Characterization of the threshold for NAD(P)H:quinone oxidoreductase activity in intact sulforaphane-treated pulmonary arterial endothelial cells.
Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5μM, 24h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP(+) in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP(+) ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K(m) for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold.
Department of Pulmonary Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Robert D
RD
Bongard
Gary S
GS
Krenz
Adam J
AJ
Gastonguay
Carol L
CL
Williams
Brian J
BJ
Lindemer
Marilyn P
MP
Merker
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D017879:Isothiocyanates; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*
D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D016660:NAD(P)H Dehydrogenase (Quinone); C016766:sulforaphane
Free Radic Biol Med. 2006 Jun 15;40(12):2206-13
16785034
Toxicol In Vitro. 2006 Aug;20(5):594-600
16314070
Methods Enzymol. 2004;382:320-51
15047110
Diabetes. 1995 May;44(5):520-6
7729609
Mol Vis. 2003 Nov 03;9:594-600
14627957
Drug Metab Rev. 2004 Oct;36(3-4):639-54
15554240
Chem Biol Interact. 1991;80(1):1-41
1913977
J Clin Invest. 1986 Aug;78(2):462-71
3734101
Free Radic Biol Med. 1998 May;24(7-8):1130-8
9626567
Antioxid Redox Signal. 2006 Jul-Aug;8(7-8):1315-27
16910779
Biochem Pharmacol. 2002 Dec 1;64(11):1597-603
12429349
Lung Cancer. 2009 Aug;65(2):129-37
19185948
Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L788-97
15994278
Angiogenesis. 2006;9(2):83-91
16821112
Br J Cancer. 1999 Jun;80(8):1223-30
10376975
Antioxid Redox Signal. 2011 Jun;14(11):2151-62
21083422
Clin Immunol. 2009 Mar;130(3):244-51
19028145
Pharmacol Rev. 2004 Mar;56(1):53-102
15001663
Org Biomol Chem. 2008 Feb 21;6(4):637-56
18264564
Mol Pharmacol. 1996 Oct;50(4):728-35
8863816
J Cereb Blood Flow Metab. 1995 Sep;15(5):814-26
7673374
Cardiovasc Toxicol. 2010 Jun;10(2):139-45
20405237
Biochem Pharmacol. 2001 Jun 15;61(12):1509-16
11377380
Biochim Biophys Acta. 2009 Oct;1790(10):1191-7
19595745
J Biol Chem. 1986 Mar 25;261(9):4039-45
3949801
Clin Cancer Res. 2005 Dec 15;11(24 Pt 1):8866-71
16361576
Clin Cancer Res. 1998 Sep;4(9):2065-70
9748120
Inflamm Res. 2009 Aug;58(8):513-21
19277846
J Biol Chem. 1999 Jan 29;274(5):2750-7
9915806
Free Radic Biol Med. 2004 Jul 1;37(1):86-103
15183197
Proc Natl Acad Sci U S A. 2010 May 25;107(21):9590-5
20439747
Free Radic Biol Med. 2005 Jul 15;39(2):257-65
15964517
Chem Biol Interact. 2009 Jan 27;177(2):115-20
18823965
J Pharmacol Exp Ther. 2004 Jul;310(1):263-71
14988420
Am J Clin Nutr. 2005 Dec;82(6):1283-91
16332662
Cancer Chemother Pharmacol. 2005 Sep;56(3):307-16
15877230
Mol Pharmacol. 2006 Oct;70(4):1194-203
16825487
Chem Biol Interact. 2000 Dec 1;129(1-2):77-97
11154736
Mol Cancer Ther. 2006 Mar;5(3):575-85
16546971
Am J Physiol Lung Cell Mol Physiol. 2003 Nov;285(5):L1116-31
12882764
J Exp Biol. 2011 Jan 15;214(Pt 2):254-62
21177945
Biochem Pharmacol. 1995 Jan 18;49(2):127-40
7530954
J Biol Chem. 2003 Aug 22;278(34):32100-6
12777375
Eur J Pharmacol. 2007 Jun 14;564(1-3):158-66
17397824
Free Radic Biol Med. 2000 Aug;29(3-4):246-53
11035253
Arterioscler Thromb Vasc Biol. 2003 Mar 1;23(3):411-7
12615686
Mol Carcinog. 2005 Aug;43(4):215-24
16003741
Free Radic Biol Med. 2009 Jan 1;46(1):25-32
18848878
Cancer Res. 1996 Jun 15;56(12):2823-6
8665520
Cancer Lett. 2008 Oct 8;269(2):291-304
18504070
Toxicol In Vitro. 2003 Aug;17(4):423-31
12849725
J Surg Res. 1990 Sep;49(3):222-7
2395368
Protoplasma. 2003 May;221(1-2):129-35
12768350
PLoS One. 2008 Jul 16;3(7):e2682
18628998
Biochim Biophys Acta. 1962 Apr 9;58:171-88
13890666
Biochem Pharmacol. 1989 Aug 15;38(16):2631-7
2764986
J Appl Physiol (1985). 2008 Oct;105(4):1114-26
18703762
Mol Pharmacol. 1997 Aug;52(2):300-5
9271353
Diabetes. 2008 Oct;57(10):2809-17
18633117
Biochem J. 1969 Dec;115(5):1009-29
5360673
Am J Respir Crit Care Med. 2008 Sep 15;178(6):592-604
18556627
Am J Respir Crit Care Med. 2009 Jan 15;179(2):138-50
18931336
Cancer Treat Rev. 2004 Aug;30(5):437-49
15245776
J Agric Food Chem. 2009 Jun 24;57(12):5615-22
19456137
J Nutr. 2010 Aug;140(8):1404-10
20554899
J Biol Chem. 2004 Jul 23;279(30):31606-12
15155746
Am J Physiol Lung Cell Mol Physiol. 2006 Mar;290(3):L607-19
16243901