Regulation of endothelial nitric oxide formation

Nitric oxide (NO), a widespread signal and effector molecule, is synthesized from L-arginine by different NO synthase (NOS) isozymes, which differ in their subcellular distribution and in the mechanisms of their regulation. Endothelial NOS (eNOS) is unique among the three known NOS isoforms in being targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae. This targeting is promoted by direct interaction of eNOS with the caveolae structural protein caveolin- 1, which renders the enzyme inactive. Ligand-induced rises of intracellular Ca2+ promote a reversible Ca2+/calmodulin (CaM)-mediated dissociation of the inhibitory eNOS/caveolin complex resulting in a transient activation of eNOS. In addition to Ca2+, eNOS is also activated via a Ca2+-independent pathway, which is induced by shear stress and involves phosphorylation of eNOS by protein kinase Akt. Although both pathways have been extensively studied in the last few years, there are still a number of open questions remaining concerning the regulation of NO formation in endothelial cells. This especially accounts for the Ca2+-independent pathway, as little is known how phosphorylation of eNOS promotes enzyme activation. Moreover it has been suggested, that activation of Akt may also occur in response to Ca2+- mobilizing agonists, but an involvement of eNOS phosphorylation. in the Ca2+-dependent pathway has not been demonstrated so far. It is also unclear whether phosphorylation of eNOS by Akt affects uncoupled NADPH oxidation, which leads to generation of superoxide and H202 at low concentrations of L-arginine and/or tetrahydrobiopterin. Very recently is has been demonstrated, that stimulation of endothelial cells with bradykinin leads to phosphorylation of eNOS by the NLAP kinase ERK. Since in vitro phosphorylation of eNOS by ERK was reported to diminish eNOS activity, it has been proposed that the MAP kinase pathway may serve as a negative feedback loop to attenuate Ca2+induced eNOS activation. However, a functional role of ERK in the modulation of endothelial NO synthesis has not been demonstrated so far in intact cells or tissues. Moreover, the molecular mechanisms underlying the reported inhibition of eNOS by ERK-catalyzed phosphorylation. are also unclear. The proposed project is aimed at clarifying these selected aspects of eNOS regulation and should provide answers to the following open questions: A) How does phosphorylation of eNOS by Akt promote enzyme activation? B) Does phosphorylation of eNOS by Akt contribute to the activation of the enzyme by Ca2+-mobilizing agonists? Q Does phosphorylation of eNOS by Akt affect uncoupled NADPH oxidation? D) How does phosphorylation of eNOS by ERK promote enzyme inactivation? E) Does phosphorylation of eNOS by ERK serve as a negative feedback mechanism to attenuate Ca2+-dependent eNOS activation in intact cells?

Endothelial cells play an important role in the regulation of vascular tone. Upon stimulation with hormones or shear stress, they respond with the formation of nitric oxide (NO), which relaxes neighboring vascular smooth muscle cells leading to a decrease in blood pressure. The biosynthesis a NO is catalyzed by an enzyme termed endothelial nitric oxide synthase (eNOS) and regulated in by changes in the intracellular calcium concentration and phosphorylation reactions. The aim of the project was to investigate molecular mechanisms underlying the formation and inactivation of NO in endothelial cells and to contribute to better understanding of the regulation of blood flow under physiological and pathophysiological conditions. In the first part of the project we focused on the regulation of NO formation by the endogenous transmitters bradykinin and ATP and demonstrated that activation of NO synthase by these compounds is solely dictated by an increase in the intracellular calcium concentration and not enzyme phosphorylation. Moreover, we provided evidence for the existence of two distinct eNOS pools, which are independently regulated by bradykinin and ATP. We also observed that activation of eNOS by these agonists leads to a rapid desensitization of the enzyme, which in addition to the transient calcium signal contributes to the termination of NO formation in endothelial cells. We then focused on the mechanisms leading to NO inactivation and identified a novel enzymatic pathway by which endothelial cells convert NO to nitrate. Based on vitro data, showing that purified eNOS generates superoxide instead of NO in the absence of the pterin- cofactor tetrahydrobiopterin (BH4), we investigated in the second part of the project whether BH4 also controls NO/superoxide formation in intact endothelial cells. In the course of these studies we observed that inhibition of BH4 biosynthesis leads to a pronounced increase of superoxide formation that is blocked upon inhibition of eNOS or supplementation of the cells with BH4. In contrast to superoxide formation, biosynthesis of NO was impaired in BH4-depleted and restored upon addition of BH4. The data clearly demonstrate that BH4 plays an importing role in controlling endothelial NO vs. superoxide formation. Since cardiovascular diseases are often associated with reduced levels of BH4, these data suggest that eNOS-catalyzed superoxide formation due to impaired availability of BH4 may contribute to endothelial dysfunction in vivo.