MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca2+-dependent MLC kinase or Ca2+-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP ( 10 muM) increased ERK2 phosphorylation from basal 17 +/- 3 to 53 +/- 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 ( 20 muM) or U0126 ( 10 muM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca2+ rise, because it was unaltered when this was suppressed by the Ca2+ chelator BAPTA ( 10 muM) or xestospongin C ( 3 muM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido) adenosine (1 muM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 muM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca2+-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase.