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. Methods and 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.