NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.

Abstract-Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 mol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O 2 Ϫ was not significantly reduced by 10 mol/L oxypurinol or 30 mol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 mol/L) as well as the O 2 Ϫ scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 mol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O 2 Ϫ scavenger Tiron (10 mmol/L), concomitant with 1 mol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O 2 Ϫ . Incubation with 100 mol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. Key Words: cyclosporin Ⅲ bradykinin Ⅲ nitric oxide Ⅲ cyclic GMP Ⅲ antioxidants C yclosporin (CsA) is an important immunosuppressant used in improving the chances of whole organ transplant and graft survival. 1,2 However, cyclosporin treatment has been linked to several significant nephrotoxic side effects. The side effects range from afferent arteriolar constriction 3 and a reduction in glomerular filtration rate 4 to interstitial fibrosis 5 and ultimately hypertension. Although the toxic effects are well established, the exact mechanisms that lead to the pathology and hypertension are not agreed on. The proposed mechanisms for the development of hypertension focus on induction of vasoconstrictive pathways as well as obstruction of vasodilative pathways examined in patients, rat models, and endothelial cells. The pathways examined as possibly affected by CsA include the renal sensory nerve endings, 6 the renin-angiotensin system, 7 endothelin-1, 8,9 thromboxane, 9,10 and the renal kallikrein-kinin system. 11,12 Bradykinin is an important vasodilating peptide involved in the renal kallikrein-kinin system. The bradykinin peptide exerts its effects by binding to its receptor, activating a heterotrimeric G protein complex, phospholipase C, and then nitric oxide synthase (eNOS), which generates nitric oxide (NO). NO then binds to the heme group of soluble guanylate cyclase (sGC), thereby activating the enzyme to produce cGMP. 13,14 The NO synthase family and the NO radical have been shown to play an important role in many ...

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Resveratrol, found in wine and grapes, has cardioprotective and anticarcinogenic properties. The guanylate cyclase‐cyclic GMP (cGMP) system is known to mediate the effects of vasoactive hormone and ligands such as atrial natriuretic factor and nitric oxide. It is possible that resveratrol exerts its effects by activating guanylate cyclase. This study was conducted to examine whether resveratrol can affect the guanylate cyclase‐cGMP system in PC12 cells. The results showed that resveratrol dose‐dependently increased cGMP levels. It inhibited membrane‐bound guanylate cyclase activity stimulated by atrial natriuretic factor, but had no effect on soluble guanylate cyclase activity stimulated by sodium nitroprusside. It also directly stimulated membrane‐bound guanylate cyclase activity in PC12 cell membranes. These results show that resveratrol increases cGMP formation by activating membrane‐bound, but not soluble, guanylate cyclase.