Abstract
Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.
Highlights
Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase
We examined the reaction of ONOOϪ with BH4, ascorbate, and thiols using electron spin resonance (ESR) and the spin probe 1-hydroxy-3-carboxy-2, 2,5-tetradrobiopterin radical; ESR, electron spin resonance; CPH, 1-hydroxy3-carboxy-2, 2,5-tetramethyl-pyrrolidine; bovine aortic endothelial cells (BAECs), bovine aortic endothelial cell; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; DAHP, 2,4-diamino-6-hydroxypyrimidine; L-NAME, L-nitro arginine methyl ester; ROS, reactive oxygen species; CP1⁄7, 3-carboxy-proxyl; CM1⁄7, 3-methoxycarbonyl-proxyl; O2., superoxide radical; SIN-1, 3-morpholinosydnonimine
In the absence of any scavenger, the reaction of ONOOϪ with CPH resulted in formation of CP1⁄7 that could be detected as a strong ESR signal (Fig. 1A)
Summary
Cal was simulated as a combination of five nitrogens with four protons with following hyperfine coupling constants (aN ϭ 8.05 G, aN ϭ 2.31 G, aN ϭ 1.79 G, aN ϭ 1.16 G, aN ϭ 0.93 G, aN ϭ 8.41 G, aN ϭ 9.50 G, aN ϭ 2.50 G, aN ϭ 1.06 G). Statistical Analysis—Data are presented as means Ϯ S.E. Analysis with linear regression was done with the software Sigma Plot. For comparison of two groups, a one-tailed t test was employed using Excel software. Statistical significance was assumed when p Ͻ 0.05
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