Kinase Regulation of Angiotensin II-Mediated Activation of Phospholipase A 2 and Growth in Vascular Smooth Muscle Cells

Abstract

P149 Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via multiple signaling mechanisms, including activation of tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, we have reported that cytosolic phospholipase A 2 (cPLA 2 )-dependent release of arachidonic acid (AA) is linked to Ang II-induced VSMC growth independent of tyrosine kinase. Ang II-mediated activation of cPLA 2 involves translocation to the nucleus, as well as serine phosphorylation. Furthermore, VSMC growth stimulated by Ang II has been shown to involve phosphoinositol 3-kinase (PI3K). Thus, Ang II-mediated cPLA 2 activation may require a MAPK-dependent phosphorylation, while additional kinases may couple Ang II to the activation of MAPK. Therefore, we used cultured rat VSMC to examine the role of PI3K and MAPK in the phosphorylation of cPLA 2 , release of AA and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased 3 H-thymidine incorporation and cell number by 82% and 75%, respectively when compared to controls (5976 ± 861 cpm/well; 1.9 ± 0.25 x 10 5 cells/mL, respectively). Ang II increased 3 H-AA release by 145% when compared to controls (411 ± 81 cpm/well). The PI3K inhibitor LY294002 (10 μM) blocked the Ang II-induced thymidine incorporation completely and the release of AA by 66%. Similarly, the MAPK-kinase (MEK) inhibitor PD98059 (10 μM) abolished Ang II-mediated growth and attenuated AA release by 35%. Exogenous AA restored VSMC growth in the presence of PD98059 but not in cells treated with LY294002. Western blot analysis revealed that inhibition of PI3K or MEK reduced the Ang II-mediated increase in phosphorylation of MAPK, as well as the amount of phosphoserine-cPLA 2 associated with the nucleus. Thus, it appears that cPLA 2 -dependent release of AA is required for Ang II-induced growth of VSMC and may involve the PI3K-dependent phosphorylation of MAPK and subsequent phosphorylation-dependent activation of cPLA 2 .