Tetrahydrobiopterin and eNOS dimer/monomer ratio?a clue to eNOS uncoupling in diabetes?

See article by Cai et al.[14](pages 823–831) in this issue. Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) critically determines vascular tone as well as vascular wall homeostasis [1]. Reduced NO bioavailability, which is associated with an increase in the formation of reactive oxygen species within the vascular wall, is the key determinant of endothelial dysfunction. The paradoxical finding of a concomitant increase in eNOS expression and reduced endothelium-dependent vasodilatation/relaxation [2–4] has drawn attention to the fact that eNOS itself in pathological states may be the source of superoxide anions, a so-called “eNOS uncoupling” (for review, see [5]). eNOS uncoupling has been linked to a reduced tetrahydrobiopterin (BH4) availability, and supplementation of BH4 is generally able to restore eNOS-mediated NO formation and endothelial function in hypertension, hypercholesterolemia and diabetes [6–8]. Endothelial dysfunction occurs already in pre-diabetic stages of insulin resistance and subsequently contributes to smooth muscle cell proliferation and platelet and leukocyte adhesion as well as atherogenesis. eNOS uncoupling and endothelial dysfunction are apparent in experimental diabetes and in diabetic patients [9,10] despite the fact that eNOS expression is actually increased. The uncoupling of eNOS has been linked to its monomerisation [11]: treating endothelial cells with peroxynitrite was associated with reduced eNOS activity and disruption of eNOS dimers. Similar observations were made in endothelial cells exposed to high glucose or in organs from diabetic mice [11]. While peroxynitrite-induced eNOS uncoupling was not attenuated in the presence of exogenous BH4 [11], in Apo E knock-out mice, peroxynitrite-mediated BH4 oxidation was identified as a pathogenic cause of eNOS uncoupling [12]. Furthermore, in human endothelial cells, gene transfer of the GTP cyclohydrolase 1 (GTPCH), the rate-limiting enzyme of BH4 synthesis, not only augmented intracellular BH4 levels and eNOS activity, but also eNOS homodimerisation [13]. The advantage of GTPCH gene transfer vs. pharmacological interventions such as BH4 supplementation is that it avoids nonspecific (e.g. antioxidant) effects and achieves high intracellular concentrations of BH4. In this issue of Cardiovascular Research, Cai et al. [14] extend these observations to eNOS uncoupling in hyperglycaemic human endothelial cells: Using GTPCH gene transfer, they demonstrated an essential role of BH4 for both eNOS-mediated NO formation and dimerisation. However, there is no direct evidence from cells or tissues that eNOS is ever present in a monomeric form in vivo. This is difficult to address as SDS-PAGE and Western blotting necessarily subject the protein to artificial conditions. Nevertheless, the results of Cai et al. show that BH4 has a major effect not only on NOS activity (which could be due to non-specific effects of BH4) but also on the eNOS dimer/monomer ratio, suggesting an effect of BH4 on eNOS structure. However, it is important to state that changes in the dimer/monomer ratio are not directly related to the functional uncoupling of eNOS, because only the dimeric form is biochemically active and able to generate either NO or superoxide. Also in the uncoupled state, eNOS is not a monomer. So the influences of BH4 on ‘dimer stabilisation’ and coupling of eNOS are not necessarily one and the same effect. Moreover, as reduced eNOS activity in hyperglycaemic endothelial cells was associated with glycation and reduced phosphorylation of eNOS at Ser1177[15], the influence of GTPCH gene transfer on eNOS phosphorylation status would have been of interest. Nevertheless, the present study by Cai et al. [14] nicely demonstrates reduced eNOS dimer/monomer ratio in combination with increased oxidative stress and reduced NO formation in hyperglycaemic endothelial cells, changes that were prevented by overexpression of GTP cyclohydrolase 1. These data obtained in cultured cells raise the question whether gene transfer of GTPCH induces changes in the eNOS dimer/monomer ratio in animal models of diabetes. While the same authors reported beneficial effects of GTPCH overexpression in vivo [16] on vascular function and NO formation in diabetic mice, the eNOS dimer/monomer ratio in vessels taken from these animals has not yet been investigated.