Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes.
[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.
Yang
C M
CM
Department of Pharmacology, Chang Gung Medical College, Tao-Yuan, Taiwan, Republic of China.
Chen
F F
FF
Sung
T C
TC
Hsu
H F
HF
Wu
D
D
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D009116:Muscarine / Q000037:antagonists & inhibitors; D018727:Muscarinic Antagonists; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019832:N-Methylscopolamine; D010880:Piperidines / Q000378:metabolism; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism* / Q000502:physiology; D012602:Scopolamine Derivatives / Q000378:metabolism
D007295:Inositol Phosphates; D018727:Muscarinic Antagonists; D010880:Piperidines; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D010890:Pirenzepine; D009116:Muscarine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D000242:Cyclic AMP; C048593:otenzepad; D019832:N-Methylscopolamine