Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5'-monophosphate-activated protein kinase-mediated guanosine 5'-triphosphate cyclohydrolase 1 preservation.

AIMS: Guanosine 5'-triphosphate cyclohydrolase-1 (GTPCH-1) is a rate-limiting enzyme in de-novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) coupling. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is crucial for GTPCH-1 preservation, and tumor suppressor kinase liver kinase B1 (LKB1), an upstream kinase of AMPK, is activated by NAD-dependent class III histone deacetylase sirtuin 1 (SIRT1)-mediated deacetylation. ß-Lapachone has been shown to increase cellular NAD/NADH ratio via NAD(P)H: quinone oxidoreductase 1 (NQO1) activation. In this study, we have evaluated whether ß-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model. METHODS AND RESULTS: Spontaneously hypertensive rats (SHRs), primary aortic endothelial cells, and endothelial cell line were used to investigate the hypotensive effect of ß-lapachone and its action mechanism. ß-Lapachone treatment dramatically lowered BP and vascular tension in SHRs and induced eNOS activation in endothelial cells. Consistent with these effects, ß-lapachone treatment also elevated levels of both aortic cGMP and plasma nitric oxide in SHRs. Meanwhile, ß-lapachone-treated SHRs showed significantly increased levels of aortic NAD, LKB1 deacetylation, and AMPK Thr phosphorylation followed by increased GTPCH-1 and tetrahydrobiopterin/dihydrobiopterin ratio. In-vitro study revealed that AMPK inhibition by overexpression of dominant-negative AMPK nearly abolished GTPCH-1 protein conservation. Enhanced LKB1 deacetylation and AMPK activation were also elicited by ß-lapachone in endothelial cells. However, inhibition of LKB1 deacetylation by blocking of NQO1 or SIRT1 blunted AMPK activation by ß-lapachone. CONCLUSION: This is the first study demonstrating that eNOS coupling can be regulated by NQO1 activation via LKB1/AMPK/GTPCH-1 modulation, which is possibly correlated with relieving hypertension. These findings provide strong evidence to suggest that NQO1 might be a new therapeutic target for hypertension.

NQO1 activation regulates angiotensin-converting enzyme shedding in spontaneously hypertensive rats.

AIMS: Angiotensin-converting enzyme (ACE) plays a key role in blood pressure (BP) homeostasis via regulation of angiotensin II. Active ACE ectodomain is enzymatically cleaved and released into body fluids, including plasma, and elevated plasma ACE levels are associated with increased BP. ß-lapachone (ßL) has been shown to increase cellular NAD(+)/NADH ratio via activation of NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, we evaluated whether NQO1 activation by ßL modulates BP through regulation of ACE shedding in an animal model of hypertension. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) and a human ACE-overexpressing rat lung microvascular endothelial cell line (RLMVEC-hACE) were used to investigate the mechanism by which ßL exerts a hypotensive effect. In vitro studies revealed that ßL significantly increased intracellular Ca(2+) ([Ca(2+)]i) levels and CaMKII Thr(286) phosphorylation, followed by diminished ACE cleavage secretion into culture media. Inhibition of ßL-induced [Ca(2+)]i level changes through intracellular Ca(2+) chelation, Nqo1-specific siRNA or ryanodine receptor blockade abolished not only ßL-induced increase in [Ca(2+)]i levels and CaMKII phosphorylation, but also ßL-mediated decrease in ACE shedding. The effect of ßL on ACE shedding was also blocked by inhibition of CaMKII. In SHR, ßL reduced BP following increase of CaMKII Thr(286) phosphorylation in the lung and decrease of ACE activity and angiotensin II levels in plasma. CONCLUSION: This is the first study demonstrating that ACE shedding is regulated by NQO1 activation, which is possibly correlated with relieving hypertension in SHR. These findings provide strong evidence suggesting that NQO1 might be a new target for ACE modulation and BP control.