Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.     

An application of electron paramagnetic resonance to evaluate nitric oxide and its quenchers

Electron paramagnetic resonance (EPR) spectrometry has been used for detection of free radicals, including nitric oxide (NO). The method was applied to evaluate if lazaroids, nonspecific scavengers of oxygen-derived free radicals, can reduce another radical species, namely, NO. When solutions containing 0 to 10 microM lazaroids (U78517F, U83836E, and U74500A) were mixed with NO (approximately 8.0 microM), and with a spin-trap agent specific to NO (10 microM carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide, c-PTIO), the spectrum of NO-c-PTIO determined by the EPR decreased with increasing concentrations of lazaroids. An identical study with 1 to 1000 microM methylprednisolone or dexamethasone, which are prototypes of lazaroids, failed to reduce NO. In a separate study, rat red blood cells preloaded with U78517F were exposed to NO. EPR spectrometry demonstrated that hemoglobin in these cells were less nitrosylated compared with the cells not preloaded with U78517F. An ability of lazaroids to quench NO in vivo was evaluated by an intravenous injection of sodium nitrite (4 mumol/kg body weight) into rats pretreated with 10 mg/kg lazaroids (U78517F and U74500A). The nitrosylation of RBC hemoglobin was markedly attenuated in rats pretreated with lazaroids compared with those with vehicle. Thus, these lipophilic compounds protected cells from NO-induced nitrosylation both in vitro and in vivo. Lazaroids may be applicable as anti-NO agents.     

The active site of the bacterial nitric oxide reductase is a dinuclear iron center

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.     

Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1- B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.     

Dysregulated cytokine expression in vivo in prediseased and diseased autoimmune-prone MRL mice

Macrophages (m?) from prediseased autoimmune-prone MRL/ + and MRL/lpr mice produce markedly decreased levels of IL-1 in vitro in response to LPS. In contrast, tissues from diseased MRL/lpr mice overexpress IL-1 in vivo. To determine whether IL-1 underproduction in the MRL strains is solely an in vitro phenomenon, we compared in vivo cytokine mRNA expression from prediseased age-matched MRL/ + and MRL/lpr mice to that from normal BALB/c and C3HeB/FeJ mice. Like m? in vitro, whole organ RNA from the spleen, liver, and kidney of MRL/ + and MRL/lpr mice showed down-regulation of IL-1 RNA following intraperitoneal injection of LPS. This abnormality in inducible IL-1 expression was present in all MRL mice, irrespective of disease stage or the presence of the lpr gene. On the other hand, only diseased MRL/lpr mice displayed elevated and constitutive expression of IL-1 in their livers and kidneys. We suggest that inducible expression is most indicative of the intrinsic, or genetic, capacity of cells to produce cytokine, whereas constitutive expression reflects extracellular disease-related inflammatory stimuli present only in the diseased MRL/lpr strains. By restricting our studies to prediseased MRL mice, we have tried to eliminate the effects of disease and to focus on the predisposing genetic background. The existence both in vitro and in vivo of a defect in inducible IL-1 expression by prediseased MRL mice suggests that the molecular abnormality underlying this defect may be a part of this predisposing background to autoimmunity.     

B cell selection and allelic exclusion of an anti-DNA Ig transgene in MRL-lpr/lpr mice

We have used an Ig transgene (VH3H9) that increases the frequency of anti-DNA autoantibodies to address whether the production of antinuclear Abs in systemic lupus erythematosus is the consequence of a breakdown of B cell tolerance. We have shown that nonautoimmune mice regulate anti-DNA B cells, and that lupus-prone MRL-lpr/lpr mice are defective in this regulation. Here we show that a subset of anti-DNA B cells, namely those that stain nuclei in a homogeneous fashion, not only fail to be deleted in MRL-lpr/lpr mice, but undergo preferential clonal expansion. In addition, we describe a surprising finding: the VH3H9 transgene is less efficient at inhibiting endogenous heavy chain gene rearrangement on the autoimmune-prone MRL-lpr/lpr genetic background than on the nonautoimmune BALB/c background.     

Detection and evaluation of NO stores in awake rats

We demonstrated and substantiated the possibility of detection and evaluation of NO stores in freely moving awake rats. NO stores were created by administering NO donor or by heat shock and were then detected by hypotensive reaction to diethyldithiocarbamate (blood pressure monitoring) under conditions of NO synthase inhibition. Electron paramagnetic resonance revealed NO release from its stores by incorporation into paramagnetic mononitrosyl-iron complexes with diethyldithiocarbamate. Five hours after administration of NO donor or heat shock diethyldithiocarbamate induced a blood pressure drop and the appearance of electron paramagnetic resonance signals from the mononitrosyl-iron-diethyldithiocarbamate complex in rat heart, liver, kidneys, and brain. The hypotensive reaction to diethyldithiocarbamate and electron paramagnetic resonance signals were absent in control rats.     

Murine models of autoimmune disease and Sj?gren's syndrome

Murine models of autoimmune disease exhibit heterogenous features that are now being understood in terms of their basic molecular defects. Defects in apoptosis genes or genes that contribute to B-cell activation, T-cell tolerance loss, and development of autoimmune disease, including glomerulonephritis, have been identified. The interaction of primary and secondary predisposing genes leads to a diverse spectrum of autoimmune features that is different in different strains of mice. The challenge in the future will be to correlate these primary and secondary autoimmune genes with specific environmental triggers to explain the diverse spectrum of autoimmune disease. The ability to correct the apoptosis defect and autoimmune disease in CD2-Fas transgenic MRL-lpr/lpr mice brings hope that genetic therapy will eventually be possible in humans.     

Genetic determinants of autoimmune disease and coronary vasculitis in the MRL-lpr/lpr mouse model of systemic lupus erythematosus

MRL-lpr/lpr (MRL/lpr) mice are a model of human autoimmune disease. They exhibit a number of characteristics of systemic lupus erythematosus, including anti-DNA Abs, anti-cardiolipin Abs, immune complex-mediated vasculitis, lymphadenopathy, and severe glomerulonephritis. Although the autoimmune disorder is mediated primarily by mutation of the Fas gene (lpr), which interferes with lymphocyte apoptosis, MRL/lpr mice also have other predisposing genetic factors. In an effort to identify these additional factors, we have applied quantitative trait locus (QTL) mapping using an intercross between MRL/lpr mice and the nonautoimmune inbred strain BALB/cJ. A complete linkage map spanning the entire genome was constructed for 189 intercross progeny, and genetic loci contributing to features of the autoimmunity were identified using statistical analytic procedures. As expected, the primary genetic determinant of autoimmune disease in this cross was the Fas gene on mouse chromosome 19, exhibiting a lod score of 60. In addition, two novel loci, one on chromosome 2 (lod score, 4.3) and one on chromosome 11 (lod score, 3.1), were found to contribute to levels of anti-DNA Abs. Interestingly, the chromosome 19 and chromosome 11 QTLs, but not the chromosome 2 QTL, also exhibited associations with anti-cardiolipin Abs (lod scores, 38.4 and 2.6). We further examined the effects of these QTLs on the development of coronary vasculitis in the F2 mice. Our results indicate that the QTLs on chromosomes 11 and 19 also control the development of vasculitis, demonstrating common genetic determinants of autoantibody levels and vasculitis.     

Macrophage growth factors introduced into the kidney initiate renal injury

BACKGROUND: CSF-1 expression precedes renal injury in autoimmune MRL-lpr mice and is responsible for macrophage (M phi) proliferation and survival in the kidney. By comparison, C3H-lpr mice do not express CSF-1 in the kidney, and despite the lpr mutation, kidneys remain normal. The purpose of this study was to test the capacity of local and systemic expression of M phi growth factor, CSF-1 to initiate renal injury in normal (C3H-(++), MRL-(++) and autoimmune (C3H-lpr, MRL-lpr) mice. MATERIALS AND METHODS: We designed a gene transfer system to deliver cytokines into the kidney by transducing renal tubular epithelial cells (TEC) using retroviral vectors expressing CSF-1 or another M phi growth factor, GM-CSF. We placed transduced syngeneic cytokine-TEC under the renal capsule of normal and autoimmune prone mice prior to renal injury and evaluated renal pathology at 3, 7, 14, 28, and 90 days postimplant. RESULTS: CSF-1-TEC and GM-CSF-TEC, but not uninfected TEC, caused extensive local renal injury in strains with the lpr mutation. At 3-7 days the infiltrating cells were mainly M phi, and by 28 days they were predominantly lymphocytes. By comparison, the kidneys of MRL-(++) and C3H-(++) mice remained normal. Implanted genetically modified TEC caused a sustained increase of CSF-1 or GM-CSF in the circulation which did not modify the contralateral kidney. CONCLUSIONS: Gene transfer of M phi growth factors into the kidney initiates severe local renal injury in autoimmune prone mice with the lpr mutation, but does not compromise the kidney in nonautoimmune hosts. Of note, introduction of M phi growth factors into the kidney of C3H-lpr mice which do not spontaneously develop renal injury incites renal damage. These studies offer a gene transfer approach to explore the impact of local and systemic cytokine production on renal injury.     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Regional blood flow alterations after bovine fumaryl beta beta-crosslinked hemoglobin transfusion and nitric oxide synthase inhibition

OBJECTIVES: a) To determine whether isovolemic exchange transfusion with cell-free, bovine fumaryl beta beta-crosslinked hemoglobin results in a different pattern of regional blood flow distribution than transfusion with a poor oxygen-carrying, colloidal solution. b) Because of potential nitric oxide scavenging by plasma-based hemoglobin, to determine whether blood flow differences are reduced after nitric oxide synthase inhibition. DESIGN: A prospective, randomized design with repeated blood flow measurements within groups. SETTING: Experimental physiology laboratory in a university medical center. SUBJECTS: Pentobarbital-anesthetized female cats. INTERVENTIONS: Three groups of eight cats were studied: a) a control group with no transfusion (hematocrit of 32%); b) an anemia group in which exchange transfusion with an albumin-containing solution reduced hematocrit to 18% over a 40- to 50-min period; and c) a group in which cell-free hemoglobin was exchanged transfused to reduce hematocrit to 18%, without a proportional reduction in oxygen-carrying capacity. Bovine hemoglobin was covalently crosslinked intramolecularly between the 81-lysine residues on the beta-subunits to stabilize the tetramer. Regional blood flow was measured by the radiolabeled microsphere technique before transfusion and at 10, 100, and 180 mins from the start of transfusion. At 190 mins, N omega-nitro-L-arginine methyl ester (L-NAME; 10mg/kg) was infused to inhibit nitric oxide synthase and blood flow was measured 30 mins later. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure was unchanged in the control and albumin-transfused groups. However, mean arterial pressure increased rapidly in the hemoglobin-transfused group. With hemoglobin transfusion, there were marked reductions in blood flow to the intestines, kidneys and adrenal glands. Administration of L-NAME after hemoglobin transfusion failed to increase arterial pressure or cause further reductions in intestinal, renal, or adrenal blood flow. Administration of L-NAME to the control and albumin-transfused groups increased arterial pressure and reduced intestinal, renal, and adrenal blood flows to values attained with hemoglobin transfusion. In contrast, in skeletal muscle and left ventricle, blood flow rates increased in the albumin-transfused group and were greater than those values found in the control group and hemoglobin-transfused group. The greater flow in the albumin-transfused group persisted after L-NAME administration. There was no difference in renal sodium, potassium, or osmolar excretion, or in urine flow between groups. CONCLUSIONS: Transfusion with cell-free, bovine crosslinked hemoglobin in cats can selective reductions in blood flow in the intestines, kidneys, and adrenal glands without evidence of renal dysfunction by a mechanism consistent with nitric oxide scavenging. In skeletal and cardiac muscle, the increase in blood flow persisted after nitric oxide inhibition in the albumin group relative to the hemoglobin-transfused group at equivalent hematocrit values. This finding is consistent with compensatory vasoconstriction with hemoglobin transfusion due to improved oxygenation by this oxygen carrier.     

Genotype effects on the antioxidant enzymes activity and mRNA expression in liver and kidney tissues of autoimmune-prone MRL/MpJ-lpr/lpr mice

Congeneic pairs of MRL/lpr and MRL/++ (+/+) mice differ in incidence of autoantibodies, lymphoproliferative disease and survival, characteristics that are linked to immunological abnormalities. MRL/lpr mice have a significantly shorter life span compared to +/+ mice. Because a weak antioxidant defense and an increased generation of free radicals are generally implicated in the severity of many autoimmune disease, the present study was undertaken to compare the influence of genotype on lipid composition, lipid peroxidation and expression of mRNA, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in the livers and kidneys of these mice. The expression of SOD, GSH-Px and CAT mRNAs was significantly higher (P < 0.05) in the livers of +/+ mice, while in the kidneys only SOD expression was found significantly higher in +/+ mice when compared to MRL/lpr mice. Further, the activity of cytosolic SOD and GSH-Px was also found significantly higher (P < 0.001) in the livers of +/+ mice. Both livers and kidneys of MRL/lpr mice exhibited significantly higher levels of arachidonic acid (20:4(n-6)), significantly higher generation of thiobarbituric acid reactive substances (TBARS) and higher estimated peroxidation index than the +/+ mice. In addition, the MRL/lpr mice had higher levels of serum anti-cardiolipin antibodies. In summary, the results from the present study indicate that besides several immune-related abnormalities, the MRL/lpr mice may exhibit their inability to cope with oxidative stress due to a poor antioxidant defense system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rats maintained on high-fat diets exhibit reduced satiety in response to CCK and bombesin

Mycophenolate mofetil (MMF) is the morpholinoethyl ester of mycophenolic acid, which is its active metabolite. MMF is effective in prolonging survival of allografts and xenografts. However, little is known about the effects and the main mechanism of action of MMF in autoimmune diseases. In this study, the effect of MMF on the spontaneous disease progression in the MRL/lpr mouse model of lupus was examined. Eight-week-old MRL/lpr mice (n=18) were orally treated with MMF dissolved in a vehicle (90 mg/kg) once a day. Control animals received vehicle alone (n=17). The incidence of albuminuria (>300 microg/18 h) was significantly reduced by MMF treatment compared with vehicle-treated controls (cumulative incidence of albuminuria at 23 wk in MMF-treated mice; 22% versus 88% in controls; P=0.0001). The glomerulonephritis was histologically less severe in MMF-treated mice than in control mice (P=0.005). Furthermore, in immunofluorescence studies the amount of immunoglobulin and C3 deposits in the glomerular capillary wall was significantly less in MMF-treated mice (P < or = 0.002). Surprisingly, in vivo no clear-cut immune-modulating effects were observed because there were no differences between MMF-treated and control animals with regard to autoantibody formation. Also, spleen enlargement and numbers of CD3+, CD4+, and CD8+ T cells in spleen, lymph nodes, and peripheral blood were not different between both groups. Furthermore, no immunosuppressive properties of 90 mg/kg MMF were found in BALB/c mice on delayed-type hypersensitivity and primary antibody response to methylated bovine serum albumin. Interestingly, renal perfusion experiments revealed that binding of nucleosome/antinucleosome complexes to the glomerular basement membrane is decreased in MMF-treated mice compared with control mice. It is concluded that MMF suppresses the development of lupus glomerulonephritis and albuminuria in MRL/ lpr mice. The observed reduction of glomerular immunoglobulin deposits in MMF-treated mice and the renal perfusion studies indicate that MMF treatment leads to a decreased binding of immune complexes in the glomerular capillary wall in lupus nephritis.     

Direct detection of radicals in intact soybean nodules: presence of nitric oxide-leghemoglobin complexes

Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO. may involve regulation of gene activity.     

Host modifier genes affect mouse autoimmunity induced by the lpr gene

A defect in apoptotic signal transmission through CD95 is an essential genetic mechanism for lymphoproliferation and autoimmunities in lpr or gld mice. However, disease manifestations are largely affected by the host genetic background. To identify and map such host genes modifying lpr gene effect, ie, the lpr modifier (Lprm) genes, 82 MRL/lpr x (MRL/lpr x C3H/lpr) F1 mice were subjected to immunopathological and genetical analyses. High-grade vasculitis and glomerulonephritis among backcross mice were observed in separate groups of mice. Microsatellite analysis revealed that there were two host genes affecting the occurrence of vasculitis, Lprm1 (chromosome 4) and Lprm2 (chromosome 3). A recessive MRL allele at Lprm1 enhanced vasculitis to occur in both sexes, whereas that of Lprm2 inhibited its development selectively in females. Genotype combinations of these two genes explained the severity of vasculitis in crosses of MRL/lpr and C3H/lpr mice and also the vasculitis-prone recombinant inbred strain McH5/lpr. A recessive MRL allele at Lprm3 (chromosome 14) suppressed glomerulonephritis. The weight of the spleen was increased by a recessive MRL allele at Lprm4 (chromosome 5) yielding a logarithm of odds score of 2.02 in a quantitative trait locus analysis. In contrast, the weight of axillary lymph nodes was increased by a recessive MRL allele at a locus on chromosome 2, but its presence was not supported by the quantitative trait locus analysis. The titer of anti-dsDNA autoantibody was controlled by the locus Lprm5 on chromosome 16, which had an logarithm of odds score of 3.41. Possible candidate genes for Lprm genes deduced from their map locations are discussed and compared with the autoimmunity genes reported thus far. In conclusion, autoimmune disease manifestations by the lpr mutation are affected by multiple host genes separately.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.     

Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation

BACKGROUND: Inhaled nitric oxide gas selectively decreases pulmonary artery pressure without affecting systemic arterial pressure. To determine if the selective pulmonary vasodilating effect of inhaled nitric oxide gas is due to inactivation by hemoglobin, we studied the ability of whole blood to inhibit the vasodilator activity of effluent from isolated lungs exposed to inhaled nitric oxide. METHODS AND RESULTS: The effluent from ventilated, Krebs-perfused rabbit lungs was passed directly over 3- to 4-mm rabbit aortic rings. Inhaled nitric oxide (150 ppm for 3 minutes) reduced pulmonary perfusion pressure, elevated by a continuous infusion of U46619, by 35 +/- 7% (mean +/- SEM, n = 5). Lung effluent from this series of experiments caused 40 +/- 13% relaxation of phenylephrine-preconstricted aortic rings. When blood was added to the combined lung/ring perfusion cascade (final hemoglobin concentration, 1 g/dL), inhaled nitric oxide again significantly reduced pulmonary perfusion pressure, but the effluent now failed to relax the aortic rings (30 +/- 6% [control] versus 1.5 +/- 1% [blood]). Both reduction in pulmonary perfusion pressure and relaxation of the rings during nitric oxide exposure were unchanged from control values after discontinuing the blood infusion. CONCLUSIONS: The presence of hemoglobin, even in extremely small amounts, restricts the vasodilating effect of inhaled nitric oxide gas to the pulmonary circulation.