Regulation Of Nitric Oxide Synthase Activity Research Articles

Identify novel gene signatures in atrial fibrillation by comprehensive bioinformatics analysis

Background: Atrial fibrillation (AF), one of the most prevalent heart rhythm disorders, may lead to thromboembolism, heart failure, and sudden death. However, the mechanism of AF has not yet been fully explained. Objective: This study aims to identify novel gene signatures and to investigate the potential therapeutic targets of AF with an integrated bioinformatic approach. Methods: The gene expression and methylation datasets of AF were obtained through the Gene Expression Omnibus (GEO) database. Subsequently, a set of differentially expressed genes and differential methylation sites were identified. Gene functional annotation analysis was conducted to explore the potential function of differentially-methylated/expressed genes. Then, we constructed a PPI network and TF–miRNA–mRNA network. Finally, weighted gene co-expression network analysis (WGCNA) was presented to study critical modules of AF. Results: Seven hypomethylated-high expression genes and nine hypermethylated-low expression genes were acquired from AF patients. Functional enrichment results indicated that the differentially-methylated/expressed genes were mainly concentrated in decidualization, maternal placenta development, regulation of nitric-oxide synthase activity, and osteoclast differentiation. Based on the results of the PPI, we defined 4 key genes namely FHL2, STC2, ALPK3, and RAP1GAP2 as the core genes, playing essential roles in the TF-miRNA-mRNA network. In the end, we constructed two co-expression modules that highly correlated with AF-related phenotype. Conclusion: In our study, we found critical genes for AF that might help understand the molecular changes in AF.

Interleukin-1 beta is a potential mediator of airway nitric oxide deficiency in cystic fibrosis

Airway nitric oxide (NO) deficiency is a hallmark of cystic fibrosis (CF), but the reasons for the reduced NO production in CF airways are unclear. Interleukin (IL)-1 pathway activation plays a role in early CF lung disease and is also involved in the regulation of NO synthase activity. Treatment of CF patients with the CFTR-targeting drug ivacaftor, among other beneficial effects, results in an increase in airway NO levels. In this longitudinal observational trial, we show that ivacaftor therapy leads to a significant reduction in sputum IL-1β concentration but not in other IL-1- or Th17-associated cytokines. IL-1β concentrations were closely linked to improvement in pulmonary function, measures of NO metabolism in sputum and exhaled NO. These data therefore suggest a potential interaction between transepithelial chloride conductance, IL-1β and airway NO production.

Involvement of 5-Hydroxytryptamine Receptor 2A in the Pathophysiology of Medication-Overuse Headache.

BackgroundRecent studies indicated that analgesic overuse upregulated 5-hydroxytryptamine receptor 2A (5-HT2AR) and subsequently activated nitric oxide synthase (NOS) and thus induced latent sensitization, which provided a mechanistic basis for medication-overuse headache (MOH). Moreover, glycogen synthase kinase-3β (GSK-3β) was regulated by serotonin receptors and the phosphorylation of GSK-3β affected NOS activity, indicating that GSK-3β could be involved in the regulation of NOS activity by 5-HT2AR in MOH pathophysiology. Herein, we performed this study to investigate the role of 5-HT2AR in MOH pathophysiology and the role of GSK-3β in the regulation of NOS activity by 5-HT2AR.Materials and MethodsWistar rats were daily administered with paracetamol (200 mg/kg) for 30 days to set animal models for pre-clinical MOH research. After the rat MOH models were successfully established, the expression of 5-HT2AR and NOS, GSK-3β activity in trigeminal nucleus caudalis (TNC) were assayed. Then, 5-HT2AR antagonist ketanserin and agonist DOI were applied to investigate the effect of 5-HT2AR on NOS activity in TNC of MOH rats, and GSK-3β antagonist LiCl and agonist perifosine were applied to explore the role of GSK-3β in the activation of NOS by 5-HT2AR.ResultsWe found that the expression of 5-HT2AR and NOS, GSK-3β activity were enhanced in TNC of MOH rats. 5-HT2AR modulator regulated the activity of NOS and GSK-3β in TNC of MOH rats, and drugs acting on GSK-3β affected NOS activity.ConclusionThese data suggest that GSK-3β may mediate the activation of NOS by 5-HT2AR and underline the role of 5-HT2AR in MOH pathophysiology.

Arginase II and nitric oxide synthase uncoupling – a culprit for oocyte quality deterioration in older patients?

Utilizing nitric oxide (NO) selective electrode, recently we have shown that the intraoocyte NO concentration of oocytes obtained from young are significantly higher than those from old animals. NO supplementation of old and young oocytes not only resulted in sustained the ability of oocytes to undergo normal fertilization and development, but also prevented the onset of apoptosis in the embryos created from aged oocytes. NO is generated enzymatically by three distinct isoforms of NO synthase (NOS): neuronal, inducible, and endothelial. All three isoforms typically require molecular oxygen, NADPH, tetrahydrobiopterin (H4B), and oxygen to convert L-arginine (L-Arg) to NO and citrulline. Deficiency of L-Arg or H4B, leads NOS to generate O2•- instead of NO, in a process called NOS uncoupling. Arginase II is a critical enzyme in the urea cycle that converts arginine to ornithine and urea and, by modulating the availability of L-Arg for NOS, influences NO release in a variety of cells. Based on the above we hypothesized that enhancement in arginase II levels/activity in old oocytes mediates the decrease in NO and L-Arg levels and the increase in superoxide generation due NOS uncoupling, being a major cause of oocyte deterioration and infertility. To test our hypothesis, we first measured NO levels in young (8-14 weeks) vs. old mice (40-45 weeks) in normal uninjured ovaries. NO measurements were performed by monitoring NO concentration with an NO-electrode that was placed gently over the surface of the ovary, as far as possible from any visible blood vessels and in the oviducts of the mice. In order to verify the role of arginase II in regulating NOS activity, we next measured the expression of arginase II in both young and old ovaries. Gene expression levels in the ovaries of young vs old mice were determined for the ARG1 gene, which regulates expression of arginase I and II. Real-time qPCR was run in triplicate. To see the impact of this on the actual oocytes, we next measured the concentration of ornithine, final product of arginase II, in freshly harvested oocytes obtained from young (n=100; 6-10 week) as compared to old (n=100; 48-60 week) mice utilizing high-performance liquid chromatography (HPLC). Normal tissue NO levels of the uninjured ovary in young animals were in the 20 ± 1.5 nmol ranges, as opposed to, in old animals, the NO levels were markedly reduced by ∼60%. Comparable results were obtained when the NO-selective electrode was inserted in the oviducts of young and old mice. The levels of ornithine were dramatically increased in old compared to young oocytes. In contrast, arginine levels decreased by 50% in oocytes obtained from old mice as compared to young. There was also an enhancement of arginase II expression in old vs young mice as a ratio. These results suggest that arginase II activity is higher in old compared to young oocytes. Thus, the catalytic activity of NOS in old oocytes may be mediated by increased levels and activity of arginase II, thereby influencing NO bioavailability.

Metformin and vildagliptin combination: A new approach of endothelial nitric oxide synthase activity regulation and metabolism in diabetes mellitus type 2

This review analyzes the research results of additional effects and mechanisms of action of sugar-reducing drugs. Hence it has been suggested that asymmetric dimethylarginine may be a target molecule for metformin, vildagliptin, and combinations thereof. (For citation: Urakov AL, Gurevich KG, Sorokina IA, et al. Metformin and vildagliptin combination: a new approach of endothelial nitric oxide synthase activity regulation and metabolism in diabetes mellitus type 2. Reviews on Clinical Pharmacology and Drug Therapy. 2018;16(2):5-12. doi: 10.17816/RCF1625-12).

Study on Yueju Wan for "different diseases with same treatment" based on integrative pharmacology

The traditional Chinese medicine prescription Yueju Wan has the function of regulating qi-flowing for activating stagnancy and resolving food stagnation, which is now used for the treatment of depression and gastrointestinal related diseases. In this study, an integrative pharmacological method was adopted to predict the targets and pathways of Yueju Wan and explore its molecular mechanism for depression and gastrointestinal dysfunction with the same treatment. First, disease targets were collected from Human Phenotype Ontology database, 201 targets related to depression and 474 targets related to gastrointestinal dysfunction, including 95 common targets of these two. Then, the integrative pharmacology platform of traditional Chinese medicine (TCMIP) was used to predict and analyze the drug targets, GO function, KEGG pathway, core targets network of Yueju Wan and heterogeneous network of TCM-chemical components-key drug targets-pathway. According to the integrative analysis, it is found that ATP1A1, KRAS, and PRKAA1 were key targets, and neuron apoptotic process, neurotrophin signaling pathway, serotonergic synapse and regulation of nitric-oxide synthase activity were key pathways which played important roles in molecular mechanism of Yueju Wan for depression and gastrointestinal dysfunction. In conclusion, speculated serotonergic synapse and regulation of nitric-oxide synthase activity maybe the common process in depression and gastrointestinal dysfunction. This paper provided an overall understanding on Yueju Wan based on TCMIP, helping to elucidate the mechanism of the same treatment for different diseases, depression and gastrointestinal dysfunction.

Chronic intermittent hypoxia-induced neuronal apoptosis in the hippocampus is attenuated by telmisartan through suppression of iNOS/NO and inhibition of lipid peroxidation and inflammatory responses

Obstructive sleep apnea syndrome (OSAS) plays a critical role in the initiation and progression of Alzheimer׳s disease (AD), but little is known about the precise mechanism of OSAS-induced AD. Nitric oxide synthase (NOS) and nitric oxide (NO) are known to play key roles in the development of AD. Several studies have confirmed that an angiotensin II type 1 receptor blocker, telmisartan, beneficially regulates NOS and NO. Here, we examined the neuroprotective effects of telmisartan against hippocampal apoptosis induced by chronic intermittent hypoxia (CIH), the most characteristic pathophysiological change of OSAS. Adult male Sprague Dawley rats were subjected to 8h of intermittent hypoxia per day with or without telmisartan for eight weeks. Neuronal apoptosis in the hippocampal CA1 region, NOS activity, NO content, and the presence of inflammatory agents and radical oxygen species in the hippocampus were determined. The results showed that CIH activated inducible nitric oxide synthase (iNOS), increased NO content, and enhanced lipid peroxidation and inflammatory responses in the hippocampus. Treatment with telmisartan inhibited excessive iNOS and NO generation and reduced lipid peroxidation and inflammatory responses. In addition, telmisartan significantly ameliorated the hippocampal apoptosis induced by CIH. In conclusion, Pre-CIH telmisartan administration attenuated CIH-induced hippocampal apoptosis partly by regulating NOS activity, inhibiting excessive NO generation, and reducing lipid peroxidation and inflammatory responses.

Mechanism of the Protective Effect of Yulangsan Flavonoid on Myocardial Ischemia/Reperfusion Injury in Rats

Aims: Effect and mechanism of Yulangsan flavonoid (YLSF) on rat myocardial ischemia/reperfusion injury (MI/RI) has been investigated. Methods: Sprague-Dawley (SD) rats were randomly divided into seven groups (sham group, model group and NS group: 2 mL of normal saline/kg body weight was administered; diltiazem group: 5 mg of diltiazem hydrochloride/kg body weight was administered; YLSFL, YLSFM and YLSFH groups: 20, 40 and 80 mg of YLSF/kg body weight was administered) and the MI/RI model was established. Myocardial infarct area, levels of myocardial enzymes and nitric oxide synthase (NOS) were measured. Caspase-3 and adenine nucleotide translocator-1 (ANT1) mRNA expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Pathological structure and cardiocyte ultrastructure were also analysed. Results: Compared with the MI/RI group, pretreatment with YLSF or diltiazem hydrochloride decreased the infarct area, levels of inducible nitric oxide synthase (iNOS), caspase-3 as well as the leakage of myocardial enzyme and increased activities of total nitric oxide synthase (tNOS) as well as constitutive nitric oxide synthase (cNOS). Cellular edema and the infiltration of inflammatory cells were alleviated. Conclusions: The experiment showed that YLSF protected the heart against MI/RI, possibly by reducing lipid peroxidation damage, regulating NOS activity and modulating the apoptosis genes expression.

Dissecting regulation mechanism of the FMN to heme interdomain electron transfer in nitric oxide synthases

Nitric oxide synthase (NOS), a flavo-hemoprotein, is responsible for biosynthesis of nitric oxide (NO) in mammals. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their biological functions by tight control of interdomain electron transfer (IET) process through interdomain interactions. In particular, the FMN−heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O2 activation at the catalytic heme site. Emerging evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS by a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the FMN and heme domains in the three NOS isoforms. In the absence of a structure of full-length NOS, an integrated approach of spectroscopic, rapid kinetic and mutagenesis methods is required to unravel regulation mechanism of the FMN−heme IET process. This is to investigate the roles of the FMN domain motions and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in this area that are driven by the combined approach are the focuses of this review. A better understanding of the roles of interdomain FMN/heme interactions and CaM binding may serve as a basis for the rational design of new selective modulators of the NOS enzymes.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamusin vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on - l-[ 14C]arginine-to- l-[ 14C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN 2-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN 2-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN 2-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN 2-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN 2-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.

Therapeutic Use of Nitric Oxide Scavengers in Shock and Sepsis

Nitric oxide (NO) is a reactive radical produced by the enzyme nitric oxide synthase (NOS) and it plays an important role in a large number of biological pathways. NO can be produced under normal physiologic conditions and contribute to homeostasis but, when produced in excess, it may lead to tissue injury and organ dysfunction. The regulation of NOS activity and expression is becoming increasingly understood. NOS enzyme inhibitors as tools to decrease excessive NO synthesis have received the most attention and have been subjected to the greatest experimental study. Compounds that scavenge excessive NO have been developed and have shown promise in a number of experimental models but have received considerably less attention as potential therapeutic agents. In this article, the use of NO scavengers in two conditions in which excessive NO appears to be pathophysiologically significant, shock and sepsis, is reviewed. The relevant biology of NO in these disease states is presented and the therapeutic potential of NO scavengers in clinical care is explored.

High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase and arginine decarboxylase activities

Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method, we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC.

Quantification of ADMA: analytical approaches

Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantitative determination of N-monomethyl-L-arginine (NMMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in human blood and urine. These assays revealed elevated circulating levels of ADMA in various diseases and gave accumulating evidence of the usefulness of ADMA as a cardiovascular risk factor. However, the methods used are hampered by the fact that NMMA, ADMA and SDMA can be distinguished from L-arginine only by means of chromatographic separation. This has promoted the development of alternatives that involve mass spectrometry (MS) technology. Today, various MS-based approaches such as liquid chromatography (LC)-MS, LC-MS/MS, gas chromatography (GC)-MS, and GC-MS/MS are available. L-arginine and its analogs have been subjected to LC-MS analysis with and without further derivatization to their o-phthaldialdehyde derivatives. For these methods, labelled L-arginine was used as the internal standard. The first MS-based method that distinguishes NMMA, ADMA, SDMA and L-arginine by mass-to-charge (m/z)-ratio has been reported by Tsikas et al. This GC-MS approach has been further improved by Albsmeier et al by introducing labelled ADMA as an internal standard. As an alternative to existing methods, a commercially available ELISA kit has recently been developed and validated.

Quantification of ADMA: analytical approaches

Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantitative determination of N-monomethyl-L-arginine (NMMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in human blood and urine. These assays revealed elevated circulating levels of ADMA in various diseases and gave accumulating evidence of the usefulness of ADMA as a cardiovascular risk factor. However, the methods used are hampered by the fact that NMMA, ADMA and SDMA can be distinguished from L-arginine only by means of chromatographic separation. This has promoted the development of alternatives that involve mass spectrometry (MS) technology. Today, various MS-based approaches such as liquid chromatography (LC)-MS, LC-MS/MS, gas chromatography (GC)-MS, and GC-MS/MS are available. L-arginine and its analogs have been subjected to LC-MS analysis with and without further derivatization to their o-phthaldialdehyde derivatives. For these methods, labelled L-arginine was used as the internal standard. The first MS-based method that distinguishes NMMA, ADMA, SDMA and L-arginine by mass-to-charge (m/z)- ratio has been reported by Tsikas et al. This GC-MS approach has been further improved by Albsmeier et al by introducing labelled ADMA as an internal standard. As an alternative to existing methods, a commercially available ELISA kit has recently been developed and validated.

Expression of the protein inhibitor of nitric oxide synthase in the adult rat retina before and after optic nerve lesion

The molecular messenger nitric oxide (NO) not only serves a number of physiologic functions, but is also involved in the pathophysiology of neurodegeneration. It is produced by the nitric oxide synthase (NOS) isoenzymes. One of the many players regulating NOS activity is the Protein Inhibitor of NOS, PIN. To gain further insight into the mechanisms of NOS regulation and NO-mediated cell death after nerve trauma, we examined PIN expression in a standard model of lesion-induced neurodegeneration, the rat optic nerve transsection model. In both the axotomized retinae and the control retinae PIN expression was predominantly observed in the retinal ganglion cell layer. Optic nerve lesion did neither change the amount of PIN mRNA, as determined by in situ hybridization and real-time RT-PCR, nor did it change the amount of PIN as determined by immunohistochemistry and Western blot analysis. These results suggest that in our model, NOS activity is not regulated by altered PIN levels, which contributes to our understanding of apoptotic mechanisms in injured neurons.

Posttranslational regulation of NO synthase activity in the renal medulla of diabetic rats

Shear stress increases nitric oxide (NO) production by endothelial cells, inner medullary collecting duct cells, and thick ascending limb. We postulated that the osmotic diuresis accompanying type 1 diabetes is associated with increased NO synthase (NOS) activity and/or expression in the renal medulla. Diabetes was induced by injection of streptozotocin, with insulin provided to maintain moderate hyperglycemia (Hyp) or euglycemia (Eug) for 3 wk. Sham rats received vehicle treatments. A separate group of rats (Phz) received phlorizin to produce a glucose-dependent osmotic diuresis. Renal medullary NOS1 and NOS2 activities did not differ between groups, whereas NOS3 activity was significantly increased in Hyp. Neither NOS1 nor NOS3 protein levels differed significantly between groups. Reduced phosphorylation of NOS3 at Thr(495) and Ser(633) was evident in medullary homogenates from Hyp rats, with no difference apparent at Ser(1177). Immunohistochemical analysis indicated prominent expression of pThr(495)NOS3 in the thick ascending limb and collecting duct of Sham and Phz rats. Hyp rats displayed staining in the collecting duct but minimal thick ascending limb staining. Immunostaining with anti-pSer(1177)NOS3 was evident only in the thick ascending limb, with no apparent differences between groups. In summary, glucose-dependent osmotic diuresis alone did not alter NOS activity or expression in the renal medulla. Diabetic hyperglycemia increased medullary NOS3 activity without a concomitant increase in NOS3 protein levels; however, NOS3 phosphorylation was reduced at Thr(495) and Ser(633). Thus changes in the phosphorylation of NOS at known regulatory sites might represent the primary mechanism underlying increased renal medullary NOS activity in diabetic hyperglycemia.

Functional tetrahydrobiopterin synthesis in human platelets.

Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells.

Nitric oxide (NO) is produced by NO synthase (NOS) from L-arginine (L-Arg). Alternatively, L-Arg can be metabolized by arginase to produce L-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with L-valine (L-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-alpha (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of L-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM L-Val, EGM with 10 mM L-Arg, or EGM with both 10 mM L-Arg and 30 mM L-Val. In both control and L/T bPAEC, treatment with L-Val decreased urea production and increased NO production. Treatment with L-Arg increased both urea and NO production. The addition of the combination L-Arg and L-Val decreased urea production compared with the addition of L-Arg alone and increased NO production compared with L-Val alone. These data suggest that competition for intracellular L-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.