Fig. 5.

Opposite role of Akt on iNOS expression in GK diabetic VSMC. Equal amounts of total RNA from WKY and GK VSMC post to stimulation with high glucose or low glucose for 24 h were lysed and subject to quantitative real-time PCR. A: basal iNOS gene expression were measured by real time PCR for gene expression. B: nitrite/nitrate production from the culture media of WKY and GK VSMC were measured by the Greiss methods as described in materials and methods. WKY and GK VSMC cells transfected with siAKTc (C) or Ad-myr-Akt (D) for 48 h were serum starved and pulsed with insulin (10 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II (5 min) followed by insulin (10 nM). Equal amounts of protein from each cell lysate were resolved by SDS-PAGE and transferred to nitrocellulose membrane, and the levels of iNOS, pAkt, and Akt compared with β-actin as an internal control. pAkt level was determined after total Akt amount adjusted. Densitometric analyses of four separate experiments with GK VSMC are given below each graph. *P < 0.05 siCON vs. siAKTc (C); *P < 0.05 β-gal vs. Ad-myr-Akt (D).