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. β-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 β-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model.
Spontaneously hypertensive rats (SHRs), primary aortic endothelial cells, and endothelial cell line were used to investigate the hypotensive effect of β-lapachone and its action mechanism. β-Lapachone treatment dramatically lowered BP and vascular tension in SHRs and induced eNOS activation in endothelial cells. Consistent with these effects, β-lapachone treatment also elevated levels of both aortic cGMP and plasma nitric oxide in SHRs. Meanwhile, β-lapachone-treated SHRs showed significantly increased levels of aortic NAD+, LKB1 deacetylation, and AMPK Thr172 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 β-lapachone in endothelial cells. However, inhibition of LKB1 deacetylation by blocking of NQO1 or SIRT1 blunted AMPK activation by β-lapachone.
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.
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aLaboratory Animal Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon
bKorea Bioactive Natural Material Bank, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul
cDiabetes and Metabolic Disease Research Center, Lee Gil Ya Cancer & Diabetes Institute, Gachon University of Medicine, Incheon
dKT&G Life Sciences Corporation/R&D Center, Suwon
eHormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju
fDepartment of Internal Medicine, Kyungpook National University School of Medicine, Daegu, South Korea
Correspondence to Chul-Ho Lee, DVM, PhD, Laboratory Animal Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea. Tel: +82 42 860 4637; fax: +82 42 860 4609; e-mail: chullee@kribb.re.kr
Abbreviations: [Ca2+]i, intracellular Ca2+; AMPK, adenosine 5′-monophosphate-activated protein kinase; bEnd.3 cell, mouse brain endothelial cells; BH4, tetrahydrobiopterin; BP, blood pressure; CaMKKβ, Ca2+/calmodulin-dependent protein kinase kinase β; GTPCH-1, guanosine 5′-triphosphate cyclohydrolase-1; HAEC, human aortic endothelial cell; LKB1, tumor suppressor kinase liver kinase B1; L-NAME, ω-nitro-L-arginine methyl ester; NQO1, NAD(P)H:quinone oxidoreductase 1; SHR, spontaneously hypertensive rat; SIRT1, NAD+-dependent class III histone deacetylase sirtuin 1
Received 24 December, 2012
Revised 21 March, 2013
Accepted 2 May, 2013
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