Figure 2.

Effect of 12(S)-HETE on AT1R or angiotensin II type II receptor (AT2R) mRNA expression in RMC. (A) 12(S)-HETE-induced AT1R expression: RMC were serum starved for 48 h, then treated with 12(S)-HETE (10⁻⁷ M) for 1 to 24 h. Expression of AT1R (A) was determined by quantitative reverse transcriptase PCR (QRT-PCR) using β-actin as an internal control. Data represent mean ± SEM of three independent experiments (*P < 0.05; ^#P < 0.01; ^$P < 0.001 versus control). (B) No change in AT2R expression seen under the same conditions. (C). RMC were pretreated with vehicle or with 2 μg/ml actinomycin D for 1 h and then stimulated with 12(S)-HETE (10⁻⁷ M) for 2 h and QRT-PCR with gene-specific primers performed with total RNA isolated from these cells. Bar graph represents fold over control AT1R mRNA induction by 12(S)-HETE in cells treated without or with actinomycin D. (D) Quiescent RMC were pretreated with either vehicle or 12(S)-HETE (10⁻⁷ M) for 6 h, then exposed to 2 μg/ml actinomycin D. Total RNA was then extracted at the indicate time points and real time QRT-PCR performed. The mRNA level of AT1R in vehicle-treated RMC before addition of actinomycin D was set as 100%. Each point represents relative AT1R mRNA level (mean ± SEM) of three independent experiments (*P < 0.05; ^#P < 0.01 versus vehicle by two-way ANOVA) and shows decreased rate of disappearance of AT1R mRNA in 12(S)-HETE-treated cells relative to vehicle control.