Figure 7

Exogenous NE and clonidine activate PI3Ks in canine isolated mesenteric vein. Immunoblots of Akt and phospho-Akt (P-Akt) in untreated control tissues (CT), and in tissues treated with NE (1 μM, 3 min) alone or after 60-min preincubation with 100 nM wortmannin, Wm, or 10 μM LY-294002, LY (A). Immune blots of P-Akt in veins incubated with clonidine (1 μM, 3 min) in the absence or after 60-min preincubation with 0.1 μM yohimbine, Yoh (C). The phospho-Akt (P-Akt) band densities were normalized to the respective Akt band densities, and the P-Akt/Akt ratios of treated tissues were presented relative to the ratios of non-treated tissue controls (bar graphs in A and C, n = 3). Thin-layer chromatography (TLC) separation of PI3P, obtained upon in vitro phosphorylation of PI by immunoprecipitated PI3Kγ. PI3Kγ activation was assessed in untreated (control) tissue (CT) and in tissues incubated for 3 min with 1 μM NE (B) or 1 μM clonidine (D), without or after 1-h preincubation with LY-294002 (LY, 10 μM) or yohimbine (Yoh, 0.1 μM). Phosphorylated PIP3 was quantified by radiography and densitometry, and PI3Kγ activation in treated tissues was expressed relative to non-treated tissue controls (bar graph, n = 3). *, P < 0.05 versus controls, #, P < 0.05 versus veins treated with NE or clonidine.