Apelin stimulates myosin light chain phosphorylation in vascular smooth muscle cells.
OBJECTIVE
Physiological roles of apelin and its specific receptor APJ signaling were investigated in vascular smooth muscle cells (VSMCs). The present study determined whether apelin activates myosin light chain (MLC), a major regulatory event in initiating smooth muscle contraction.


RESULTS
To assess MLC activation, we performed Western blot and immunohistochemical studies using an antibody against the phospho-MLC. In VSMCs, apelin induces the phosphorylation of MLC in a concentration-dependent manner with a peak at 2 minutes. Pretreatment of VSMCs with pertussis toxin abolishes the apelin-induced phosphorylation of MLC. Inhibition of protein kinase C (PKC) with GF-109203X markedly attenuated the apelin-induced MLC phosphorylation. In addition, methylisobutyl amiloride, a specific inhibitor of the Na+/H+ exchanger (NHE), and KB-R7943, a potent inhibitor for the reverse mode of the Na+/Ca2+ exchanger (NCX), significantly suppressed the action of apelin. In wild-type mice, apelin phosphorylates MLC in vascular tissue, whereas it had no response in APJ-deficient mice by Western blot and immunohistochemistry. Apelin-induced phosphorylation of MLC was accompanied with myosin phosphatase target subunit phosphorylation.


CONCLUSIONS
These results provide the first evidence to our knowledge for apelin-mediated MLC phosphorylation in vitro and in vivo, which is a potential mechanism of apelin-mediated vasoconstriction.
Hashimoto
Tatsuo
T
Division of Cellular Pathobiology, Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama, 236-0004, Japan.
Kihara
Minoru
M
Ishida
Junji
J
Imai
Nozomi
N
Yoshida
Shin-ichiro
S
Toya
Yoshiyuki
Y
Fukamizu
Akiyoshi
A
Kitamura
Hitoshi
H
Umemura
Satoshi
S
D054392:Adipokines; D000818:Animals; D001013:Aorta, Thoracic; D000073861:Apelin; D000075244:Apelin Receptors; D002352:Carrier Proteins / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007474:Ion Exchange; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
D054392:Adipokines; D000073861:Apelin; D000075244:Apelin Receptors; C402826:Apln protein, mouse; C491285:Aplnr protein, mouse; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D008301:Maleimides; D018994:Myosin Light Chains; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D043562:Receptors, G-Protein-Coupled; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; C070515:bisindolylmaleimide I