Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line

Abstract

1. The effects of AM on expression of muscarinic (M) receptors from P19-derived cardiomyocytes were examined. 2. 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 (pA(2): 8.1). Inhibition of [(3)H]-NMS binding by atropine (pK(i): -8.4+/-0.2) and methoctramine (pK(i): -8.3+/-0.2) suggests that M(2) is the functional expressed isoform. 3. [(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. 4. 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 nM) plus factor H (50 nM) 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 micro M) but not by CGRP antagonist, CGRP(8-37) (1 micro M). 5. The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. 6. 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.

Figure 1

Characterization of muscarinic receptors on P19-derived cardiomyocytes. (A) RT–PCR analysis of mRNA expression for muscarinic receptors M₁–M₄ in P19 differentiated cardiomyocytes. D9 and D15 indicate 9 and 15 days of differentiation respectively. (−) is a control PCR without reverse transcription of the mRNA. Band sizes are indicated. (B) Inhibition of the isoprenaline-induced increase in beating rate by increasing concentrations of carbachol alone (n=8, open squares) or in the presence of a fixed concentration (10 μM) of atropine (n=8, filled squares), methoctramine (n=12, filled circles) or pirenzepine (n=8, open circles). (C) Inhibition of [³H]-NMS binding by atropine (n=6, filled line), methoctramine (n=6, hatched line) and pirenzepine (n=6, dotted line). (D) Top: Colocalization of M₂ muscarinic receptor and desmin by immunofluorescent staining of P19 differentiated cardiomyocytes (representative example of four experiments). M₂ is revealed by a rhodamine-coupled antibody (a) and desmin is revealed by a FITC-coupled antibody (b). Bottom: negative controls: c and d.

Figure 2

Time course expression of M₂ muscarinic receptor mRNA in P19 cells during differentiation. Semiquantitative RT–PCR were performed from day 1 (D1) to day 15 (D15). 18S mRNA are used as internal standard. Values of M2/18S ratio are depicted on bottom line of the figure.

Figure 3

Effects of AM with or without factor H on M₂ muscarinic receptor mRNA expression in P19 cardiomyocytes and rat atrial cardiomyocytes. Mean±s.e.m. of six separate experiments using real-time quantitative PCR with 18S mRNA used as standard for normalization. *P<0.05 vs control values. (a) Effect of increasing AM concentrations on 15 days differentiated P19 cardiomyocytes. Statistical analysis was performed using ANOVA. (b) Effect of two concentrations of factor H added to a fixed concentration of AM. (c) Effect of AM alone or associated with factor H on isolated rat atrial cardiomyocytes.

Figure 4

RT–PCR analysis of mRNA expression from L1/G10D and CRLR receptors and RAMP_1–3 proteins. Sizes of the bands are indicated between parentheses.

Figure 5

Effect of antagonists of L1/G10D (AM_22–52) and CRLR (CGRP_8-37) receptors on the induction of M₂ mRNA expression by AM plus factor H. Experiments were monitored by real-time PCR in P19 cardiomyocytes (D15 of differentiation) using 18S RNA as standard for normalization. Mean±s.e.m. of three independent experiments.*P<0.05.

Figure 6

Temporal evolution of L1/G10D and CRLR receptors and of GATA4 mRNAs in P19 cells during culture from day 1 (D1) to day 15 (D15). Results of semiquantitative RT–PCR analysis using 18S mRNA as internal standard. For L1/G10D and CRLR the value of ratio of expression over 18S expression is provided at each time.