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With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC)2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC)2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45 ± 0.13 vs 0.73 ± 0.13 au (arbitrary units)]. The signal increased linearly with incubation time (r2=0.93), but was abolished by addition of NG-nitro-l-arginine methyl ester (l-NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14 ± 0.06 vs 3.17 ± 0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17 ± 0.76 vs 3.37 ± 0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels. [Copyright &y& Elsevier]

The hph-1 ENU-mutant mouse provides a model of tetrahydrobiopterin deficiency for studying hyperphenylalaninaemia, dopa–response dystonia, and vascular dysfunction. We have successively localized the hph-1 mutation to a congenic interval of 1.6–2.8 Mb, containing the GCH gene encoding GTP cyclohydrolase I (GTP-CH I). We used these data to establish a PCR method for genotyping wild type, hph-1 and heterozygote mice, and found that heterozygote animals have partial tetrahydrobiopterin deficiency. These new findings will extend the utility of the hph-1 mouse in studies of GTP-CH I deficiency. [Copyright &y& Elsevier]

Introduction GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero , and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. Methods and results Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2–3 of the Gch1 gene. Gch1 fl/fl mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1 −/− embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1 −/− embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l -DOPA. Conclusion These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4. [ABSTRACT FROM AUTHOR]

Abstract: The exogenous administration of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase (NOS), has been shown to reduce left ventricular hypertrophy, fibrosis, and cardiac dysfunction in mice with pre-established heart disease induced by pressure-overload. In this setting, BH4 re-coupled endothelial NOS (eNOS), with subsequent reduction of NOS-dependent oxidative stress and reversal of maladaptive remodeling. However, recent studies suggest the effective BH4 dosing may be narrower than previously thought, potentially due to its oxidation upon oral consumption. Accordingly, we assessed the dose response of daily oral synthetic sapropterin dihydrochloride (6-R-l-erythro-5,6,7,8-tetrahydrobiopterin, 6R-BH4) on pre-established pressure-overload cardiac disease. Mice (n=64) were administered 0–400mg/kg/d BH4 by ingesting small pre-made pellets (consumed over 15–30min). In a dose range of 36–200mg/kg/d, 6R-BH4 suppressed cardiac chamber remodeling, hypertrophy, fibrosis, and oxidative stress with pressure-overload. However, at both lower and higher doses, BH4 had less or no ameliorative effects. The effective doses correlated with a higher myocardial BH4/BH2 ratio. However, BH2 rose linearly with dose, and at the 400mg/kg/d, this lowered the BH4/BH2 ratio back toward control. These results expose a potential limitation for the clinical use of BH4, as variability of cellular redox and perhaps heart disease could produce a variable therapeutic window among individuals. This article is part of a special issue entitled ‘‘Key Signaling Molecules in Hypertrophy and Heart Failure.’’ [Copyright &y& Elsevier]

Abstract: Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor-l-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues. [Copyright &y& Elsevier]