Modulation of cardiac M 2 muscarinic receptor binding by progesterone-related steroids

Abstract

We have previously shown that progesterone, but not estradiol or testosterone, can compete with [ 3H]N-methyl scopolamine (NMS) for the cardiac M 2 muscarinic binding site. Experiments have been carried out to investigate the inhibitory effects of a large variety of progesterone-like steroids on the M 2 muscarinic receptor. These studies were performed with the aid of a new binding assay which uses intact tissue in the form of ventricular micropunches. Our data show that synthetic, clinically-used progestins such as Provera, norgesterel and cyproterone are largely ineffective at the M 2 binding site whereas the naturally occurring progesterone derivatives 17a hydroxy progesterone and Reichstein's Substance S are highly active. (K 1 values 5 × 10 −6M and 1 × 10 −6M, respectively). Minor structural modifications such as acetylation of the 17α hydroxy group abolishes activity. Steroids known to exert cell membrane effects, such as alfaxalone and pregnenolone sulfate, had no influence on [ 3H] NMS binding. The progesterone antagonist RU-486 did not block the inhibitory effect of progesterone. Moreover, this putative receptor may be located in the cardiomyocyte membrane since 17α hydroxy progesterone induces rapid dissociation of [ 3H] NMS from its binding site (30% reduction in 5 min). We attempted to further localize the inhibitory locus to the M 2 receptor itself by means of the irreversible antagonist propylbenzilylcholine mustard (PrBCM). Ninety percent of the M 2 receptor could be blocked by PrBCM (10 −6 M), an effect reversible by the specific muscarinic antagonist scopolamine methyl bromide but not by progesterone. These results suggested that progeserone does not interact directly with the [ 3H] NMS labelled M 2 binding site. Atropine-induced dissociation of [ 3H] NMS binding was not affected by progesterone, suggesting the lack of an allosteric effect. We conclude that (a) some naturally-occurring, but not synthetic, progesterone-like molecules inhibit [ 3H] NMS binding to cardiac M 2 receptors (b) this inhibition is not mediated via the [ 3H] NMS binding site nor at an allosteric site (c) the modulatory effect is possibly non-genomic.