Abstract-It has been suggested that tetrahydrobiopterin (H 4 B), a cofactor of NO synthase, can reverse endothelial dysfunction caused by cardiovascular diseases, including atherosclerosis, coronary artery disease, and hypertension. Moreover, an impairment of H 4 B biosynthesis in spontaneously hypertensive rats (SHR) was observed. Thus, we hypothesized that the defect of the H 4 B synthesis system may play an important role in the development of hypertension in SHR. In the present study H 4 B (10 mg/kg per day IP) was used to treat SHR and Wistar-Kyoto rats (WKY) from the age of 5 through 16 weeks. Results demonstrated that chronic treatment with H 4 B significantly improved the impaired vascular responses to acetylcholine and suppressed the development of hypertension in SHR but did not affect WKY. The increase of inducible NO synthase expression, nitrotyrosine immunostaining, NO production, and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with H 4 B. In contrast, H 4 B had no effect on WKY.In conclusion, this study demonstrated that H 4 B significantly attenuated the development of hypertension in SHR. The antihypertensive effect of H 4 B might be mediated through its direct antioxidant activity and/or decreasing oxygen free radical production from NO synthase, thereby reducing inducible NO synthase expression and peroxynitrite formation. Thus, the present study proposed that supplementation with H 4 B might be beneficial in preventing pathological conditions such as essential hypertension. Key Words: nitric oxide synthase Ⅲ peroxynitrite Ⅲ rats, inbred SHR Ⅲ superoxide Ⅲ biopterin I ncreased superoxide formation occurs in spontaneously hypertensive rats (SHR). 1 Superoxide may induce inducible NO synthase (iNOS) and then cause overproduction of NO. 2 The formation of massive amounts of NO via iNOS has a potentially cytotoxic effect, whereas relatively small amounts of NO formed via endothelial NO synthase (eNOS) have a cytoprotective action on the cardiovascular system. 3 In addition, the parallel time course of the generation of superoxide anion and iNOS indicates an efficient simultaneous reaction: NOϩO 2 *3 ONOOϪ (peroxynitrite). 4 Peroxynitrite is a short-lived and potently damaging oxidant that contributes significantly to pathological oxidative stress in living tissues. 5 Moreover, the unique chemical reactions of peroxynitrite, such as protein nitration, DNA single-strand breakage, and guanidine nitration, are not only cytotoxic but also mutagenic. 6 These findings suggest that increased production of superoxide in SHR may lead to the development of hypertension through chronic damage of the cardiovascular system, especially endothelium, induced by massive amounts of NO and peroxynitrite. Because peroxynitrite formation was previously identified through immunostaining of nitrotyrosine at the local site of infected organs, 4 in the present study we used a Western blotting assay to detect nitrotyrosine expression in the thoracic aorta as indirect eviden...

Endothelin-1 (ET-1) has been implicated in fibroblast proliferation. However, the mechanism involving ET-1 is not clear. The present study was performed to examine the role of endogenous ET-1 in ET-1-stimulated fibroblast proliferation and to investigate the regulatory mechanism of ET-1-induced ET-1 gene expression in cardiac fibroblasts. Both ET A receptor antagonistand endothelin-converting enzyme inhibitor (phosphoramidon) inhibited the increased DNA synthesis caused by ET-1. ET-1 gene was induced by ET-1, as revealed with Northern blotting and ET-1 promoter activity assay. ET-1 increased intracellular reactive oxygen species (ROS), which were significantly inhibited by BQ485 and antioxidants. Antioxidants suppressed ET-1 gene expression and DNA synthesis stimulated by ET-1. ET-1 activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase, which were significantly inhibited by antioxidants. Only ERK inhibitor U0126 could inhibit ET-1-induced transcription of the ET-1 gene. Cotransfection of dominant-negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced increase in ET-1 transcription, suggesting that the Ras-Raf-ERK pathway is required for ET-1 action. Truncation and mutational analysis of the ET-1 gene promoter showed that the activator protein-1 (AP-1) binding site was an important cis-element in ET-1-induced ET-1 gene expression. Antioxidants attenuated the ET-1-stimulated AP-1 binding activity. Our data suggest that ROS were involved in ET-1-induced fibroblast proliferation and mediated ET-1-induced activation of ERK pathways, which culminated in ET-1 gene expression.

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Tanshinone IIA extracted from danshen, a popular medicinal herb used in traditional Chinese medicine, exhibits cardio-protective effects. However, the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II, cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation, and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effect of tanshinone IIA on nitric oxide (NO) production, and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation, and ERK phosphorylation. In addition, tanshinone IIA was found to increase the NO generation, and eNOS phosphorylation. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.