Adrenomedullin upregulates M 2 ‐muscarinic receptors in cardiomyocytes from P19 cell line

The effects of AM on expression of muscarinic (M) receptors from P19‐derived cardiomyocytes were examined. RT–PCR experiments revealed expression of M 1 –M 4 receptor genes. Immuno‐histochemistry indicated that M 2 expression is restricted to contractile cells. Carbachol inhibition of isoprenaline‐induced increase in beating rate was prevented by atropine and methoctramine (p A 2 : 8.1). Inhibition of [ 3 H]‐NMS binding by atropine (p K i : −8.4±0.2) and methoctramine (p K i : −8.3±0.2) suggests that M 2 is the functional expressed isoform. [ 3 H]‐NMS binding and semiquantitative RT–PCR studies showed a dome shaped time course of M 2 expression with a maximum at 7 days of differentiation followed by a progressive decline. AM concentration‐dependently upregulated M 2 receptor mRNA during late differentiation stages in P19 cells but also in rat atrial cardiomyocytes. This effect was potentiated by factor H. AM (100 n M ) plus factor H (50 n M ) treatment of P19 cells for 24 h significantly increased [ 3 H]‐NMS‐specific binding ( B max : 81±7 vs 31±6 fmol mg −1 prot). The effect of AM on mRNA levels was prevented by AM receptor antagonist AM 22–52 (1 μ M ) but not by CGRP antagonist, CGRP 8–37 (1 μ M ). The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. Our findings demonstrate that AM regulates M 2 receptors expression in cardiomyocytes probably through a mechanism involving L1/G10D receptors. The ‘ in vivo ’ significance of this phenomenon remains to be demonstrated.British Journal of Pharmacology (2003) 139 , 1219–1227. doi: 10.1038/sj.bjp.0705350