Cao, Sheng

Tetrahydrobiopterin is an essential cofactor required for the synthesis of NO. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrated that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial NO synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a 2-fold increase in GTPCH I activity was detected in the aortas of caveolin-1–deficient mice, suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by the disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells, and tetrahydrobiopterin production occurs in close proximity to endothelial NO synthase. In addition, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.

Endothelial cell-based angiogenesis requires activation of survival signals that generate resistance to external apoptotic stimuli, such as tumor necrosis factor-alpha (TNF-α), during pathobiologic settings. Mechanisms by which this is achieved are not fully defined. Here, we use a model in which the multifunctional cytokine nitric oxide counterbalances TNF-α-induced apoptosis, to define a role for membrane trafficking in the process of endothelial cell survival signaling. By perturbing dynamin GTPase function, we identify a key role of dynamin for ensuing downstream endothelial cell survival signals and vascular tube formation. Furthermore, nitric oxide is directly demonstrated to promote dynamin function through specific cysteine residue nitrosylation, which promotes endocytosis and endothelial cell survival signaling. Thus, these studies identify a novel role for dynamin as a survival factor in endothelial cells, through a mechanism by which dynamin S-nitrosylation regulates the counterbalances of TNF-α-induced apoptosis and nitric oxide-dependent survival signals, with implications highly relevant to angiogenesis.

The Ca2+ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO)production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases,particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production. This was associated with the mobilization of eNOS-GFP protein into Triton X-100-insoluble fractions of cell lysates, and an internalization of plasmalemmal eNOS-GFP in live and fixed ECV 304 cells. Incubation of digitonin-permeabilized ECV304 cells with the non-hydrolyzed GTP analog,GTP-γ-S, abrogated the BK-mediated internalization of eNOS-GFP as assessed by confocal microscopy. Conversely, inhibition of clathrin-dependent endocytosis, via overexpression of AP 180 or pretreatment of cells with chlorpromazine, did not influence BK-mediated eNOS redistribution. Furthermore, specific inhibition of dynamin-2 GTPase function by overexpression of a dominant negative construct, K44A, prevented the BK-mediated enrichment of eNOS-GFP within low buoyant density,caveolin-enriched fractions of eNOS-GFP ECV304 cell lysates. Dynamin-2 K44A overexpression also markedly impaired BK-dependent, L-NAME-inhibited NO production as did incubation of permeabilized cells with GTP-γ-s. These studies demonstrate that disruption of dynamin- and GTP-dependent, but clathrin-independent, vesicle trafficking pathways impairs BK-dependent cellular NO production, via inhibition of the internalization of eNOS-containing plasmalemmal vesicles.