PubMed Health⌕ Search

Biomedical subjects

D E Heck

Publications and source records attributed to D E Heck.

35 records · Page 2Linked to original sources
PubMed1998

Increased nitric oxide synthase in the lung after ozone inhalation is associated with activation of NF-kappa B.

Acute inhalation of ozone is associated with a inflammatory response characterized by the accumulation of macrophages at sites of tissue injury. These cells, along with resident alveolar epithelial cells, become activated and release cytotoxic and proinflammatory mediators, such as nitric oxide (.NO), that we speculate contribute to toxicity. In these studies we analyzed mechanisms regulating increased .NO synthase activity in lung macrophages and type II cells after ozone inhalation. Brief exposure of rats to ozone (2 ppm for 3 hr) resulted in an increase in .NO production by alveolar macrophages as well as type II cells in response to the inflammatory mediators lipopolysaccharide and interferon gamma. These effects were apparently due to increased expression of inducible .NO synthase (iNOS) protein and mRNA, which were evident in vitro and in situ in histologic sections. .NO production and iNOS protein expression by both macrophages and epithelial cells were blocked by pyrrolidine dithiocarbamate (PDTC), an agent that inhibits activity of nuclear transcription factor kappa B (NF-kappa B). Cells from ozone-treated animals were less sensitive to the effects of PDTC than cells from control animals. Using electrophoretic mobility shift assays, we measured NF-kappa B binding activity in nuclear extracts of cells from control and ozone-exposed animals. Treatment of rats with ozone resulted in a time-dependent increase in NF-kappa B binding activity in both cell types, reaching a maximum in cells isolated 12 to 24 hr after ozone inhalation. Taken together, these data suggest that changes in the activity of NF-kappa B signaling are important in the response of lung macrophages and type II epithelial cells to cytokines after ozone inhalation.

Animals↗
PubMed1997

Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: multiple modes of action of nitric oxide synthase inhibitors.

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.

Animals↗
PubMed1996

5-Fluorouracil suppresses nitric oxide biosynthesis in colon carcinoma cells.

Nitric oxide is an important cellular mediator that plays a role in regulating cellular proliferation of both normal and tumor cells. In the present study, we characterized nitric oxide production by the human colon adenocarcinoma cell line DLD-1 and examined the effects of 5-fluorouracil (5-FUra), an antimetabolite effective against colon tumors, on nitric oxide production. IFN-gamma was found to be a potent inducer of nitric oxide production in DLD-1 cells. This effect was dependent on L-arginine and blocked by the nitric oxide synthase inhibitors NG-monomethyl-L-arginine, nitroarginine, and aminoguanidine. Production of nitric oxide by DLD-1 cells was due to the expression of the inducible (type II) form of nitric oxide synthase. mRNA for the nitric oxide synthase was present in both untreated and IFN-gamma-stimulated cells, as determined by RT-PCR, suggesting that expression of enzyme is regulated posttranscriptionally. Treatment of DLD-1 cells with concentrations of 5-FUra that are not growth inhibitory or cytotoxic strongly inhibited their ability to express nitric oxide synthase and produce nitric oxide in response to IFN-gamma. This effect was not reversed with thymidine, indicating that inhibition of nitric oxide production was due to incorporation of 5-FUra into RNA. However, pretreatment of DLD-1 cells with 5-FUra before stimulation with IFN-gamma also suppressed nitric oxide production. Thus, inhibition of nitric oxide production was not due directly to incorporation of 5-FUra into the mRNA for nitric oxide synthase. Taken together, these data suggest that inhibition of nitric oxide biosynthesis in colon tumor cells by 5-FUra may underlie, at least in part, the efficacy of this antitumor agent.

Antimetabolites, Antineoplastic↗
PubMed1996

Role of nitric oxide in hematosuppression and benzene-induced toxicity.

It is becoming increasingly apparent that nitric oxide plays a multifunctional role in regulating inflammatory processes in the body. Although nitric oxide and its oxidation products are cytotoxic toward certain pathogens, they can also cause tissue injury and suppress proliferation. Cytokines and growth factors released at sites of inflammation or injury stimulate both immune and nonimmume cells to produce nitric oxide. Nowhere in the body is this more detrimental than in the bone marrow, for the continuous production of hematopoietic precursors is essential for normal blood cell maturation. Our laboratories have discovered that, in response to inflammatory mediators, bone marrow cells readily produce nitric oxide. Nitric oxide production is enhanced by hematopoietic growth factors including interleukin-3, macrophage colony stimulating factor, and granulocyte-macrophage colony-stimulating factor. When bone marrow cells produce nitric oxide, hematopoiesis is impaired, an effect that is potentiated by colony-stimulating factors. Treatment of mice with benzene, which suppresses bone marrow cell development, was found to markedly enhance the ability of bone marrow cells to produce nitric oxide in response to inflammatory mediators alone and in combination with hematopoietic growth factors. Taken together, these data suggest that nitric oxide may be an important mediator of benzene-induced bone marrow suppression.

Animals↗
PubMed1995

Inhibition of macrophages with gadolinium chloride abrogates ozone-induced pulmonary injury and inflammatory mediator production.

Acute inhalation of toxic doses of ozone (O3) induces macrophage accumulation in the lung and the release of cytotoxic and proinflammatory mediators. To evaluate the role of macrophages and their mediators in the pathophysiologic response of the lung to O3, we examined the effects of the macrophage inhibitor, gadolinium chloride (GdCl3), on O3-induced inflammation, mediator production, and lavage fluid protein levels. Rats were pretreated with GdCl3 (7 mg/kg, intravenously) or control 24 h prior to exposure to air or O3 (2 parts per million, 3 h). Animals were killed 48 h after exposure. GdCl3 pretreatment of rats was found to abrogate O3-induced increases in the number of cells, as well as the amount of protein recovered in bronchoalveolar lavage fluid. Following GdCl3 pretreatment of rats, lung lavage cells consisting of > 90% macrophages were found to produce significantly less nitric oxide and express less inducible nitric oxide synthase (iNOS) when compared to cells from rats exposed to O3. O3-induced alterations in superoxide anion production by alveolar macrophages, both in vitro and in situ, were also attenuated by GdCl3 pretreatment of rats. In addition, increases in tumor necrosis factor alpha (TNF-alpha) and fibronectin in lung tissue induced by O3 were reduced. Taken together, these data provide support for the hypothesis that macrophages contribute to the pathogenesis of O3-induced lung injury.

Amino Acid Oxidoreductases↗
PubMed1995

Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression.

In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin.

Acute Disease↗
PubMed1994

Role of nitric oxide in hepatic injury following acute endotoxemia.

Hepatocytes from control and endotoxemic rats were cultured for 40 hours in 96-well dishes in medium containing 0-5 micrograms/ml of lipopolysaccharide in the presence or absence of 50 U/ml IFN gamma. Nitric oxide production was quantified by the accumulation of nitrite in the culture medium by the Greiss reaction. Hepatocyte proliferation and protein synthesis were measured by (3H)thymidine (TdR) and (3H)leucine (Leu) incorporation, respectively. Results are the mean +/- standard error of triplicate wells from four experiments.

Acute Disease↗
PubMed1994

Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediators in mouse kidney epithelial cells.

We report that osteopontin (OPN), a secreted, Arg-Gly-Asp-containing phosphoprotein expressed at high levels in the kidney, suppresses nitric oxide (NO) synthesis induced by the inflammatory mediators gamma-interferon and lipopolysaccharide in primary mouse kidney proximal tubule epithelial cells. Northern blot and immunofluorescence analyses of inducible nitric oxide synthase (iNOS) expression revealed that the inflammatory mediators increased iNOS mRNA and protein levels. Recombinant human OPN (purified from both mammalian cells and from Escherichia coli) inhibited this response by a process that was blocked by anti-OPN antiserum and by the peptide GRGDS, but not GRGES. The data suggest that inhibition of NO synthesis by OPN in these kidney cells is mediated by an integrin, possibly the alpha v beta 3 integrin, which is known to be an OPN receptor. NO is believed to control blood flow through the glomerulus, regulating salt and water balance, and to be important as a defense against tumor cells and infecting microorganisms. The ability of OPN to inhibit the induction of iNOS suggests that OPN may be an important regulator of the NO signaling pathway and NO-mediated cytotoxic processes.

Amino Acid Oxidoreductases↗
PubMed1994

Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin.

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Oxidoreductases↗
PubMed1992

Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.

Amino Acid Oxidoreductases↗
PubMed1992

Production of nitric oxide by murine bone marrow cells. Inverse correlation with cellular proliferation.

The present studies were designed to assess the ability of primary cultures of bone marrow cells to produce nitric oxide. We found that two inflammatory stimuli, IFN-gamma and LPS, were potent inducers of nitric oxide production by bone marrow cells. In addition, the CSF granulocyte-macrophage (GM)-CSF and IL-3 as well as TNF-alpha, while inactive by themselves, were synergistic with LPS and IFN-gamma in inducing nitric oxide production. Maximal effects were observed with combinations of GM-CSF and LPS. Nitric oxide production by bone marrow cells was found to be dependent on the presence of L-arginine in the culture medium and inhibitable by NG-monomethyl-L-arginine and L-canavanine, two nitric oxide synthase inhibitors. Nitric oxide produced by the cells was also suppressed by TGF-beta 1 and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Separation of bone marrow cells by density gradient centrifugation and flow cytometry revealed that the granulocyte-containing fraction was largely responsible for nitric oxide production. In additional experiments we found that treatment of bone marrow cells with GM-CSF significantly stimulated bone marrow cell growth. In contrast, the combination of GM-CSF and LPS or IFN-gamma markedly suppressed cellular proliferation. This suppression was completely reversed by treatment of the cells with NG-monomethyl-L-arginine. Taken together, these data demonstrate that various inflammatory stimuli and cytokines induce nitric oxide production by primary cultures of bone marrow cells and that this mediator may play a role in the regulation of bone marrow cell growth and development.

Animals↗
PubMed1991

Production of hydrogen peroxide by cutaneous T-cell lymphoma following photopheresis with psoralens and ultraviolet light.

Treatment of peripheral blood mononuclear cells with 8-methoxypsoralen (8-MOP) and ultraviolet light, a procedure known as PUVA, has been found to be useful in the management of systemically disseminated cutaneous T-cell lymphoma (CTCL). In the present study we used a highly sensitive flow cytometric assay in conjunction with the hydroperoxide-sensitive dye 2',7'-dichlorofluorescein diacetate to measure intracellular hydrogen peroxide in normal lymphocytes and CTCL following PUVA treatment. Based on their laser light-scattering properties, lymphocytes were separated into three major subpopulations. We found that ultraviolet light alone caused an increase in the hydrogen peroxide content of each of the subpopulations, a response that was augmented when the cells were pretreated with 8-MOP (50 ng/ml). Cells from CTCL patients were more sensitive to the effects of 8-MOP than were normal lymphocytes. In both cell types, the production of hydrogen peroxide was found to be inhibitable by catalase. We noted an increase in hydrogen peroxide production following photopheresis; however, this was observed only 24 h after treatment. In addition, a further increase in hydrogen peroxide production was observed in lymphocytes isolated from peripheral blood that had been obtained from patients at 15 min after a second photopheresis treatment. Hydrogen peroxide is known to modulate the action of cytokines as well as the immunological responses of leukocytes. Our data suggest that the production of hydrogen peroxide by lymphocytes may be important in the action of PUVA in CTCL.

Catalase↗