Owen W. Griffith

BIOLOGIC AND PHARMACOLOGIC REGULATION OF MAMMALIAN GLUTATHIONE SYNTHESIS

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) is synthesized from its constituent amino acids by the sequential action of gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase. The intracellular GSH concentration, typically 1-8 mM, reflects a dynamic balance between the rate of GSH synthesis and the combined rate of GSH consumption within the cell and loss through efflux. The gamma-GCS reaction is rate limiting for GSH synthesis, and regulation of gamma-GCS expression and activity is critical for GSH homeostasis. Transcription of the gamma-GCS subunit genes is controlled by a variety of factors through mechanisms that are not yet fully elucidated. Glutathione synthesis is also modulated by the availability of gamma-GCS substrates, primarily L-cysteine, by feedback inhibition of gamma-GCS by GSH, and by covalent inhibition of gamma-GCS by phosphorylation or nitrosation. Because GSH plays a critical role in cellular defenses against electrophiles, oxidative stress and nitrosating species, pharmacologic manipulation of GSH synthesis has received much attention. Administration of L-cysteine precursors and other strategies allow GSH levels to be maintained under conditions that would otherwise result in GSH depletion and cytotoxicity. Conversely, inhibitors of gamma-GCS have been used to deplete GSH as a strategy for increasing the sensitivity of tumors and parasites to certain therapeutic interventions.

N5-(1-IMINO-3-BUTENYL)-L-ORNITHINE : A NEURONAL ISOFORM SELECTIVE MECHANISM-BASED INACTIVATOR OF NITRIC OXIDE SYNTHASE

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion ofl-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-l-NIO (N 5-(1-imino-3-butenyl)-l-ornithine;l-VNIO) binds to and inhibits nNOS in competition with l-arginine (K i = 100 nm); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2,l-VNIO irreversibly inactivates nNOS (k inact = 0.078 min−1;K I = 90 nm); inactivation is Ca2+/calmodulin-dependent. The cytochromec reduction activity of the enzyme is not affected by such treatment, but the l-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by l-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated byl-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain ∼75% of the rate of inactivation seen with nNOS. Among the NOS inactivating l-arginine derivatives,l-VNIO is the most potent and nNOS-selective reported to date.

Design of isoform-selective inhibitors of nitric oxide synthase

Nitric oxide synthase, the mammalian enzyme catalyzing the oxidation of L-arginine to L-citrulline and nitric oxide, is present in three isoforms that have distinct physiological roles. Overstimulation or overexpression of individual nitric oxide synthase isoforms plays a role in a wide range of disorders including septic shock, arthritis, diabetes, ischemia-reperfusion injury, pain and various neurodegenerative diseases. Animal studies and early clinical trials suggest that nitric oxide synthase inhibitors could be therapeutic in many of these disorders, but preservation of physiologically important nitric oxide synthase functions might require use of isoform-selective inhibitors. Within the past few years both amino acid and nonamino acid nitric oxide synthase inhibitors with pharmacologically useful isoform selectivity have been reported. Selectivity has been achieved on the basis of initial binding affinity and, for mechanism-based inactivators, on the basis of isoform-dependent catalytic activation; particularly interesting are N5-(1-imino-3-butenyl)-L-ornithine, ARL 17477, 1400W and S-(2-aminoethyl)isothiourea.

Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3.

Nitric oxide synthase (NOS) is believed to play an important role in protecting the myocardium against ischemia. Chronic hypoxia from birth increases NOS activity in the myocardium resulting in enhanced nitric oxide production and increased resistance to ischemia. We examined the effects of chronic hypoxia on NOS gene and protein expression and on NOS protein association with caveolin-3. Rabbits were raised from birth in a normoxic (F(I)O(2) = 0.21) or a hypoxic (F(I)O(2) = 0.12) environment for 9 d, and then the hearts were isolated. Ribonuclease protection assays revealed that chronic hypoxia did not alter NOS transcript levels for NOS1, NOS2, or NOS3. The most abundant transcript was NOS3. Western analysis revealed NOS3 was the only isoform detected. Immunoblots of NOS3 immunoprecipitates showed that chronic hypoxia increases NOS3 protein by 2.0 +/- 0.4-fold and decreases the amount of caveolin-3 that can be coprecipitated with NOS3 by 5.5 +/- 0.9-fold. Immunoblots of normoxic and hypoxic hearts showed that chronic hypoxia decreases the amount of caveolin-3 in heart homogenates by 2. 2 +/- 0.5-fold. These data suggest that a decrease in caveolin-3 plays a role in the mechanisms by which chronic hypoxia increases NOS3 activity in the myocardium.

Adaptation to Chronic Hypoxia Confers Tolerance to Subsequent Myocardial Ischemia by Increased Nitric Oxide Production

Abstract: Chronic exposure to hypoxia from birth increased the tolerance of the rabbit heart to subsequent ischemia compared with age‐matched normoxic controls. The nitric oxide donor GSNO increased recovery of post‐ischemic function in normoxic hearts to values not different from hypoxic controls, but had no effect on hypoxic hearts. The nitric oxide synthase inhibitors L‐NAME and L‐NMA abolished the cardioprotective effect of hypoxia. Message and catalytic activity for constitutive nitric oxide synthase as well as nitrite, nitrate, and cGMP levels were elevated in hypoxic hearts. Inducible nitric oxide synthase was not detected in normoxic or chronically hypoxic hearts. Increased tolerance to ischemia in rabbit hearts adapted to chronic hypoxia is associated with increased expression of constitutive nitric oxide synthase.

Measurements of total plasma nitrite and nitrate in pediatric patients with the systemic inflammatory response syndrome

OBJECTIVESnThe systemic inflammatory response syndrome (SIRS) is typified by the presence of fever, hemodynamic changes, and end organ dysfunction. Endothelial cell activation leads to overproduction of nitric oxide, which results in sustained vasodilation and hypotension. This study was undertaken to determine the sensitivity, specificity, and positive and negative predictive values of plasma nitrite/nitrate measurements in identifying patients with clinical characteristics of SIRS, as defined by criteria based on physician diagnosis.nnnDESIGNnProspective cohort study with consecutive sampling of patients.nnnSETTINGnTertiary, multidisciplinary, pediatric intensive care unit (ICU) at Childrens Hospital of Wisconsin.nnnPATIENTSnPatients were divided into five groups. There were 16 pediatric controls undergoing elective surgery and 177 pediatric ICU patients without and 46 pediatric ICU patients with physician-diagnosed sepsis, septic shock, SIRS, or sepsis syndrome documented in the medical record (all considered physician-diagnosed sepsis). The 223 pediatric ICU patients included 195 pediatric ICU patients not meeting and 28 pediatric ICU patients meeting predetermined physiologic criteria for SIRS (considered criteria-based sepsis).nnnINTERVENTIONSnBlood samples were obtained for quantitative nitrite/nitrate analysis at the time of admission to the pediatric ICU and daily until discharge.nnnMEASUREMENTS AND MAIN RESULTSnMean plasma nitrite/nitrate concentrations in the controls were 34.5 +/- 12 microM (95th percentile 54 microM). In pediatric ICU patients without and with physician-diagnosed sepsis, mean plasma nitrite/nitrate concentrations were 39 +/- 24 microM (p > .05 compared with controls) and 127 +/- 91 microM (p < .0001 compared with both controls and patients without physician-diagnosed sepsis), respectively. In pediatric ICU patients without and with criteria-based sepsis, the mean total plasma nitrite/nitrate concentrations were 56 +/- 59 microM (p = .008 compared with controls) and 80 +/- 64 microM (p = .003 compared with patients without criteria-based sepsis), respectively. The ability of plasma nitrite/nitrate > 54 microM to identify patients with physician-diagnosed sepsis is characterized as follows: 87% sensitivity, 77% specificity, 50% positive predictive value, and 96% negative predictive value. The ability of plasma nitrite/nitrate > 54 microM to identify patients with criteria-based sepsis is characterized as follows: 61% sensitivity, 68% specificity, 21% positive predictive value, and 92% negative predictive value.nnnCONCLUSIONSnClinical diagnosis of SIRS is strongly associated with increased total plasma nitrite/nitrate concentrations in pediatric patients in the pediatric ICU. Many patients with increased nitrite/nitrate concentrations have inflammation without having a clinical diagnosis of SIRS. Our data suggest that increased plasma nitrite/nitrate concentrations are the standard for identifying patients with inflammation in the pediatric ICU.

Non-heme iron protein: A potential target of nitric oxide in acute cardiac allograft rejection

We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis → Lewis) and mismatched (allografts: Wistar–Furth → Lewis) rats. On postoperative days (POD) 4–6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl–iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to ≈50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl–iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl–iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N6-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.

Nitric oxide synthesis from L-arginine modulates renal vascular resistance in isolated perfused and intact rat kidneys

This study tested the effects on renal hemodynamics of blockage of nitric oxide (NO) synthesis from L-arginine with N G -methyl-L-arginine (L-NMMA) using both intact and isolated perfused rat kidneys. Infusion of L-NMMA into anesthetized rats increased the mean arterial pressure and reduced the glomerular filtration rate and renal plasma flow only when the renal perfucion pressure was maintained at control levels. In isolated kidneys, L-NMA increased vascular resistance; this was attenuated by coadministration of L-arginine.

L-ARGININE BINDING TO NITRIC-OXIDE SYNTHASE : THE ROLE OF H-BONDS TO THE NONREACTIVE GUANIDINIUM NITROGENS

Nitric-oxide synthase (NOS) catalyzes the oxidation of l-arginine to nitric oxide andl-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of thel-arginine-binding site. We report here studies withl-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the –NH2, =NH, or =NH2 + groups of l-arginine. Replacement groups include –NH-alkyl, –alkyl, =O, and =S. Binding ofl-canavanine, an analog unable to form hydrogen bonds involving a N 5-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the l-arginine-binding site and use IC50 or K i values and optical difference spectra to quantitate their affinity relative tol-arginine. We find that the non-reactive guanidinium nitrogen of l-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3–6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design.

INHIBITION OF ENDOTHELIAL CELL AMINO ACID TRANSPORT SYSTEM Y+ BY ARGININE ANALOGS THAT INHIBIT NITRIC OXIDE SYNTHASE

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.