Cardiac cholinergic muscarinic receptors: changes in multiple affinity forms with down-regulation.

The isolated working rat heart exhibits dynamic changes in the cholinergic muscarinic receptor in response to perfusion with the acetylcholine congener methacholine. For example, perfusion with 4 microM methacholine for 2.5 hr mediates a statistically significant and reversible 10 to 15% decrease in receptor content in right atrium and left atrium and ventricle. The muscarinic receptor exhibits a single affinity state for antagonists but multiple affinity states for receptor agonists. When agonist 3H-antagonist competition experiments are performed, the concentration dependence of displacement by agonists is flattened and extends over more than 2 log units. From such curves, it is difficult to extract values for the relative proportions of the multiple receptors or their KD values by inspection. We have developed a procedure for plotting the competition curves so that the KD values and proportion of multiple receptor forms can be estimated graphically. We determined these more accurately by computer using a nonlinear least-squares analysis. After perfusion of methacholine for 1 hr, there were increases in the KD of the low and high affinity forms of the receptor in the right and left atria. After 2.5 hr of perfusion, the KD of the high-affinity form increased further in the left atrium. In the right atrium, the two affinity states were converted into a single state of low affinity. Although there was a significant decrease in the amount of receptor in left ventricle, there were no changes in the KD values or proportions of the two states. All changes in receptor reversed during an additional 2.5 hr of perfusion without methacholine.