Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells.
Adrenomedullin (AM) is a multifunctional peptide hormone, which plays a significant role in vasodilation and angiogenesis, implicating it in hypertension as well as in carcinogenesis. AM exerts its effects via the calcitonin receptor-like receptor (CRLR, now known as CL) complexed with either receptor activity modifying protein (RAMP) 2 or 3. We have investigated the effect of AM on immortalized human microvascular endothelial cells 1, since endothelial cells are a major source as well as a target of AM actions in vivo. Cells treated with AM showed elevated cAMP in a time (5-45 min)-dependent and dose (10(-6)-10(-14) M)-dependent manner. Pre-treatment with the AM receptor antagonist AM(22-52) partially suppressed the AM-induced increase in cAMP levels. An increase in extracellular signal-regulated kinase 1/2 phosphorylation was observed after 5 min of treatment with 10(-8) M AM. This phosphorylation was specific, since we were able to block the AM-induced effect with 1 microM U0126, a specific mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Using real-time PCR, we were able to show for the first time that AM upregulates peptide and mRNA expression of vascular endothelial growth factor (VEGF). However, AM treatment of cells did not result in increased cell proliferation. Instead, we observed that AM and VEGF induced cell migration, which could be inhibited by the AM(22-52) and anti-VEGF antibody respectively. AM also significantly elevated mRNA levels of CL (after 2 and 24 h treatment) and RAMP2 (after 1 and 24 h treatment). The upregulation of the AM receptor at two time points reflects possibly different cellular responses to short- and long-term exposure to AM.
Schwarz
Nele
N
Centre of Molecular Endocrinology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary University, London, UK.
Renshaw
Derek
D
Kapas
Supriya
S
Hinson
Joy P
JP
D053607:Adrenomedullin; D000704:Analysis of Variance; D015153:Blotting, Western / Q000379:methods; D058286:Calcitonin Receptor-Like Protein; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000032:analysis; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008833:Microcirculation; D010455:Peptides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013268:Stimulation, Chemical; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C545503:RAMP2 protein, human; D012333:RNA, Messenger; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D042461:Vascular Endothelial Growth Factor A; D053607:Adrenomedullin; D000242:Cyclic AMP