Abstract
Exposure of cultured embryonic chicken cardiac cells to the muscarinic agonist carbachol results in a 70–80% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed on the surface of the cells. Removal of the agonist results in a gradual increase in mAChR number because of the accumulation of newly synthesized receptors, reaching the control level in 14 hr. Measurements of increases in K+ permeability elicited by carbachol show that even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The EC50 for carbachol is 13-fold higher in cells that have been exposed to carbachol and allowed to recover for 18 hr than in control cells, but is not significantly different from the EC50 for control cells 24 hr after agonist removal. The sensitivity of the mAChR- mediated inhibition of adenylate cyclase is also decreased at 18 hr, and recovers by 24 hr. These increases in sensitivity of mAChR-mediated responses are not blocked by administration of cycloheximide, and thus do not require de novo protein synthesis. The number of surface mAChR available for ligand binding can be reduced by 85–100% by treatment with the affinity-alkylating antagonist propylbenzilylcholine mustard. Newly synthesized mAChR that appear following affinity alkylation are also poorly coupled to mAChR-mediated increases in K+ permeability, indicating that decreased physiological sensitivity is not due to a nonspecific effect of long-term agonist exposure on general cellular function, but reflects, rather, an intrinsic property of newly synthesized mAChR. The decrease in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of mAChR on the surface nor to reduced agonist affinity of the mAChR. Cells exhibiting decreased responsiveness contain GTP-binding proteins, which function normally in the inhibition of adenylate cyclase and appear to be identical to pertussis toxin substrates from control cells using gel electrophoresis; therefore, the decreased sensitivity does not appear to be the result of an alteration in coupling proteins. These cells also contain mAChR that do not differ from those in control cells either by molecular weight or isoelectric point. Thus, the diminished sensitivity observed in cells containing newly synthesized receptors is either caused by a small change in mAChR not detected by these electrophoretic techniques or by a change in an as-yet-undefined component of mAChR transduction system in the heart.