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J R Lancaster

Publications and source records attributed to J R Lancaster.

At least 55 records · Page 3Linked to original sources
PubMed1993

Tetrahydrobiopterin-dependent nitrite oxidation to nitrate in isolated rat hepatocytes.

We have found that isolated rat hepatocytes and cell extracts catalyze the stoichiometric conversion of nitrite (either as a product of the oxidation of endogenously synthesized nitric oxide or added as sodium nitrite) to nitrate, which in extracts requires the presence of tetrahydrobiopterin and is inhibited by cyanide but apparently not by carbon monoxide. The reaction is sensitive to heat denaturation and does not involve oxidation of NO2- by a peroxidative or radical oxygen mechanism. These results indicate the presence of a hitherto undescribed mammalian mechanism of inorganic nitrogen oxide oxidation that may be a protective mechanism against one potentially damaging effect of endogenous .NO production (NO2- formation), and also that assays of .NO formation based on NO2- determination alone may be an inaccurate measurement of this activity.

Amino Acid Oxidoreductases↗
PubMed1993

Induction of nitric oxide synthesis and its reactions in cultured human and rat hepatocytes stimulated with cytokines plus LPS.

We have examined the time course of appearance of mRNA for nitric oxide synthase (NOS), intracellular nonheme iron-nitrosyl complexes (NHFeNO, detected by EPR spectroscopy), and rates of medium appearance of NO2- + NO3- in cultured rat and human hepatocytes stimulated with a combination of cytokines (TNF-alpha, IFN-gamma, IL-1 beta) and LPS. In both cells types, NOS mRNA precedes NHFeNO formation which in turn precedes maximum rates of NO2- + NO3- (NOx) formation. This profile occurs earlier in human hepatocytes than rat hepatocytes and the appearance of NOS mRNA is also more transient. These results indicate that (1) NOS is stable intracellularly (peak NOx production occurs substantially after peak mRNA levels), (2) intracellular iron is an early target (preceding maximum NOx production) for NO in both human and rat hepatocytes, and (3) decline in NHFeNO in the face of maximum NOx production indicates the presence of a "repair" or "removal" mechanism for these intracellular iron-nitrosyl complexes.

Amino Acid Oxidoreductases↗
PubMed1993

Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans.

Cytokines have been implicated as immunological effector molecules that mediate beta cell destruction associated with insulin-dependent diabetes mellitus. In this report we demonstrate that the cytokine combination of human recombinant interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) induces the formation of nitric oxide by human islets. This combination of cytokines stimulates both the formation of the nitric oxide derivative, nitrite, and the accumulation of cGMP by human islets. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine prevents formation of both cGMP and nitrite. IL-1 beta and IFN-gamma are sufficient to induce nitric oxide formation by human islets, whereas TNF-alpha potentiates nitrite production. This combination of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) also influences insulin secretion by human islets. Pretreatment of human islets with low concentrations of this cytokine combination (IL-1 beta at 15 units/ml, 0.7 nM TNF-alpha, and IFN-gamma at 150 units/ml) appears to slightly stimulate insulin secretion. Higher concentrations (IL-1 beta at 75 units/ml, 3.5 nM TNF-alpha, and IFN-gamma at 750 units/ml) inhibit insulin secretion from human islets, and the inhibitory effect is prevented by NG-monomethyl-L-arginine. This higher concentration of cytokines also induces the formation of an electron paramagnetic resonance-detectable g = 2.04 axial feature by human islets that is characteristic of the formation of an iron-dithio-dinitrosyl complex. The formation of this complex is prevented by NG-monomethyl-L-arginine, thus confirming that this cytokine combination induces the formation of nitric oxide by human islets. These results indicate that nitric oxide mediates the inhibitory effects of cytokines on glucose-stimulated insulin secretion by human islets and suggest that nitric oxide may participate in beta-cell dysfunction associated with insulin-dependent diabetes mellitus.

Arginine↗
PubMed1993

A 1-hour pulse with IL-1 beta induces formation of nitric oxide and inhibits insulin secretion by rat islets of Langerhans: evidence for a tyrosine kinase signaling mechanism.

Nitric oxide has been implicated as the effector molecule that mediates interleukin-1 beta (IL-1 beta)-induced inhibition of glucose-stimulated insulin secretion by rat islets. Brief exposures of islets (1 h) to IL-1 beta have been shown to inhibit glucose-stimulated insulin secretion at 8 or 18 h after removal of this cytokine. The purpose of this investigation was to determine if brief exposures of islets to IL-1 beta are sufficient to induce the formation of nitric oxide and to examine the signaling process associated with IL-1 beta-induced expression of nitric oxide synthase. We demonstrate that a 1-h pretreatment of islets with IL-1 beta followed by an 8-h incubation in the absence of this cytokine results in inhibition of glucose-stimulated insulin secretion (50%), which is completely prevented by pretreatment of islets with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (NMMA). The production of nitric oxide by islets under these pulse conditions is demonstrated by IL-1 beta-induced nitrite and electron paramagnetic resonance-detectable iron-nitrosyl complex formation, both of which are prevented by NMMA. IL-1 beta initiates a signal transduction process resulting in the expression of nitric oxide synthase. The signaling process appears to require the activation of a tyrosine kinase, since the tyrosine kinase inhibitor genistein prevents both IL-1 beta-induced inhibition of insulin secretion by islets and formation of nitric oxide by the insulinoma cell line RINm5F. These results show that short exposures of islets to IL-1 beta are sufficient to induce the formation of nitric oxide resulting in inhibition of glucose-stimulated insulin secretion and that a tyrosine kinase may participate in the early signaling events required for IL-1 beta to induce the expression of nitric oxide synthase.

Animals↗
PubMed1993

Nonheme iron-nitrosyl complex formation in rat hepatocytes: detection by electron paramagnetic resonance spectroscopy.

Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and lipopolysaccharide [LPS]) in vitro, after in vivo immune activation by Corynebacterium parvum, and after exposure to .N = O and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-gamma, TNF-alpha, IL-1 beta, and LPS demonstrated the appearance of a g = 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of .N = O synthase, prevented the appearance of the signal. The g = 2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated from C. parvum-treated rats exhibited a modest g = 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the .N = O produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system .N = O is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous .N = O or nitroprusside generated an identical g = 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and .N = O. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of .N = O in these two systems.

Animals↗
PubMed1992

Nitric oxide and cyclic GMP formation induced by interleukin 1 beta in islets of Langerhans. Evidence for an effector role of nitric oxide in islet dysfunction.

Treatment of pancreatic islets with interleukin 1 (IL-1) results in a time-dependent inhibition of glucose-stimulated insulin secretion which has recently been demonstrated to be dependent on the metabolism of L-arginine to nitric oxide. In this report IL-1 beta is shown to induce the accumulation of cyclic GMP (cGMP) in a time-dependent fashion that mimics the time-dependent inhibition of insulin secretion by IL-1 beta. The accumulation of cGMP is dependent on nitric oxide synthase activity, since NG-monomethyl-L-arginine (a competitive inhibitor of nitric oxide synthase) prevents IL-1 beta-induced cGMP accumulation. cGMP formation and nitrite production induced by IL-1 beta pretreatment of islets are also blocked by the protein synthesis inhibitor, cycloheximide. The formation of cGMP does not appear to mediate the inhibitory effects of IL-1 beta on insulin secretion since a concentration of cycloheximide (1 microM) that blocks IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and nitric oxide formation does not prevent cGMP accumulation, thus dissociating the two events. By using e.p.r. spectroscopy, IL-1 beta is shown to induce the formation of a g = 2.04 iron-nitrosyl feature in islets which is prevented by cycloheximide, demonstrating the requirement of protein synthesis for IL-1 beta-induced nitric oxide formation. Iron-nitrosyl complex-formation by islets confirms that IL-1 beta induces the generation of nitric oxide by islets, and provides evidence indicating that nitric oxide mediates destruction of iron-sulphur clusters of iron-containing enzymes. Consistent with the destruction of iron-sulphur centres is the finding that pretreatment of islets with IL-1 beta results in an approx. 60% inhibition of mitochondrial oxidation of D-glucose to CO2. Inhibition of islet glucose oxidation appears to be mediated by nitric oxide since both NMMA and cycloheximide prevent IL-1 beta-induced inhibition of glucose oxidation. These results show that IL-1 beta-induced nitric oxide formation parallels the ability of IL-1 beta to inhibit glucose-stimulated insulin secretion by islets, and that protein synthesis is required for IL-1 beta-induced nitric oxide formation. These results also suggest that nitric oxide mediates IL-1 beta-induced inhibitory effects on the pancreatic beta-cell by functioning as an effector molecule responsible for the destruction of iron-sulphur centres of iron-containing proteins, resulting in an impairment of mitochondrial function.

Amino Acid Oxidoreductases↗
PubMed1992

EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft.

The paramagnetic molecule nitric oxide (NO), produced from L-arginine by a specific enzyme (NO synthase), has been shown to be involved in a surprising variety of mammalian cellular responses, including the regulation of T cell immunity to alloantigens in vitro. In cytotoxic activated macrophages, NO production results in a characteristic pattern of alteration of iron-containing enzyme function that is mimicked by exposure to NO. Electron paramagnetic resonance (EPR) studies have shown the formation of iron-nitrosyl species during macrophage activation and also during sepsis, indicating that alteration of iron-containing protein function may be the result of the well-documented tendency of NO to bind to metal ions. We have recently shown that the NO synthesis induced during alloantigenic activation of rat splenocytes inhibits lymphocyte proliferation and cytotoxic T-lymphocyte generation. This report demonstrates that iron-nitrosyl EPR signals similar to those observed in macrophages and during sepsis are present in the blood and in the grafted tissue of rats during the rejection of allogeneic (but not syngeneic) heart grafts. These signals are found in the blood and at the site of allograft rejection, but are not found in other tissues (such as spleen and lung), and are obliterated by administration of the immunosuppressant FK506. These results directly demonstrate the formation of iron-nitrosyl complexes during vascularized allograft rejection and suggest that consequent destruction of iron-containing protein function plays an important role in the rejection response.

Animals↗
PubMed1992

Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide.

Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.

Aconitate Hydratase↗
PubMed1992

Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction.

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO.) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 beta-induced 1) nitrite formation (an oxidation product of NO.) and cGMP accumulation by the rat beta-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is approximately 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO. in the pathogenesis of diabetic vascular complications.

Amino Acid Oxidoreductases↗
PubMed1992

Detection of nitric oxide by electron paramagnetic resonance spectroscopy during rejection and graft-versus-host disease after small-bowel transplantation in the rat.

BACKGROUND: Our previous observation that nitric oxide (NO) is synthesized during antigen-specific immune reactions in vitro led us to investigate whether NO is produced during the in vivo immune response to a vascularized organ allograft. METHODS: Orthotopic small-bowel transplantation in the rat was performed by standard microsurgical techniques in the LBNF1 to Lewis (rejection alone), Lewis to LBNF1 (graft-versus-host disease [GVHD] alone), and a syngeneic strain combination with and without immunosuppressive therapy with FK 506. The recipient serum NO2-/NO3- levels (stable end products of NO metabolism) were measured and erythrocytes were evaluated for the presence of nitrosylferrohemoglobin (specific for NO bound to hemoglobin). RESULTS: Animals that acutely rejected small-bowel allografts or suffered from acute GVHD showed significantly elevated serum NO2-/NO3- levels on days 6 and 9, and nitrosylferrohemoglobin electron paramagnetic resonance signals of different intensity were detected on days 3, 6, and 9. FK 506-treated allograft recipients and recipients of syngeneic grafts showed normal serum NO2-/NO3- levels and lacked nitrosylferrohemoglobin signals at all time points. CONCLUSIONS: This study indicates that NO is produced early during the course of small-bowel allograft rejection and GVHD and might therefore serve as a simple marker to detect such immune reactions.

Animals↗
PubMed1991

Interleukin-1 beta-induced formation of EPR-detectable iron-nitrosyl complexes in islets of Langerhans. Role of nitric oxide in interleukin-1 beta-induced inhibition of insulin secretion.

The molecular mechanism by which interleukin (IL)-1 inhibits insulin secretion and ultimately causes destruction of the pancreatic beta-cell remains unknown. Evidence is presented which suggests that IL-1 beta-induced inhibition of insulin secretion is dependent on the metabolism of L-arginine to nitric oxide. NG-Monomethylarginine, a competitive inhibitor of the L-arginine-dependent enzyme nitric oxide synthase, completely prevents IL-1-induced inhibition of glucose-stimulated insulin secretion as well as nitrite production by islets. It is further shown that IL-1 beta induces nitric oxide formation in islets as evidenced by an electron paramagnetic resonance feature at g = 2.04 which is similar to previously reported iron-nitrosyl complexes formed from the destruction of iron-sulfur centers by nitric oxide. Inhibition of the nitric oxide synthase by NG-monomethylarginine completely prevents the formation of this EPR signal in islets. These results show that IL-1-induced inhibition of insulin secretion is mediated through formation of nitric oxide and suggest that the generation of nitric oxide may represent the cellular mechanism responsible for beta-cell destruction.

Animals↗
PubMed1991

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.

Agaricales↗
PubMed1990

EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages.

Activated macrophage cytotoxicity is characterized by loss of intracellular iron and inhibition of certain enzymes that have catalytically active nonheme-iron coordinated to sulfur. This phenomenon involves the oxidation of one of the terminal guanidino nitrogen atoms of L-arginine, which results in the production of citrulline and inorganic nitrogen oxides (NO2-, NO3-, and NO). We report here the results of an electron paramagnetic resonance spectroscopic study performed on cytotoxic activated macrophage (CAM) effector cells, which develop the same pattern of metabolic inhibition as their targets. Examination of activated macrophages from mice infected with Mycobacterium bovis (strain bacillus Calmette-Guérin) that were cultured in medium with lipopolysaccharide and L-arginine showed the presence of an axial signal at g = 2.039, which is similar to previously described iron-nitrosyl complexes formed from the destruction of iron-sulfur centers by nitric oxide (NO). Inhibition of the L-arginine-dependent pathway by addition of NG-monomethyl-L-arginine (methyl group on a terminal guanidino nitrogen) inhibits the production of nitrite, nitrate, citrulline, and the g = 2.039 signal. Comparison of the hyperfine structure of the signal from cells treated with L-arginine with terminal guanidino nitrogen atoms of natural abundance N14 atoms or labeled with N15 atoms showed that the nitrosyl group in this paramagnetic species arises from one of these two atoms. These results show that loss of iron-containing enzyme function in CAM is a result of the formation of iron-nitrosyl complexes induced by the synthesis of nitric oxide from the oxidation of a terminal guanidino nitrogen atom of L-arginine.

Animals↗
PubMed1989

Inhibition of target cell mitochondrial electron transfer by tumor necrosis factor.

Using digitonin permeabilization to assay mitochondrial electron transfer, we have found that respiratory activity (succinoxidase and cytochrome oxidase) in three mouse fibroblast lines is completely eliminated by incubation with human recombinant tumor necrosis factor-alpha (hrTNF). As with cytotoxicity, hrTNF-induced mitochondrial dysfunction occurs in resistant cells upon inhibition of protein synthesis, whereas sensitive cells exhibit spontaneous respiratory inhibition. In C3HA cells, inhibition is detectable 1.5-2 h after hrTNF addition, preceding cell lysis by at least 5 h (as measured by dye exclusion), and is approximately coincidental with morphological changes we have previously reported for this cell line. LM cells also exhibit inhibition of electron transfer, coincidental with morphological changes. These results suggest that bioenergetic dysfunction may be involved in the cytotoxic mechanism of TNF.

Animals↗