Angiotensin II induces activation of phosphatidylinositol 3-kinase in cardiomyocytes

Background Phosphatidylinositol 3-kinase phosphorylates membrane lipids at the third position of the inositol ring producing phosphoinositides, not on the pathway for production of 1,4,5-triphosphate. Objective To test the hypotheses that angiotensin II (Ang II) activates phosphatidylinositol 3-kinase in cardiomyocytes and that this pathway is involved in Ang II-induced protein synthesis. Methods Cardiomyocytes, in culture, from 7-day-old chick embryonic hearts were treated with Ang II and the activation of phosphatidylinositol 3-kinase was assessed after immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase by the conversion of PI (phosphatidylinositol) to phosphatidylinositol 3-monophosphate (PIP) in the presence of gamma-[P-32]-ATP and analyzed by thin-layer chromatography. Western blotting was performed after antiphosphotyrosine immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase. Protein synthesis was assessed by [S-35]-methionine incorporation and polyacrylamide gel electrophoresis. Results Ang II stimulated phosphatidylinositol 3-kinase activity dramatically with 4.5- and 3.5-fold increases in PIP formation after 1 and 5 min, respectively. The involvement of tyrosine kinases was demonstrated by Western blotting in which Ang II increased tyrosine phosphorylation of a protein recognized by antibodies to the 85 kDa subunit of phosphatidylinositol 3-kinase. Furthermore, the tyrosine kinase inhibitor lavendustin A blocked Ang II-stimulated phosphatidylinositol 3-kinase activity and conversion of phosphatidylinositol to PIP, Ang II increased new protein synthesis as reflected by the significantly (P < 0.05) greater incorporation of [S-35]-methionine into cardiomyocytes treated with Ang II, The link between Ang II and protein synthesis was mediated in part through phosphatidylinositol 3-kinase because the phosphatidylinositol 3-kinase inhibitor wortmannin blocked the effect of Ang II on protein synthesis. Increased production both of nuclear and of cytosolic proteins was demonstrated by agarose gel electrophoresis of these cellular components of Ang II-treated cardiomyocytes. Wortmannin produced a general inhibition of the synthesis of nuclear and cytosolic proteins, with a greater effect on nuclear proteins. The action of wortmannin on nuclear protein synthesis was confirmed by similar findings with another phosphatidylinositol 3-kinase inhibitor, LY294002. Conclusion Phosphatidylinositol 3-kinase activation by Ang II occurs through a pathway utilizing tyrosine phosphorylation, Furthermore, this pathway is involved in cardiomyocyte protein synthesis and the possibility that it is operative in Ang II-mediated cardiac hypertrophy arises.