Targeted Increases in Endothelial Cell Superoxide Anion Production Stimulate eNOS-Dependent Nitric Oxide Production, Not Uncoupled eNOS Activity
Every once in a while a paper comes along that makes us question our understanding of cell biology. Current theory holds that oxidative stress increases oxidation of tetrahydrobiopterin (BH4), which in turn uncouples endothelial nitric oxide synthase (eNOS) activity. Numerous publications, using a variety of experimental approaches, provide strong support for the BH4 oxidation hypothesis. Indeed, nearly 10 years ago it was shown, using purified recombinant eNOS, that loss of this critical cofactor promoted eNOS generation of superoxide anion (O2·−) rather than nitric oxide (·NO) on activation.1
See accompanying article on page 1627
The report by Zhang et al,2 however, demonstrates that when vascular endothelial cells in vivo are transduced with NOX5 they responded by increasing ·NO production rather than eNOS becoming uncoupled as might be predicted. However, these findings are consistent with an earlier study showing that hypercholesterolemia increases vascular production of nitrogen oxides but not vasodilation.3 At the time, the loss in vascular ·NO activity in hypercholesterolemic rabbits was considered the result of marked increases in production of O2·−, which inactivates ·NO rather than impairing ·NO production.3 Although hypercholesterolemia adversely impacts the vessel wall in numerous ways, transducing NOX5 provides essentially a singular means of increasing vascular endothelial O2·− generation. The advantage of this approach should be clear, that the increase in O2·− represents a critical signaling event in the vessel wall that is separate from the confounding variable effects of oxidized lipids, inflammatory cells, or impaired high density lipoprotein function that are noted in disease.
The fact that O2·− stimulates eNOS-dependent ·NO production in Zhang’s system rather than uncoupled eNOS activity is a finding that should not be overlooked. When supplying BH4 did not improve vascular EC responses, this result was interpreted to mean that the BH4 content in the vascular endothelium was sufficient for maintaining eNOS ·NO production even during times of increased O2·− production. If O2·− stimulates eNOS-dependent ·NO production then logically, other mechanisms must come into play before oxidation of BH4 dominates eNOS function. Although the exact mechanisms by which O2·− from NOX5 stimulates eNOS-dependent ·NO production remain to be determined, cell biology studies indicate that differences in eNOS phosphorylation or monomer/dimer formation are likely not involved. Interestingly, what does seem to be involved in maintaining coupled eNOS activity is an increase in hsp90 association. This observation is consistent with a recent report showing that when hsp90 associates with eNOS the targeted chaperone activity increases ·NO production, which can be confirmed by disrupting hsp90 association to increase uncoupled eNOS activity in either cells or vascular endothelium.4
The studies by Zhang et al2 raise important new questions about the role of O2·− in vascular function (Figure). If BH4 oxidation is not involved in this model, then what are the signaling mechanism(s) activated by O2·− that increase coupled eNOS activity? Does O2·− activate specific kinases that direct hsp90 to bind to eNOS or influence hsp90 acetylation to modulate chaperone-dependent signaling with eNOS? Are other chaperones required for eNOS activation and optimal coupled activity? Will other radical nitrogen- or lipid-derived species induce eNOS to generate ·NO? Is it just a matter of the degree of oxidative stress? And, if so, how much is required to oxidize BH4 before uncoupled eNOS can be detected? Or more importantly, have we simply misinterpreted the findings implicating BH4 oxidation and overlooked other potential mechanisms for governing eNOS activation and function?
Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998; 95: 9220–9225.
Zhang Q, Malik P, Pandey D, Gupta S, Jagnandan D, de Chantemele EB, Banfi B, Marrero MB, Rudic DR, Stepp DW, Fulton DJR. Arterioscler Thromb Vasc Biol. 2008; 28: 1627–1633.
Xu H, Shi Y, Wang J, Jones D, Weihrauch D, Ying R, Wakim B, Pritchard KA Jr. A heat shock protein 90 binding domain in endothelial nitric-oxide synthase influences enzyme function. J Biol Chem. 2007; 282: 37567–37574.