Objective: We have previously shown an enhanced expression of Gialpha proteins in spontaneously hypertensive rats (SHR) that precedes the development of hypertension. Since oxidative stress has been shown to be increased in SHR, the present studies were undertaken to examine the role of oxidative stress in enhanced expression of Gialpha proteins in SHR.
Methods: Aortic vascular smooth muscle cells (VSMC) from 12-week-old SHR and Wistar-Kyoto (WKY) rats were used for the present studies. The levels of inhibitory guanine nucleotide regulatory proteins (Gialpha-2 and Gialpha-3) and stimulatory proteins (Gsalpha) were determined by western blotting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [alpha-32P]ATP.
Results: VSMC from SHR exhibited enhanced expression of Gialpha-2 and Gialpha-3 proteins as compared with age-matched WKY rats; however, the levels of Gsalpha proteins were not different between the two groups. The levels of superoxide anion (O2-) were also increased in SHR as compared with WKY rats, and losartan, an AT1 receptor antagonist, restored the enhanced levels to control WKY rat levels. Treatment of VSMC with antioxidants such as N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) for 24 h decreased the enhanced expression of Gialpha-2 and Gialpha-3 proteins in a concentration-dependent manner in VSMC from SHR. In addition, the inhibition of forskolin-stimulated enzyme activity by low concentrations of GTPgammaS (receptor-independent Gi functions) and C-ANP4-23-mediated inhibition of adenylyl cyclase (receptor-dependent Gi functions) that were significantly enhanced in SHR were restored to WKY rat levels by NAC and DPI treatments. Similarly, diminished stimulation of adenylyl cyclase by GTPgammaS, isoproterenol and sodium fluoride in SHR was also restored towards control WKY rat levels by NAC and DPI treatments. Furthermore, PD98059, a selective inhibitor of mitogen-activated protein kinase, was able to restore the enhanced expression of Gialpha proteins in VSMC from SHR towards WKY rat levels. In addition, the enhanced activity of extracellular signal-regulated kinase 1/2 in SHR as compared with WKY rats, as demonstrated by enhanced phosphorylation of extracellular signal-regulated kinase 1/2, was also restored to WKY rat levels by NAC or DPI.
Conclusions: These results suggest that enhanced levels of Gialpha proteins and associated functions in SHR may be attributed to the enhanced oxidative stress present in SHR, which exerts its effects through the mitogen-activated protein kinase signaling pathway.