Biomedical subjects
R G Knowles
Publications and source records attributed to R G Knowles.
3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine.
This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.
Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose.
Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I-III. Both GSH (IC50 =10 microM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 microM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity.
Myometrial arginase activity increases with advancing pregnancy in the guinea pig.
OBJECTIVES: Arginase has been suggested to play an important role in cellular growth and development, particularly important to the fetus, by supplying L-ornithine for the synthesis of polyamines. The purpose of this investigation was to determine whether pregnancy alters myometrial arginase activity and whether estradiol was responsible for the change. STUDY DESIGN: Myometrium and kidney were obtained from nonpregnant and pregnant guinea pigs of known gestational age. Arginase activity was measured under physiologic conditions by the conversion carbon 14-labeled guanidino-L-arginine to carbon 14-labeled urea. The concentrations of the enzyme's substrate, L-arginine, and its principal metabolite, L-ornithine, were measured in myometrium from near-term pregnant animals by use of an amino acid analyzer. Finally, a group of random cycle guinea pigs received 500 microgram/kg estradiol for 5 days before the myometrium was removed. RESULTS: Myometrial arginase activity in pregnant animals was more than double that of myometrium from nonpregnant animals by the time the first measurement was made at 0.14 gestation. It continued to rise, peaking at values >25-fold higher than the nonpregnant activity by 0.90 gestation. Arginase activity in the myometrium underlying the placental implantation site was >25 fold higher (p<0.05) than myometrium from nonpregnant animals when first studied at 0.63 gestation and 10-fold higher than the contralateral fundal myometrium at the same time of gestation. Myometrial arginase activity in the sterile horn of six pregnant animals was half that of the horn containing one or more pups, but still five times higher than that of nonpregnant animals. Renal arginase activity also rose with advancing pregnancy, but the magnitude of the increase (up to 2-fold) was much smaller than that observed in either the fundal or placental implantation site myometrium. Estradiol had no significant effect on myometrial arginase activity. CONCLUSIONS: These studies demonstrate that pregnancy increases myometrial arginase activity and that the presence of placenta or fetus is necessary for the maximal effect.
Expression of human nitric oxide synthase isozymes.
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Metabolic control analysis of purine biosynthesis from serine in Molt-4 cells.
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The ontogeny of cerebral and cerebellar nitric oxide synthase in the guinea pig and rat.
The appearance of nitric oxide synthase (NOS, EC 1.14.13.39) activity in the brain of fetal and neonatal guinea pigs and rats was studied. In the guinea pig, NOS increased from an almost undetectable level at 0.49 of gestation (31 d), reaching adult levels before birth and peaking at 140% of the adult activity (forebrain) or 250% of the adult activity (cerebellum) in the week after birth. The rise in fetal NOS activity followed the reported rise in the estrogen receptor concentration in the brain and could be reduced by treatment of the guinea pig at full term with tamoxifen, implicating estrogens in the expression of fetal NOS activity. In the rat, brain NOS activity did not rise significantly until after birth, reaching adult levels approximately 2 wk after birth, and rising to 150 or 130% of the adult activity in the forebrain and cerebellum, respectively, at 4 wk after birth. The appearance of NOS activity in the rat also followed the reported appearance of estrogen receptors in the brain. In both species the appearance of high NOS activity in the brain immediately precedes the period in which maximal synaptogenesis occurs: immediately before birth in the guinea pig and 2-3 wk after birth in the rat. Thus the appearance of a functional estrogen-estrogen receptor system in the brain may be responsible, at least in part, for the expression of a high activity of NOS, which in turn may play important roles in promoting cerebral blood flow and synaptogenesis in the developing brain.
The role of indoleamine 2,3-dioxygenase in the anti-tumour activity of human interferon-gamma in vivo.
We have studied the relationship between L-tryptophan metabolism and the response to human IFN-gamma in 3 human ovarian cancer xenografts growing in nude mice. During IFN-gamma therapy all 3 tumours showed a profound depletion in L-tryptophan and a corresponding rise in L-kynurenine. The microenvironment surrounding the tumours was also depleted of L-tryptophan. The IFN-gamma-inducible enzyme indoleamine dioxygenase, IDO, was induced in treated tumours. While there was a variability in IDO mRNA expression in the different xenografts tested, in situ hybridization showed that the gene was induced at all levels of the tumour, and not just the periphery. These results show that induction of IDO by IFN-gamma in vivo can metabolize L-tryptophan rapidly enough for it to become depleted, despite a continued supply of L-tryptophan from the host. The IDO mRNA and protein remained induced after the L-tryptophan levels had returned to normal, suggesting that the gene may be post-transcriptionally regulated and/or the IDO co-factor supply may be limited. Another IFN-gamma-inducible gene, tryptophanyl tRNA synthetase, was also induced in the tumour. It is possible that this enzyme, which is responsible for synthesizing tryptophanyl tRNA, acts in a compensatory manner by allowing protein synthesis to continue despite low free L-tryptophan concentrations. There was no correlation of the above parameters with the anti-tumour response to IFN-gamma, suggesting that other mechanisms must play a role. L-tryptophan depletion may be a contributor to a multifactorial growth inhibition of tumour cells following IFN-gamma treatment, but cannot on its own explain their growth inhibition.
Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis.
Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-alpha (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6 x DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was covered rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrate, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71 +/- 0.04 l/kg and the clearance was 0.32 +/- 0.02 l/h-1/kg-1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.
Nitric oxide synthase activity in human breast cancer.
Nitric oxide (NO) is generated by a family of isoenzymes (NO synthases) expressed in a wide range of mammalian cells. We have recently reported NO synthase expression in human gynaecological cancers. In this study we have assessed the activity and distribution of NO synthase in a series of human breast tumours and in normal breast tissue. Calcium-dependent (constitutive) and -independent (inducible) NO synthase activity, as well as NO biosynthesis, was high in invasive tumours compared with benign or normal tissue. Furthermore, for invasive ductal carcinomas, NO biosynthesis was significantly greater for grade III compared with grade II tumours. Immunohistochemical investigations revealed immunolabelling with a monoclonal antibody to murine inducible NO synthase predominantly within tumour-associated macrophages. Immunolabelling with a polyclonal antiserum raised against rat brain NO synthase was also observed in vascular endothelial and myoepithelial cells. Thus NO synthase is expressed in human breast tumours, where its presence correlates with tumour grade.
Mechanism of inhibition of hepatic gluconeogenesis by bacterial endotoxin: a role for nitric oxide?
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Endotoxin causes reciprocal changes in hepatic nitric oxide synthesis and gluconeogenesis.
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Inhibition by lamotrigine of the generation of nitric oxide in rat forebrain slices.
Depolarization of adult rat forebrain slices with veratrine induced the release of excitatory amino acids (glutamate and aspartate), the synthesis of nitric oxide (NO), and increases in cyclic GMP (cGMP). The NO synthase inhibitors N omega-monomethyl-L-arginine and N omega-nitro-L-arginine methyl ester decreased the release of NO and the levels of cGMP without affecting the release of excitatory amino acids. In contrast, the antiepileptic drug lamotrigine inhibited the release of excitatory amino acids and of NO, and decreased the levels of cGMP without causing a significant direct inhibition of the NO synthase. Furthermore, the synthesis of NO and the increases in cGMP induced by veratrine were partially blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 but not by 6-nitro-7-sulphamobenzo (f) quinoxaline-2,3-dione, a non-NMDA receptor antagonist. Neither of these compounds inhibited directly the NO synthase or the release of excitatory amino acids. Thus, these three types of compound act as an inhibitor of voltage-sensitive sodium channels (lamotrigine), as a receptor antagonist (MK-801), or as direct inhibitors of the NO synthase, to block the pathway leading to increased cGMP after veratrine depolarization. It is likely that some of the pharmacological and therapeutic actions shared by these three types of compound are, at least in part, a consequence of inhibition of the synthesis of NO.
The formation of nitric oxide donors from peroxynitrite.
1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.
Endotoxin causes reciprocal changes in hepatic nitric oxide synthesis, gluconeogenesis, and flux through phosphoenolpyruvate carboxykinase.
Treatment of rats with bacterial endotoxin resulted in a significant induction of hepatic nitric oxide synthase within 3 hours. The response was maximal at 12 hours and was maintained over 18 hours. The induction of nitric oxide synthase correlated well with the increase in plasma nitrate plus nitrite concentrations and also with the inhibition of glucose synthesis in subsequently isolated hepatocytes. The decline in the rate of gluconeogenesis also correlated with an inhibition of flux through phosphoenolpyruvate carboxykinase but not with alterations in flux through either pyruvate kinase or 6-phosphofructo-1-kinase, suggesting that a nitric oxide-induced inhibition of phosphoenolpyruvate carboxykinase may underlie the decreased glucose production in sepsis.
Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline.
Potent and selective inhibition of neuronal nitric oxide synthase (nNOS) compared to endothelial NOS (eNOS) and inducible NOS (iNOS) may be useful to treat cerebral ischemia (stroke) and other neurodegenerative diseases. S-Methyl-L-thiocitrulline (Me-TC) and S-ethyl-L-thiocitrulline (Et-TC) inhibited the oxidation of L-arginine and the L-arginine-independent oxidation of NADPH by nNOS from human brain. Me-TC and Et-TC were slow, tight binding inhibitors of nNOS with second-order association rate constants (kon) of 2.6 x 10(5) M-1 s-1 and 1.3 x 10(5) M-1 s-1, respectively. The respective dissociation rate constants (koff) were 3 x 10(-4) s-1 and 0.7 x 10(-4) s-1. Thus, the Kd values calculated from koff/kon were 1.2 and 0.5 nM, respectively. L-Arginine was a competitive inhibitor of Me-TC and Et-TC binding with competition constant (Ks) values of 2.2 and 2.7 microM, respectively. The Km of nNOS for L-arginine was 1.6 microM. The active site concentration of nNOS was estimated by titration with Et-TC. Based on this active site concentration, a kcat of 0.4 s-1 for the oxidation of L-arginine, was calculated. Me-TC and Et-TC were less potent inhibitors of human iNOS (Ki values of 34 and 17 nM, respectively) and human eNOS (Ki values of 11 and 24 nM). Thus, Me-TC and Et-TC were 10- and 50-fold, respectively, more potent inhibitors of nNOS than eNOS. Furthermore, Me-TC was also 17-fold selective for rat nNOS in neuronal tissue compared to rat eNOS in vascular endothelium, suggesting that Me-TC may be selective for nNOS in vivo and therefore, may be therapeutically useful to treat neurodegenerative diseases.
Induction of calcium-dependent nitric oxide synthases by sex hormones.
We have examined the effects of pregnancy and sex hormones on calcium-dependent and calcium-independent nitric oxide synthases (NOSs) in the guinea pig. Pregnancy (near term) caused a > 4-fold increase in the activity of calcium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebellum. The increase in NOS activity in the cerebellum during pregnancy was inhibited by the estrogen-receptor antagonist tamoxifen. Treatment with estradiol (but not progesterone) also increased calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone increased calcium-dependent NOS only in the cerebellum. No significant change in calcium-independent NOS activity was observed either during pregnancy or after the administration of any sex hormone. Both pregnancy and estradiol treatment increased the amount of mRNAs for NOS isozymes eNOS and nNOS in skeletal muscle, suggesting that the increases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to regulation by estrogen, an action that could explain some of the changes that occur during pregnancy and some gender differences in physiology and pathophysiology.