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International Conference on Mathematical Biology and

Annual Meeting of The Society for Mathematical Biology,

July 27-30, 2009

University of British Columbia, Vancouver

.

Program

Poster PS59A
Gary Krenz
Marquette University, Zablocki VAMC and Medical College of Wisconsin
Title Evidence of a NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Reduction Activity Threshold in Intact Pulmonary Arterial Endothelial Cells: Experimental Data and Mathematical Model
Abstract One putative pulmonary endothelial metabolic function is the reduction of blood borne redox compounds via NQO1, wherein phase II enzyme induction is a potential means to optimize this function. In other cell lines, there appears to be a threshold above which increases in intact cell NQO1 enzyme does not support further increases in NQO1 substrate reduction. The goal of this study was to evaluate whether pulmonary arterial endothelial cells (PAEC) also exhibit an NQO1 activity threshold. PAECs were grown to confluence on biosilon microcarrier beads thereby providing a cellular bioreactor for the investigation of PAEC NQO1 activity. PAEC NQO1 activity in cell cytosol fraction was induced about 6 fold (measured using a DCPIP assay: 46.2 +/- 3.4 vs. 268.1 +/- 28.5 nmol/min/mg cell protein; mean +/- SEM) by exposure to sulforaphane (5 microM;24 hours). However, when intact cell NQO1 activity was measured by addition of duroquinone (0-50 microM) to the extracellular medium, the maximal reduction rate was only about 3 fold higher in sulforaphane-treated than control cells (10.0 +/- 0.6 vs. 32.2 +/- 1.3 nmol/min/mg cell protein). The possibility that NADPH availability was limiting NQO1 activity in sulforaphane-treated cells was examined via a steady-state mathematical model of NOQ1 ping pong bi-bi kinetics interacting with the cytosolic NADPH donor system and is consistent with the observation that glucose-6-phosphate dehydrogenase was induced only 1.2 fold in sulforaphane-treated cells (19.6 +/- 1.2 vs. 23.9 +/- 1.6 nmol/min/mg cell protein). This study demonstrates, via experimental evidence and modeling of NOQ1 and NADPH interaction, a threshold effect for intact PAEC NQO1 activity. This implies that pulmonary endothelial NQO1 capacity to activate blood borne cell membrane permeant anticancer drugs or detoxify xenobiotics is potentially limited by factors other than enzyme activity measured in cytosol fractions. Supported by NIH HL-65537 and the Department of Veterans Affairs.
CoauthorsRobert Bongard, Brian Lindemer, Marilyn Merker
LocationWoodward Lobby (Monday-Tuesday)