Mechanism of inhibition by methacholine of norepinephrine-stimulated ANP secretion.

We have previously reported that methacholine inhibits norepinephrine-stimulated immunoreactive atrial natriuretic peptide (ANP-IR) secretion by 65% in vitro. In the present study, we examined the mechanism by which methacholine inhibits norepinephrine-stimulated secretion using isolated, paced rat left atria superfused in vitro. Norepinephrine has beta- and alpha-adrenergic properties, both of which stimulate ANP secretion. Thus we separately examined the effect of 10 microM methacholine on ANP-IR secretion stimulated by the beta-adrenergic agonist isoproterenol (0.1 microM) and by the alpha-adrenergic agonist phenylephrine (10 microM). Methacholine lowered isoproterenol-stimulated ANP-IR secretion to base line but did not inhibit phenylephrine-stimulated ANP-IR secretion. Atria were superfused with 0.5 mM dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) to determine whether inhibition of isoproterenol-stimulated secretion by methacholine occurred by a reduction in adenylate cyclase activity or at a point distal to cAMP. Methacholine inhibited dibutyryl cAMP-stimulated ANP-IR secretion by 50%. This inhibition could not be reversed by 20 microM isobutylmethylxanthine. We conclude that 1) methacholine completely blocks isoproterenol-stimulated ANP-IR secretion; 2) inhibition appears to be primarily due to a decrease in adenylate cyclase activity; however, inhibition occurs at a point(s) distal to cAMP production; 3) methacholine does not inhibit phenylephrine-stimulated ANP-IR secretion; and 4) inhibition by methacholine of norepinephrine-stimulated ANP-IR secretion reflects a block in beta-adrenergic activity.