Agonist binding to M1 muscarinic receptors is sensitive to guanine nucleotides.
Putative M1 (high-affinity pirenzepine) muscarinic receptors in rabbit hippocampal membranes, treated with 0.1 mM N-ethylmaleimide (NEM), were selectively labeled with [3H]pirenzepine. A single class of binding sites was labeled with a Kd of 3.4 nM, consistent with the pharmacologically-defined M1 subtype of muscarinic receptors. While full muscarinic agonists bound to high- and low-affinity states of [3H]pirenzepine-labeled M1 sites with a KL/KH ratio of approximately 100, the ratio for partial muscarinic agonists was approximately 10. The high-affinity binding of all agonists tested required divalent cations, and was interconverted to low-affinity binding in the presence of the non-hydrolyzable GTP analogue, guanylyl imidodiphosphate (GppNHp). Direct labeling of the high-affinity agonist state of M1 receptors was achieved with 5 nM [3H]oxotremorine-M by selectively uncoupling the high-affinity agonist state of M2 (low-affinity pirenzepine) receptors with NEM. The rate of dissociation of [3H]Pxotremorine-M from M1 receptors was accelerated 6-fold by GppNHp. These results provide further evidence which suggests that putative M1 muscarinic receptors activate second messenger systems by coupling to NEM-insensitive guanine nucleotide-binding proteins.
Flynn
D D
DD
Department of Pharmacology, University of Miami School of Medicine, FL 33101.
Palermo
N
N
Suarez
A
A
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008688:Methacholine Compounds / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D010862:Pilocarpine / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000187:drug effects*; D015290:Second Messenger Systems / Q000187:drug effects*
D006150:Guanine Nucleotides; D007202:Indicators and Reagents; D008688:Methacholine Compounds; D011976:Receptors, Muscarinic; D010862:Pilocarpine; D006165:Guanylyl Imidodiphosphate; D010890:Pirenzepine; D010095:Oxotremorine; D002217:Carbachol