoctramine similar to those Of M2 subtYPe. T he low-affinity M2 antagonist binding constants were close to those re ported for M3 receptors and possessed high affinity for 4-diphenylacet oxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the ba sis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic m onophosphate (cAMP) inhibition with high affinity (pK(B) 7.4), while i t antagonized inositol phosphate formation with low affinity (pK(B) 6. 5). 4-DAMP possessed high affinity in blocking inositol phosphate form ation (pK(B) 9.0) and low affinity for antagonism of cAMP inhibition ( pK(B) 7.7). Although no other muscarinic receptor mRNA has been detect ed in these cells, these data suggest the presence of a second populat ion of mAChRs, which may not be identical to the classical cardiac ''M 2'' receptors.