Ernst E. van Faassen

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Whole Body UVA Irradiation Lowers Systemic Blood Pressure by Release of Nitric Oxide From Intracutaneous Photolabile Nitric Oxide Derivates

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm2) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15N-nitrite, whole body UVA irradiation (20 J/cm2) induced significant levels of 15N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R2=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (−28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia

Background Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. Methodology/Principal Findings To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg•g bodyweight−1•h−1), combined with either L-Citrulline (6.25 mg•h-1), L-Arginine (6.25 mg•h−1), or L-Alanine (isonitrogenous control; 12.5 mg•h−1) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. Conclusion/Significance L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Controlled radical polymerization of styrene in the presence of lithium molybdate(V) complexes and benzylic halides

Abstract The new lithium molybdate(V) complexes [LiMo(NAr) 2 ( C – N )R] ( C – N =C 6 H 4 (CH 2 NMe 2 )-2; R=( C – N ) ( 5 ), Me ( 6 ), CH 2 SiMe 3 ( 7 ), p -tolyl ( 8 )), have been generated in situ from reaction of the corresponding molybdenum(VI) complexes [Mo(NAr) 2 ( C – N )R] ( C – N =C 6 H 4 (CH 2 NMe 2 )-2; R=( C – N ) ( 1 ), Me ( 2 ), CH 2 SiMe 3 ( 3 ), p -tolyl ( 4 )) with n -BuLi. The nature of these radical anions was studied by EPR spectroscopy. The spectra of toluene solutions of in situ prepared complexes 5 – 8 revealed the presence of two different paramagnetic species, i.e. a molybdenum compound with distinct g iso - and A iso -values and an unidentified radical with a sometimes strong signal at g =1.986±0.001, lacking any hyperfine coupling. Extended Huckel calculations on the crystal structure of 5 showed that the single electron occupies a molybdenum centered orbital, merely d x 2 − y 2 in character. In situ prepared complexes 5 – 8 were successfully applied in the atom transfer radical polymerization (ATRP) of styrene using benzyl chloride as the initiator. The efficiency of the benzyl chloride initiator is rather poor (6–18%). Reaction of the lithium molybdate(V) complex 5 with (α-chloroethyl)benzene and (α-bromoethyl)benzene resulted in the formation of 1 , LiCl and LiBr, respectively. The molecular weights as well as the molecular weight distributions show that the catalytic system, BzCl/ 5 – 8 , catalyses styrene polymerization successfully but does not exercise much control over the polymerization reaction due to the poor initiator efficiency of benzyl chloride and probably the extreme air-sensitivity of the lithium molybdate(V) compounds. The unidentified radical ( g =1.986±0.001) is unable to initiate radical polymerization but possibly influences the ATRP activity.

Maternal Supplementation With Citrulline Increases Renal Nitric Oxide in Young Spontaneously Hypertensive Rats and Has Long-Term Antihypertensive Effects

NO deficiency is associated with development of hypertension. Defects in the renal citrulline-arginine pathway or arginine reabsorption potentially reduce renal NO in prehypertensive spontaneously hypertensive rats (SHRs). Hence, we investigated genes related to the citrulline-arginine pathway or arginine reabsorption, amino acid pools, and renal NO in 2-week-old prehypertensive SHRs. In addition, because perinatally supporting NO availability reduces blood pressure in SHRs, we supplemented SHR dams during pregnancy and lactation with citrulline, the rate-limiting amino acid for arginine synthesis. In female offspring, gene expression of argininosuccinate synthase (involved in renal arginine synthesis) and renal cationic amino acid Y-transporter (involved in arginine reabsorption) were both decreased in 2-day and 2-week SHRs compared with normotensive WKY, although no abnormalities in amino acid pools were observed. In addition, 2-week-old female SHRs had much less NO in their kidneys (0.46±0.01 versus 0.68±0.05 nmol/g of kidney weight, respectively; P<0.001) but not in their heart. Furthermore, perinatal supplementation with citrulline increased renal NO to 0.59±0.02 nmol/g of kidney weight (P<0.001) at 2 weeks and persistently ameliorated the development of hypertension in females and until 20 weeks in male SHR offspring. Defects in both the renal citrulline-arginine pathway and in arginine reabsorption precede hypertension in SHRs. We propose that the reduced cationic amino acid transporter disables the developing SHR kidney to use arginine reabsorption to compensate for reduced arginine synthesis, resulting in organ-specific NO deficiency. This early renal deficiency and its adverse sequels can be corrected by perinatal citrulline supplementation persistently in female and transiently in male SHRs.

A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type 1, is required for apical protein expression and microvillus formation in polarized epithelial cells.

Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P4 P‐type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1‐deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain‐specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco‐2 cells. Caco‐2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin‐labeled phospholipids. The polarized organization of these cells was determined by pulse‐chase analysis, cell‐fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco‐2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1‐depleted Caco‐2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency. (HEPATOLOGY 2010)

Anti-inflammatory effects of tetrahydrobiopterin on early rejection in renal allografts: modulation of inducible nitric oxide synthase.

Oxidative stress contributes to the development of early transplant failure. As nitric oxide synthases (NOS) can act as sources of superoxide, we investigated the effect of the NOS cofactor tetrahydrobiopterin (BH4) on oxyradical production and early rejection in a rat kidney transplantation model. Allograft transplantation (Brown Norway to Lewis) showed more renal superoxide production and monocyte infiltration when compared with isografts (Lewis to Lewis). Administration of the stable BH4 precursor sepiapterin had no effect on superoxide production in the isografts (51±10 vs. 69±17 cps/10 mg protein), but led to a marked decrease in superoxide production in the allografts (116±11 vs. 60±6 cps/10 mg protein; P<0.05) and was accompanied by a reduction in periarterial macrophage infiltration (3.3±0.7 vs. 1.3±0.3 cells/vessel; P<0.05) and an increase in NO production (78±22 vs. 173±12 AU/g kidney) (P<0.01). In vitro experiments confirm that iNOS can produce superoxide mainly from the heme domain, whereas BH4 administration can reverse this superoxide production in the presence of adequate anti‐oxidant defense. Our findings support the hypothesis that BH4 can be used to modulate the function of the inflammatory iNOS isoform and suggest a potential therapeutic role for sepiapterin in early allograft rejection.

An electron paramagnetic resonance study of the antioxidant properties of the nitroxide free radical tempo

Previous reports of superoxide scavenging and ferroxidase-like activity of nitroxide free radicals have greatly increased interest in the ability of these compounds to protect cells against oxidative cellular damage. In the present study we investigated the antioxidant properties of the six membered nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in various assays. TEMPO (5 mM) inhibited the hydroxyl radical mediated generation of ethylene from 2-keto-4-methylthiobutyric acid to 31.2 +/- 4.0% of control values. Furthermore, we noted that TEMPO had the ability to maintain iron in its ferric form, a finding with strong implications for the interpretation of the ferricytochrome c reduction assay. TEMPO may be reduced to an electron paramagnetic resonance silent hydroxylamine by a number of pathways. TEMPO absorption intensity decay (TAID) was monitored to investigate the effects of hydrogen peroxide on Cu,Zn-SOD. TEMPO was found to effectively scavenge or suppress formation of hydroxyl radicals inside Cu,Zn-SOD. The generation of hydroxyl radical was confirmed by employing the conventional spin trapping agent DMPO. Using radical scavengers unable to penetrate the Cu,Zn-SOD enzyme (e.g., mannitol, ethanol, albumin) or compounds with access to copper within the Cu,Zn-SOD enzyme (azide and cyanide), we could not detect hydroxyl radicals outside the enzyme. Finally, since the electron paramagnetic resonance absorption intensity is directly proportional to the concentration of TEMPO spins, loss of absorption intensity provided information concerning radical-mediated processes. Therefore, the decay kinetics of TEMPO may be used as a very sensitive alternative to conventional spin traps.

Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.