Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line.
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.
Buys
Sophie
S
INSERM U586, Laboratoire de Pharmacologie Médicale et Clinique, Faculté de Médecine, 37 allées Jules Guesde, 31073 Toulouse Cedex, France.
Smih
Fatima
F
Pathak
Atul
A
Philip-Couderc
Pierre
P
Verwaerde
Patrick
P
Montastruc
Jean-Louis
JL
Rouet
Philippe
P
Senard
Jean-Michel
JM
D053607:Adrenomedullin; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000502:physiology*
D010455:Peptides; D012333:RNA, Messenger; D043585:Receptor, Muscarinic M2; D053607:Adrenomedullin