Abstract
Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.
Highlights
Regulation of endothelial nitric oxide (NO)2 production represents a critical mechanism for the modulation of vascular homeostasis
We analyzed immunoblots probed for dihydrofolate reductase (DHFR), GTP cyclohydrolase-1 (GTPCH1), or ā¤-actin in cells transfected with DHFR small interference RNA (siRNA) and/or GTPCH1 siRNA constructs; the same quantity of siRNA was used in each transfection using nonspecific siRNA as indicated
We show the concentrations of total biopterins and BH4 in endothelial cells transfected with control, DHFR, or GTPCH1 siRNA and supplemented with control vehicle or pterins as shown. 48 h following siRNA transfections, the cells were incubated with vehicle, BH4 (10 ā®M), or BH2 (10 ā®M) for 1 h, and intracellular levels of BH4 and total biopterins were assessed by differential oxidation method and HPLC analysis, as described in the text
Summary
MaterialsāFetal bovine serum was from HyClone Laboratories (Logan, UT). LipofectamineTM 2000, AmplexTM Red reagent, and most cell culture reagents were from Invitrogen. Culture and Treatment of CellsāBovine aortic endothelial cells (BAECs) were obtained from Cambrex (Walkersville, MD) and maintained in culture on gelatin-coated 100-mm culture dishes with Dulbeccoās modified Eagleās medium supplemented with fetal bovine serum (10% v/v) as described previously [33]. Treatments with VEGF and preparation of cell lysates were performed as described previously [34], with corresponding vehicle treatments as controls. Cell culture medium was replaced with Dulbeccoās phosphate-buffered saline, and various drugs were added as indicated. Cells were harvested to determine protein concentration, permitting NO2ĻŖ production to be reported as picomoles per mg of protein. Measurement of H2O2āH2O2 production from cells was quantitated using the AmplexTM Red fluorescence assay using previously described methods [39]. Cells were harvested to determine protein concentration, permitting H2O2 production to be reported as picomoles per mg of protein. A p value of less than 0.05 was considered significant
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
