Abstract
Muscarinic receptor stimulation elicits two distinct biochemical responses in embryonic chick heart cells: inhibition of catecholamine-stimulated cyclic AMP formation and stimulation of phosphoinositide (PhI) hydrolysis. We observe two major differences in the effects of agonists on these responses. First, carbachol and oxotremorine both inhibit cyclic AMP formation, but only carbachol stimulates PhI hydrolysis. Second, the dose-response relationships for the cyclic AMP and PhI responses differ; the half-maximal concentrations of carbachol needed to inhibit cAMP accumulation and stimulate PhI hydrolysis are 2 X 10(-7) and 2 X 10(-5) M, respectively. We carried out radioligand binding studies on intact chick heart cells to determine whether these data could be explained in terms of different agonist binding states of the muscarinic receptor. In intact cells, carbachol competes for [3H]quinuclidinyl benzilate-binding sites with high and low affinity, while oxotremorine shows only high affinity binding. We suggest that the receptor state common to both agonists is the state associated with inhibition of adenylate cyclase, while the very low affinity binding site seen only with carbachol is associated with the PhI response. We also consider the possibility that both responses are caused by a single receptor state that is efficiently coupled to adenylate cyclase inhibition and inefficiently coupled to PhI hydrolysis. Whichever mechanism is correct, our findings demonstrate that muscarinic receptors coupled to adenylate cyclase and the PhI response can be differentiated by virtue of their sensitivity to agonist and the efficiency with which some agonists induce receptor change and elicit receptor-mediated biochemical responses.
Highlights
The concept that there are muscarinic receptor subtypes was first proposed by Burgen and Spero (1) in 1968 on the basis of pharmacological studies comparing the receptors that mediate K+ efflux and contraction in guinea pig intestine
Since agonist binding to membranes may not reflect the receptor state in the intact cell, we examined the relationship between binding and biochemical responses in freshly dissociated cells prepared from 13-day-oldembryonic chick hearts
Oxotremorine is apotentand complete inhibitor of cyclic AMP formation but isvirtually inactive in stimulating phosphoinositide hydrolysis in the dissociated heart cells (Fig. 4)
Summary
1-Paccumulation was measured over a longer time period and using a higher dose of carbachol. Oxotremorine produces a complete and concentration-dependent inhibition of the PhI response to carbachol (Fig. 2). The muscarinic receptor antagonist [3H]QNBwas used to study the binding properties of muscarinic receptors in membranes prepared from chick hearts Both carbachol and oxotremorine compete for all [3H]QNB-binding sites. Since agonist binding to membranes may not reflect the receptor state in the intact cell, we examined the relationship between binding and biochemical responses in freshly dissociated cells prepared from 13-day-oldembryonic chick hearts. In these studies we have confirmed the basic observations made in the chick heart slices. The basal cyclic AMP concentration was 8.0 f 0.6 pmol/mg of urotein
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