Figure 3. Inhibition by PI3K inhibitors of KCl- and NA-induced activation of Rho and phosphorylation of MYPT1 and CPI-17.

(A) Time-dependent changes of the amounts of GTP-RhoA in KCl-stimulated VSM in the presence of absence of LY. VSM was treated as in Figures 1(A) and 1(B). A portion (1/27) of tissue extracts was subjected to Western blot analysis for evaluating amounts of total RhoA in each sample. (B) Dose-dependent inhibition of KCl-induced RhoA activation by LY. VSM was treated as in Figure 1(C). (C) Inhibition of KCl (60 mM)- and NA (3 μM)-induced Rho activation at 5 min by LY (100 μM) and WMN (1 μM). (D) Inhibition of KCl- and NA-induced Thr⁸⁵⁰ MYPT1 phosphorylation by LY and WMN. (E) Inhibition of KCl- and NA-induced Thr⁶⁹⁵ MYPT1 phosphorylation by WMN. In (C) and (D), VSM was treated as in (C), and was analysed for Thr⁸⁵⁰ and Thr⁶⁹⁵ phosphorylation of MYPT1 by Western blotting using phosphorylation site-specific anti-phospho-MYPT1 antibodies. (F) Effects of Y-27632 on KCl- and NA-induced Thr⁸⁵⁰ and Thr⁶⁹⁵ MYPT1 phosphorylation. VSM was pretreated with 10 μM Y-27632 for 30 min and then stimulated as in (C). (G) Inhibition of KCl- and NA-induced Thr³⁸ CPI-17 phosphorylation by WMN. VSM was treated as in (C). * and #, P<0.05 compared with non-treated control and non-inhibitor-treated stimulation respectively.