Vol. 303, Issue 3, 1255-1264, December 2002

Stimulated Tyrosine Phosphorylation of Phosphatidylinositol 3-Kinase Causes Acidic pH-Induced Contraction in Spontaneously Hypertensive Rat Aorta

Dileep Kumar Rohra, Tohru Yamakuni, Ken-Ichi Furukawa, Noe Ishii, Takashi Shinkawa, Toshiaki Isobe and Yasushi Ohizumi

Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (D.K.R., T.Y., N.I., Y.O.); Department of Pharmacology, Hirosaki University School of Medicine, Hirosaki, Japan (K.I.F.); Integrated Proteomics System Project, Poineer Research on Genome the Frontier, MEXT, c/o Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan (T.S., T.I.); and Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan (T.I.)

Acidic pH induced a contraction (APIC) in isolated aortas from spontaneously hypertensive (SHR) and Wistar Kyoto rats, but failed to produce any response in age-matched Wistar rat aorta. This study was conducted to test the hypothesis that tyrosine phosphorylation of proteins is a molecular mechanism underlying the APIC. Tyrosine kinase inhibitors, genistein and tyrphostin 23 inhibited the APIC in a concentration-dependent manner. APIC was inhibited by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors, LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] and wortmannin. Consistent with the results from tension measurement experiments, Western blot analysis showed that acidic pH induced an appreciable increment of tyrosine phosphorylation of 85-kDa protein (p85) in SHR aorta, which was completely inhibited by tyrphostin 23, whereas in Wistar rat aorta, the protein tyrosine phosphorylation was not observed. Further investigations using immunoprecipitation followed by Western blotting confirmed an increase in the tyrosine phosphorylation of p85. Analysis by SDS-polyacrylamide gel electrophoresis followed by silver staining of the gel revealed that amounts of multiple proteins with molecular sizes of 120, 130, 210, and 225 kDa were increased at acidic pH, which were immunoprecipitated with anti-phosphotyrosine antibody. Western blotting using a specific anti-PI3-kinase antibody identified the p85 as the regulatory subunit of PI3-kinase, whereas 120-, 130-, and 225-kDa proteins were identified by mass spectrometry as pro-2 (I) collagen, collagen 1 (I) chain, and fibernectin I, respectively. As assayed by Western blotting using anti-myosin light chain (MLC) antibody, acidic pH induced a stimulation of MLC phosphorylation, and the stimulated MLC phosphorylation was abolished by tyrphostin 23 and LY-294002. These results suggest that acidic pH induces an increase in tyrosine phosphorylation of PI3-kinase, resulting in the MLC phosphorylation-dependent contraction of SHR aorta.