sepiapterin and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

Diabetic heart disease is associated with tetrahydrobiopterin oxidation and high arginase activity, leading to endothelial nitric oxide synthase dysfunction. Sepiapterin (SEP) is a tetrahydrobiopterin precursor, and L-citrulline (L-Cit) is converted to endothelial nitric oxide synthase substrate, L-arginine. Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known. The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice.. Db/db and C57BLKS/J mice at 6 to 8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks. Cardiac function was evaluated with echocardiography. Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6 to 8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, tetrahydrobiopterin concentrations, ratio of endothelial nitric oxide synthase dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice. These detrimental effects of diabetes mellitus were abrogated by co-administration of SEP and L-Cit. Myocardial infarct size was increased, and coronary flow rate and ± dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared with control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice.. Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a tetrahydrobiopterin/endothelial nitric oxide synthase/nitric oxide pathway.

Topics: Age Factors; Animals; Biopterins; Cardiotonic Agents; Cells, Cultured; Citrulline; Coronary Circulation; Coronary Vessels; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelial Cells; Isolated Heart Preparation; Mice, Inbred C57BL; Mice, Obese; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Phosphorylation; Protein Multimerization; Pterins; Time Factors; Vasodilation; Ventricular Function, Left

Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this "pharmacological" treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Topics: Animals; Cardiotonic Agents; Disease Models, Animal; Hydrogen Peroxide; Male; Mice; Mice, Knockout; Mitochondria, Heart; Myocardial Reperfusion Injury; NADPH Oxidase 4; NADPH Oxidases; Nerve Growth Factors; Netrin-1; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Pterins; RNA Interference; RNA, Small Interfering; Superoxides; Tumor Suppressor Proteins

The aim of this study was to investigate aerobic exercise training as a means to prevent erectile dysfunction (ED) and coronary artery disease (CAD) development associated with inactivity and diet-induced obesity. Male Sprague-Dawley rats were fed a Western diet (WD) or a control diet (CD) for 12 wk. Subgroups within each diet remained sedentary (Sed) or participated in aerobic interval treadmill running throughout the dietary intervention. Erectile function was evaluated under anesthesia by measuring the mean arterial pressure and intracavernosal pressure in response to electrical field stimulation of the cavernosal nerve, in the absence or presence of either apocynin, an NADPH oxidase inhibitor, or sepiapterin, a tetrahydrobiopterin precursor. Coronary artery endothelial function (CAEF) was evaluated ex vivo with cumulative doses of ACh applied to preconstricted segments of the left anterior descending coronary artery. CAEF was assessed in the absence or presence of apocynin or sepiapterin. Erectile function (P < 0.0001) and CAEF (P < 0.001) were attenuated in WD-Sed. Exercise preserved erectile function (P < 0.0001) and CAEF (P < 0.05) within the WD. Erectile function (P < 0.01) and CAEF (P < 0.05) were augmented by apocynin only in WD-Sed, while sepiapterin (P < 0.05) only augmented erectile function in WD-Sed. These data demonstrate that a chronic WD induces impairment in erectile function and CAEF that are commonly partially reversible by apocynin, whereas sepiapterin treatment exerted differential functional effects between the two vascular beds. Furthermore, exercise training may be a practical means of preventing diet-induced ED and CAD development.

Topics: Acetophenones; Animals; Biopterins; Coronary Artery Disease; Coronary Vessels; Diet, High-Fat; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Endothelium, Vascular; Enzyme Inhibitors; Erectile Dysfunction; Exercise Therapy; Male; NADPH Oxidases; Obesity; Oxidative Stress; Penile Erection; Pterins; Rats; Rats, Sprague-Dawley; Sedentary Behavior; Time Factors; Vasodilation; Vasodilator Agents

The authors have previously shown that neonatal hyperoxic stress leads to high blood pressure, impaired endothelium-mediated vasodilatation, and increased vascular production of superoxide anion by NAD(P)H oxidase in adulthood. However, it is unknown whether changes in nitric oxide (NO) production and/or bioinactivation prevail and whether NO synthase (NOS) is also a source of superoxide. The purpose of this study was to evaluate whether adult animals exposed to neonatal hyperoxic stress have impaired vascular NO production associated with NOS uncoupling participating to vascular superoxide production and vascular dysfunction. In adult male rats exposed to 80% oxygen from day 3 to 10 of life (H, n = 6) versus room air controls (CTRL, n = 6), vascular (aorta) NO production is decreased at baseline (CTRL: 21 ± 1 vs. H: 16 ± 2 4,5-diaminofluorescein diacetate fluorescence intensity arbitrary units; P < 0.05) and after carbachol stimulation (acetylcholine analog; CTRL: 26 ± 2 vs. H: 18±2; P < 0.05). Pretreatment with L-arginine (CTRL: 32 ± 4 vs. H: 31 ± 5) and L-sepiapterine [analog of key NOS cofactor tetrahydro-L-biopterin (BH4)] (CTRL: 30 ± 3 vs. H: 29 ± 3) normalizes NO production after carbachol. L-Sepiapterine also normalizes impaired vasodilatation to carbachol. Vascular endothelial NO synthase (eNOS) immunostaining is reduced, whereas total eNOS protein expression is increased in H (CTRL: 0.76 ± 0.08 vs. H: 1.76± 0.21; P < 0.01). The significantly higher superoxide generation (CTRL: 20 ± 2 vs. H: 28 ± 3 hydroethidine fluorescence intensity arbitrary units; P < 0.05) is prevented by pretreatment with the eNOS inhibitor N-nitro-L-arginine methyl ester (CTRL: 21 ± 4 vs. H: 22 ± 4). Taken together, the current data indicate a role for eNOS uncoupling in enhanced vascular superoxide, impaired endothelium-mediated vasodilatation, and decreased NO production in adult animals with programmed elevated blood pressure after a brief neonatal oxygen exposure.

Topics: Age Factors; Animals; Animals, Newborn; Aorta; Arginine; Carbachol; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Hyperoxia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Pterins; Rats; Rats, Sprague-Dawley; Superoxides; Vasodilation; Vasodilator Agents

Endothelial dysfunction is associated with atherosclerosis in mice, but it is difficult to reduce cholesterol levels enough to study regression of atherosclerosis in genetically modified mice. The goal of this study was to examine vascular structure and function before and after reducing elevated plasma lipid levels with a genetic switch in Reversa mice, and identify novel mechanisms contributing to structural and functional improvements in the vasculature after reduction of blood lipids.. After 6 months of hypercholesterolemia, endothelial function (maximum relaxation to acetylcholine) in aorta was impaired and responses to nitric oxide were unaffected. Further impairment in endothelial function was observed after 12 months of hypercholesterolemia and was associated with reductions in sensitivity to nitric oxide. Expression of dihydrofolate reductase was reduced at 6 and 12 months, and addition of the tetrahydrobiopterin precursor sepiapterin significantly improved endothelial function. Reducing cholesterol levels at 6 months normalized dihydrofolate reductase expression and prevented further impairment in endothelial function. Similar functional changes were observed after 12 months of hypercholesterolemia followed by 2 months of lipid lowering.. Our data suggest that endothelial dysfunction after prolonged hypercholesterolemia is the result of both impairment of sensitivity to nitric oxide and reduced nitric oxide synthase cofactor bioavailability. Both of these changes can be prevented by normalizing blood lipids during moderately severe or advanced atherosclerosis.

Topics: Animals; Antioxidants; Aorta; Atherosclerosis; Biopterins; Carrier Proteins; Cholesterol; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Gene Expression Regulation; Hypercholesterolemia; Immunohistochemistry; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Donors; Pterins; Receptors, LDL; Tetrahydrofolate Dehydrogenase; Time Factors; Vasodilation; Vasodilator Agents

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.

Topics: Administration, Oral; Angiogenesis Inducing Agents; Animals; Biopterins; Blood Pressure; Cardiotonic Agents; Disease Models, Animal; Enzyme Inhibitors; Fibrosis; GTP Cyclohydrolase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pterins; Recovery of Function; Stroke Volume; Time Factors; Tyrosine; Ultrasonography; Ventricular Function, Left

Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH(4)) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly (P < 0.05) decreased myocardial infarct size (46 ± 1 to 19 ± 2% of the area at risk in control and IPC rabbits, respectively) and increased BH(4) concentrations (HPLC; 7.6 ± 0.2 to 10.2 ± 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 ± 0.3 to 5.4 ± 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH(4), Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH(4) synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH(4) precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH(4) concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 ± 0.06 to 0.59 ± 0.3) and nitric oxide production (184 ± 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH(4) concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia.

Topics: Animals; Benzoquinones; Biopterins; Blood Glucose; Blotting, Western; Cells, Cultured; Coronary Occlusion; Disease Models, Animal; Endothelial Cells; HSP90 Heat-Shock Proteins; Hyperglycemia; Immunoprecipitation; Ischemic Preconditioning, Myocardial; Lactams, Macrocyclic; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Protein Multimerization; Pterins; Rabbits; Time Factors

Glucocorticoid-induced hypertension is associated with imbalance between nitric oxide (NO) and superoxide. One of the pathways that causes this imbalance is endothelial NO synthase (eNOS) uncoupling. In the present study, adrenocorticotrophic hormone (ACTH)- and dexamethasone-treated rats were further treated with sepiapterin, a precursor of tetrahydrobiopterin, or N-nitro-L-arginine (NOLA), an inhibitor of NOS, to investigate the role of eNOS uncoupling in glucocorticoid-induced hypertension.. Male Sprague-Dawley (SD) rats (n = 7-13/group) were treated with either sepiapterin (5 mg/kg/day, IP) or saline (sham) 4 days before and during ACTH (0.2 mg/kg/day, SC), dexamethasone (0.03 mg/kg/day, SC), or saline treatment. NOLA (0.4 mg/ml in drinking water) was given to rats 4 days before and during dexamethasone treatment. Systolic blood pressure (SBP) was measured by the tail-cuff method.. Both ACTH (116 +/- 2 to 135 +/- 3 mm Hg (mean +/- s.e.m.), P < 0.001) and dexamethasone (114 +/- 4 to 133 +/- 3 mm Hg, P < 0.0005) increased SBP. Sepiapterin alone did not alter SBP. Sepiapterin did not prevent ACTH- (129 +/- 4 mm Hg, NS) or dexamethasone-induced hypertension (135 +/- 3 mm Hg, NS), although plasma total biopterin concentrations were increased. NOLA increased SBP in rats prior to dexamethasone or saline treatment. NOLA further increased SBP in both saline- (133 +/- 4 to 157 +/- 3 mm Hg, P < 0.05) and dexamethasone-treated rats (135 +/- 5 to 170 +/- 6 mm Hg, P < 0.05). ACTH and dexamethasone increased plasma F(2)-isoprostane concentrations. Neither sepiapterin nor NOLA significantly affected this marker of oxidative stress.. Sepiapterin did not prevent ACTH- or dexamethasone-induced hypertension. NOLA exacerbated dexamethasone-induced hypertension. These data suggest that eNOS uncoupling does not play a major role in the genesis of glucocorticoid-induced hypertension in the rat.

Topics: Adrenocorticotropic Hormone; Animals; Biomarkers; Biopterins; Blood Pressure; Dexamethasone; Dietary Supplements; Disease Models, Animal; Enzyme Inhibitors; F2-Isoprostanes; Hypertension; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Oxidative Stress; Pterins; Rats; Rats, Sprague-Dawley

Previously we showed that Brown Norway (BN/Mcw) rats are more resistant to myocardial ischemia-reperfusion (I/R) injury than Dahl S (SS/Mcw) rats due to increased nitric oxide (x NO) generation secondary to increased heat shock protein 90 (HSP90) association with endothelial nitric oxide synthase (NOS3). Here we determined whether increased resistance to I/R injury in BN/Mcw hearts is also related to tetrahydrobiopterin (BH(4)) and GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for BH(4) synthesis. We observed that BH(4) supplementation via sepiapterin (SP) and inhibition of GCH-1 via 2,4-diamino-6-hydroxypyrimidine (DAHP) differentially modulate cardioprotection and that SP alters the association of HSP90 with NOS3. BH(4) levels were significantly higher and 7,8-dihydrobiopterin (BH(2)) levels were significantly lower in BN/Mcw than in SS/Mcw hearts. The BH(4)-to-BH(2) ratio in BN/Mcw was more than two times that in SS/Mcw hearts. After I/R, BH(4) decreased and BH(2) increased in hearts from both strains compared with their preischemia levels. However, the increase in BH(2) in SS/Mcw hearts was significantly higher than in BN/Mcw hearts. Real-time PCR revealed that BN/Mcw hearts contained more GCH-1 transcripts than SS/Mcw hearts. SP increased recovery of left ventricular developed pressure (rLVDP) following I/R as well as decreased superoxide (O(2)(x-)) and increased x NO in SS/Mcw hearts but not in BN/Mcw hearts. DAHP decreased rLVDP as well as increased O(2)(x-) and decreased x NO in BN/Mcw hearts compared with controls but not in SS/Mcw hearts. SP increased the association of HSP90 with NOS3. These data indicate that BH(4) mediates resistance to I/R by acting as a cofactor and enhancing HSP90-NOS3 association.

Topics: Animals; Biopterins; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; HSP90 Heat-Shock Proteins; Hypoxanthines; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Pterins; Rats; Rats, Inbred BN; Rats, Inbred Dahl; RNA, Messenger; Species Specificity; Superoxides; Ventricular Function, Left; Ventricular Pressure

Tetrahydrobiopterin (BH(4)) deficiency is a cause of dystonia at birth. We hypothesized that BH(4) is a developmental factor determining vulnerability of the immature fetal brain to hypoxic-ischemic injury and subsequent motor deficits in newborns.. Pregnant rabbits were subjected to 40-minute uterine ischemia, and fetal brains were investigated for global and focal changes in BH(4). Newborn kits were assessed by neurobehavioral tests following vehicle and sepiapterin (BH(4) analog) treatment of dams.. Naive fetal brains at 70% gestation (E22) were severely deficient for BH(4) compared with maternal and other fetal tissues. BH(4) concentration rapidly increased normally in the perinatal period, with the highest concentrations found in the thalamus compared with basal ganglia, frontal, occipital, hippocampus, and parietal cortex. Global sustained 40-minute hypoxia-ischemia depleted BH(4) in E22 thalamus and to a lesser extent in basal ganglia, but not in the frontal, occipital, and parietal regions. Maternal supplementation prior to hypoxia-ischemia with sepiapterin increased BH(4) in all brain regions and especially in the thalamus, but did not increase the intermediary metabolite, 7,8-BH(2). Sepiapterin treatment also reduced incidence of severe motor deficits and perinatal death following E22 hypoxia-ischemia.. We conclude that early developmental BH(4) deficiency plays a critical role in hypoxic-ischemic brain injury. Increasing brain BH(4) via maternal supplementation may be an effective strategy in preventing motor deficits from antenatal hypoxia-ischemia.

Topics: Animals; Animals, Newborn; Biopterins; Brain; Brain Chemistry; Disease Models, Animal; Dystonia; Female; Fetal Development; Fetal Hypoxia; Gestational Age; Humans; Hypoxia-Ischemia, Brain; Maternal-Fetal Exchange; Muscle Hypertonia; Nitric Oxide Synthase; Pregnancy; Pterins; Rabbits

Nitric oxide (NO*) synthesis is induced within many tumors. The time course of NO* synthesis was evaluated during intra-peritoneal Meth A fibrosarcoma progression. While increasing macrophage recruitment into ascites was noted, inducible nitric oxide synthase (iNOS) antigen and function peaked between days 3-6 after tumor implantation. The capacity of cells to respond to LPS and IFNgamma stimulation was markedly depressed on day 9 and 11. Cellular proliferation correlated in an inverse fashion with levels of NO* synthesis. Electron paramagnetic resonance spectroscopy and nitrotyrosine immunostaining failed to show accumulation of characteristic target cell lesions induced by NO*. These findings lead us to conclude that NO* production was increasingly suppressed during Meth A tumor progression. Depression of NO* production did not correlate with levels of the inhibitory cytokines TGFbeta and IL-10, but could be partially overcome by addition of sepiapterin (a tetrahydrobiopterin prodrug). Thus, depletion of essential co-factors necessary for iNOS function may contribute to depressed NO* responses during cancer progression.

Topics: Animals; Ascites; Cell Line, Tumor; Cytokines; Disease Models, Animal; Electron Spin Resonance Spectroscopy; Enzyme Repression; Fibrosarcoma; Immunohistochemistry; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Phagocytes; Pterins; Time Factors

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

Topics: Acetylcholine; Administration, Oral; Animals; Biopterins; Diabetes Mellitus; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelium, Vascular; GTP Cyclohydrolase; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Inferior; Mice; Mice, Inbred C57BL; Neopterin; Oxidative Stress; Phenylephrine; Pterins; Vasoconstriction; Vasodilation

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Topics: Animals; Arginine; Biopterins; Calcium; Calmodulin; Chromatography, High Pressure Liquid; Diabetes Mellitus; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; GTP Cyclohydrolase; Immunoblotting; Kinetics; NADP; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Protein Isoforms; Pteridines; Pterins; Rats; Rats, Mutant Strains