Modulation of cardiac M2 muscarinic receptor binding by progesterone-related steroids

We have previously shown that progesterone, but not estradiol or testosterone, can compete with [3H]N-methyl scopolamine (NMS) for the cardiac M2 muscarinic binding site. Experiments have been carried out to investigate the inhibitory effects of a large variety of progesterone-like steroids on the M2 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 M2 binding site whereas the naturally occurring progesterone derivatives 17a hydroxy progesterone and Reichsteinʹs Substance S are highly active. (K1 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 M2 receptor itself by means of the irreversible antagonist propylbenzilylcholine mustard (PrBCM). Ninety percent of the M2 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 M2 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 M2 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.