Evidence of paired M2 muscarinic receptors.
Binding assays involving various antagonists, including N-[3H] methylscopolamine, [3H]quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat "brainstem" (medulla, pons, and colliculi). However, competition curves between N-[3H]methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of [3H]-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with [3H]quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.
Potter
L T
LT
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Ballesteros
L A
LA
Bichajian
L H
LH
Ferrendelli
C A
CA
Fisher
A
A
Hanchett
H E
HE
Zhang
R
R
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001933:Brain Stem / Q000378:metabolism / Q000648:ultrastructure; D002021:Buffers; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008297:Male; D018727:Muscarinic Antagonists; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure; D019832:N-Methylscopolamine; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000378:metabolism; D010276:Parasympatholytics / Q000378:metabolism; D011813:Quinuclidinyl Benzilate; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism* / Q000502:physiology; D012602:Scopolamine Derivatives / Q000378:metabolism; D014316:Tritium
D002021:Buffers; D006150:Guanine Nucleotides; D008024:Ligands; D018727:Muscarinic Antagonists; D010276:Parasympatholytics; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D014316:Tritium; D010095:Oxotremorine; C042743:oxotremorine M; D011813:Quinuclidinyl Benzilate; D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine