Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution

Tetrahydrobiopterim (BH4) and beat shock protein 90 (hsp90) have been anticipated to regulate endothelial nitric oxide synthase (eNOS)dependent superoxide anion radical (O-2(.-)) generation in endothelial cells. It is not known, however, whether hsp90 and BH4 increase O-2(.-) in a synergistic manner, or whether this increase is a consequence of downstream changes in eNOS phosphorylation on serine 1179 (eNOS-S-1179) and changes in dimer/monomer distribution. Here O-2(.-) production from purified BH4-free eNOS and eNOS:hsp90 complexes determined by spin-trapping methodology showed that hsp90 neither inhibits O-2(.-) nor alters the requirement of BH4 to inhibit radical release from eNOS. In endothelial cells, O-2(.-) detection with the novel high-performance liquid chromatography assay of 2-hydroxyethidium showed that inhibition of hsp90 did not increase O-2(.-), while a significant increase in O-2(.-) was detected in BH4-depleted cells. Radicicol, a bsp90 inhibitor, disrupted eNOS: hsp90 association, decreased eNOS-S1179, but increased biopterin production in a dose-dependent fashion. These changes were followed by an increase in eNOS activity, demonstrating that high biopterin levels offset inhibition of eNOS phosphorylation and diminished interaction with hsp90. In contrast, depletion of biopterin did not affect hsp90 levels or interaction with eNOS or eNOS dimer/monomer ratio in bovine aorta endothelial cells (BAECs). We conclude that low BH4 but not inhibition of hsp90 increases O-2(.-) in BAECs by mechanism(s) that unlikely involve phosphorylation to eNOS-S-1179 or eNOS monomerization. (c) 2006 Elsevier Inc. All rights reserved.