Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells

被引:87
作者
Sorescu, D [1 ]
Somers, MJ [1 ]
Lassègue, B [1 ]
Grant, S [1 ]
Harrison, DG [1 ]
Griendling, KK [1 ]
机构
[1] Emory Univ, Div Cardiol, Dept Med, Atlanta, GA 30322 USA
关键词
free radicals; vascular smooth muscle; reactive oxygen species; NADPH oxidase;
D O I
10.1016/S0891-5849(00)00507-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O-2(.-) production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O-2(.-). To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O-2(.-) with DEPMPO. We confirmed that the main source of O-2(.-) from VSMC membranes is an NAD(P)H oxidase and that the O-2(.-) formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O-2(.-) when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC50's (similar to 10-50 muM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O-2(.-) (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O-2(.-). In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O-2(.-) production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme. (C) 2001 Elsevier Science Inc.
引用
收藏
页码:603 / 612
页数:10
相关论文
共 54 条
  • [1] Lucigenin is a mediator of cytochrome c reduction but not of superoxide production
    Afanas'ev, IB
    Ostrachovitch, EA
    Korkina, LG
    [J]. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1999, 366 (02) : 267 - 274
  • [3] Expression of NADH/NADPH oxidase p22phox in human coronary arteries
    Azumi, H
    Inoue, N
    Takeshita, S
    Rikitake, Y
    Kawashima, S
    Hayashi, Y
    Itoh, H
    Yokoyama, M
    [J]. CIRCULATION, 1999, 100 (14) : 1494 - 1498
  • [4] BECHLAURSEN J, 1997, CIRCULATION, V95, P588
  • [5] Investigation into the sources of superoxide in human blood vessels - Angiotensin II increases superoxide production in human internal mammary arteries
    Berry, C
    Hamilton, CA
    Brosnan, J
    Magill, FG
    Berg, GA
    McMurray, JJV
    Dominiczak, AF
    [J]. CIRCULATION, 2000, 101 (18) : 2206 - 2212
  • [6] Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells
    Bhunia, AK
    Han, H
    Snowden, A
    Chatterjee, S
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (25) : 15642 - 15649
  • [7] BOVERIS A, 1984, METHOD ENZYMOL, V105, P429
  • [8] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [9] Requirement for reactive oxygen species in serum-induced and platelet-derived growth factor-induced growth of airway smooth muscle
    Brar, SS
    Kennedy, TP
    Whorton, AR
    Murphy, TM
    Chitano, P
    Hoidal, JR
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (28) : 20017 - 20026
  • [10] Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells
    Brown, MR
    Miller, FJ
    Li, WG
    Ellingson, AN
    Mozena, JD
    Chatterjee, P
    Engelhardt, JF
    Zwacka, RM
    Oberley, LW
    Fang, X
    Spector, AA
    Weintraub, NL
    [J]. CIRCULATION RESEARCH, 1999, 85 (06) : 524 - 533