- Email: [email protected]
Prolonged nitric oxide synthesis inhibition in endothelial cells promotes neutrophil adhesion via increased oxidative stress
Session 4: Nitric Oxide and Peroxynitrite
498
4:17
PROLONGED NITRIC OXIDE SYNTHESIS ENDOTHELIAL CELLS PROMOTES
LNHIBITION IN NEUTROPHIL
ADHESION VIA INCREASED OXIDATIW STRESS Paul Kubes*, C. Wayne Smith**, Xiao-Fei Niuf Dept. of Medical Physiology, The University of Calgary, Hospital Drive N.W.
3330
Calgary, Albarta Canada
The objecctive of this study was to determine whether inhibition of nitric oxide synthesis in endothelial cells induced ncutrophil adhesion. Human umbilical vein endothelial cells were grown to confluence in 48 well microtiter plates. Exposure of endothelial cells to the nitric oxide synthesis inhibitor L-NAME 10% Ml99 did not cause neutrophil adhesion at I hr. but dd enhance adhesion at 4 hr in a dose-dependent manner. The incrtz+sd adhesion was prevented with L-arginine or nitric oxide donors and could not be reproduced with D-NAME (biologically inactive enantiomer). The L-NAME-induced neutrophil adhesion was inhibited by monoclonal antibodies dire&d against the fi-subunit of CD1 l/CD18 and ICAM-1, but not against various selecctins(E, L, and P-selectin). Platelet activating factor, receptor antagonist (WEB 2086) but not a lipoxyganase inhibitor (nordihydroguaiaretic acid) also prevented the L-NAME-induced neutrophil adhesion. Various anti-oxidants including LY,ddipyridyl, dimothyl sulfoxide and the iron chelator &feral also significantly inhibited the L-NAME-induced neutrophil adhesion when endothelial cells were pretreaatd for the entIre 4 hr with the anti-oxidants. Superoxide dismutase, catalase or a combination of Iha two reagents had no effect on L-NAME-induced adhrslon. lnhihition of endothelial cell mitochondrial function with sodium azide also significantlyattenuated L-NAME-inducdneutrophil adhesion without affecting neutrophil function diredy. These data suggest that prolonged nitric oxide synthesis inhibition in endothelial cells causes an oxidant and PAF-associated CDI8-dependent rise in neutrophil adhesion.
NITRIC OXIDE SCAVENGING ACTIVITY OF NITECAPONE. Lucia Marcocci, John J. Maguire, and Lester Packer Department of Molecular Cell Biology, University of California, Berkeley, California, 94720 USA. Nitecapone [3-(3,4-dihydroxy-Snitrobenzylidene)-2,4pentanedione] is an effective scavenger of reactive oxygen species. We report on the activity of nitecapone as a scavenger of nitric oxide. The efficiency of nitecapone is compared to that of two related compounds: entacapone [2cyano-N, N-diethyl-3-(3,4,dihydroxy-5-nitrophenyl)propenamide] and OR-1246 [3-(3,4-dihydroxy-Snitrobenzyl)2,4_pentanedione]. These compounds have the same structure of the cathecol group, but different side chain structures. The amount of nitrite formed in the reaction of nitric oxide with oxygen is decreased in the presence of nitecapone, entacapone or OR-1246 (lo-600 FM). The decrease depends on the concentration of antioxidant, and a plateau was reached at 200 FM. The rate of methemoglobin formation from nitric oxide reacting with oxyhemoglobin decreased in the presence of the compounds. These data indicate that all these compounds interact with nitric oxide. However, OR- 1246 scavenged the radical less efficiently than nitecapone or entacapone. In the presence of 200 UM nitecanone or OR-1246 the formation of nitrite was, re’spectiveli, 10% or 30 % compared to the control without antioxidant; in contrast, nitecapone and entacapone had the same efficiency. Our results show that nitecapone acts as an efficient scavenger of nitric oxide. Its activity may be modulated by the presence of a methylene group and seems to be not modulated by carbonyl groups.
ACTIVITY
IN RAT AND HUMAN
NEUTROPHILS John J. Maguire*.
**Baylor College of Medicine, Houston, Texas
4:19
NITRIC OXIDE SYNTHETASE
Hunt3,
Lucia Marcocci’,
Lester Packer’
Dan Allen3.
4:18
Thomas
& Marc Ryder4
1Dept. of Molecular and Cellular Biology and *Energy and Environment Division, Lawrence Berkeley Laboratory, University of California, Berkeley CA 94720. 3Depts. Surgery and 4Division of Periodontology, University California San Francisco, San Francisco CA 94143.
activity
Isolated neutrophils were evaluated for of inducible nitric oxide synthetase,
of of
their by
measuring arginine analog dependent formation of N02-. Human and rat neutrophils were studied and both a circulating and a “primed” preparation was isolated. The circulating neutrophils were from fresh blood, and the “primed” neutrophils from the rat were from a peritoneal wash of a rat following a peritoneal injection with a casein solution. Human “primed” neutrophils sterile were isolated from surgical drain fluid from patients undergoing reconstructive or cancer surgery and also from oral rinses. All neutrophils exhibited an oxygen burst activity following addition of phorbol myristic acetate (PMA). but only some of the the “primed” neutrophils exhibited nitric oxide production. Attempts to stimulate human circulating neutrophils, with cytokines, induce nitric oxide lipopolysaccharide or PMA, to production were not successful. As the inducible nitric oxide synthetase produces NO in the PM concentration range (extracellularly) it is believed to be involved in bactericidal activity.
EFFECTS OF TAURINE PRODUCTION OF NO, O,‘-,
CHLORAMINE TNF-cz, AND
ON
IL-l BY MURINE PERITONEAL MACROPHAGES IN CULTURE. Eunkyue Park, Chaekyun Kim, Michael R. Quinn, Charles Wright and Georgia Schuller-Levis. Depts. of Immunology and Developmental Biochemistry, NYS Institute for Basic Research, Staten Island, NY 10314 Formation of taurine chloramine (TAU-Cl) is catalyzed by halide dependent myeloperoxidase from HOC1 and taurine which is present in high concentration (20-50 mM) in leukocytes. Peritoneal macrophages (M@)were prepared from BALB/c mice 4 days after ip injection of thioglycollate and maintained in culture. Nitric oxide (NO) was measured as nitrite in the culture media using Griess reagent. TNF-a and IL-l were quantitated by ELISA. TAU-Cl inhibited production of NO and TNF-a by M@ in a concentration dependent manner with 1 mM TAU-Cl producing maximum inhibition (96-98%). TAU-Cl had no significant effect on amounts of IL-1 secreted into the media by M@. Taurine, the precursor of TAU-Cl, had no measurable effect on these parameters. Effects of 1 mM TAU-Cl on production of superoxide anion by MP and by polymorphornuclear cells (PMN) was measured in an SOD inhibitable cytochrome c assay using phorbol myristate acetate. Superoxide production was inhibited 67% in PMN, but not consistently affected in M$J. These results suggest that under certain circumstances TAU-CI may influence localized inflammatory responses.
4:20
498
4:17
PROLONGED NITRIC OXIDE SYNTHESIS ENDOTHELIAL CELLS PROMOTES
LNHIBITION IN NEUTROPHIL
ADHESION VIA INCREASED OXIDATIW STRESS Paul Kubes*, C. Wayne Smith**, Xiao-Fei Niuf Dept. of Medical Physiology, The University of Calgary, Hospital Drive N.W.
3330
Calgary, Albarta Canada
The objecctive of this study was to determine whether inhibition of nitric oxide synthesis in endothelial cells induced ncutrophil adhesion. Human umbilical vein endothelial cells were grown to confluence in 48 well microtiter plates. Exposure of endothelial cells to the nitric oxide synthesis inhibitor L-NAME 10% Ml99 did not cause neutrophil adhesion at I hr. but dd enhance adhesion at 4 hr in a dose-dependent manner. The incrtz+sd adhesion was prevented with L-arginine or nitric oxide donors and could not be reproduced with D-NAME (biologically inactive enantiomer). The L-NAME-induced neutrophil adhesion was inhibited by monoclonal antibodies dire&d against the fi-subunit of CD1 l/CD18 and ICAM-1, but not against various selecctins(E, L, and P-selectin). Platelet activating factor, receptor antagonist (WEB 2086) but not a lipoxyganase inhibitor (nordihydroguaiaretic acid) also prevented the L-NAME-induced neutrophil adhesion. Various anti-oxidants including LY,ddipyridyl, dimothyl sulfoxide and the iron chelator &feral also significantly inhibited the L-NAME-induced neutrophil adhesion when endothelial cells were pretreaatd for the entIre 4 hr with the anti-oxidants. Superoxide dismutase, catalase or a combination of Iha two reagents had no effect on L-NAME-induced adhrslon. lnhihition of endothelial cell mitochondrial function with sodium azide also significantlyattenuated L-NAME-inducdneutrophil adhesion without affecting neutrophil function diredy. These data suggest that prolonged nitric oxide synthesis inhibition in endothelial cells causes an oxidant and PAF-associated CDI8-dependent rise in neutrophil adhesion.
NITRIC OXIDE SCAVENGING ACTIVITY OF NITECAPONE. Lucia Marcocci, John J. Maguire, and Lester Packer Department of Molecular Cell Biology, University of California, Berkeley, California, 94720 USA. Nitecapone [3-(3,4-dihydroxy-Snitrobenzylidene)-2,4pentanedione] is an effective scavenger of reactive oxygen species. We report on the activity of nitecapone as a scavenger of nitric oxide. The efficiency of nitecapone is compared to that of two related compounds: entacapone [2cyano-N, N-diethyl-3-(3,4,dihydroxy-5-nitrophenyl)propenamide] and OR-1246 [3-(3,4-dihydroxy-Snitrobenzyl)2,4_pentanedione]. These compounds have the same structure of the cathecol group, but different side chain structures. The amount of nitrite formed in the reaction of nitric oxide with oxygen is decreased in the presence of nitecapone, entacapone or OR-1246 (lo-600 FM). The decrease depends on the concentration of antioxidant, and a plateau was reached at 200 FM. The rate of methemoglobin formation from nitric oxide reacting with oxyhemoglobin decreased in the presence of the compounds. These data indicate that all these compounds interact with nitric oxide. However, OR- 1246 scavenged the radical less efficiently than nitecapone or entacapone. In the presence of 200 UM nitecanone or OR-1246 the formation of nitrite was, re’spectiveli, 10% or 30 % compared to the control without antioxidant; in contrast, nitecapone and entacapone had the same efficiency. Our results show that nitecapone acts as an efficient scavenger of nitric oxide. Its activity may be modulated by the presence of a methylene group and seems to be not modulated by carbonyl groups.
ACTIVITY
IN RAT AND HUMAN
NEUTROPHILS John J. Maguire*.
**Baylor College of Medicine, Houston, Texas
4:19
NITRIC OXIDE SYNTHETASE
Hunt3,
Lucia Marcocci’,
Lester Packer’
Dan Allen3.
4:18
Thomas
& Marc Ryder4
1Dept. of Molecular and Cellular Biology and *Energy and Environment Division, Lawrence Berkeley Laboratory, University of California, Berkeley CA 94720. 3Depts. Surgery and 4Division of Periodontology, University California San Francisco, San Francisco CA 94143.
activity
Isolated neutrophils were evaluated for of inducible nitric oxide synthetase,
of of
their by
measuring arginine analog dependent formation of N02-. Human and rat neutrophils were studied and both a circulating and a “primed” preparation was isolated. The circulating neutrophils were from fresh blood, and the “primed” neutrophils from the rat were from a peritoneal wash of a rat following a peritoneal injection with a casein solution. Human “primed” neutrophils sterile were isolated from surgical drain fluid from patients undergoing reconstructive or cancer surgery and also from oral rinses. All neutrophils exhibited an oxygen burst activity following addition of phorbol myristic acetate (PMA). but only some of the the “primed” neutrophils exhibited nitric oxide production. Attempts to stimulate human circulating neutrophils, with cytokines, induce nitric oxide lipopolysaccharide or PMA, to production were not successful. As the inducible nitric oxide synthetase produces NO in the PM concentration range (extracellularly) it is believed to be involved in bactericidal activity.
EFFECTS OF TAURINE PRODUCTION OF NO, O,‘-,
CHLORAMINE TNF-cz, AND
ON
IL-l BY MURINE PERITONEAL MACROPHAGES IN CULTURE. Eunkyue Park, Chaekyun Kim, Michael R. Quinn, Charles Wright and Georgia Schuller-Levis. Depts. of Immunology and Developmental Biochemistry, NYS Institute for Basic Research, Staten Island, NY 10314 Formation of taurine chloramine (TAU-Cl) is catalyzed by halide dependent myeloperoxidase from HOC1 and taurine which is present in high concentration (20-50 mM) in leukocytes. Peritoneal macrophages (M@)were prepared from BALB/c mice 4 days after ip injection of thioglycollate and maintained in culture. Nitric oxide (NO) was measured as nitrite in the culture media using Griess reagent. TNF-a and IL-l were quantitated by ELISA. TAU-Cl inhibited production of NO and TNF-a by M@ in a concentration dependent manner with 1 mM TAU-Cl producing maximum inhibition (96-98%). TAU-Cl had no significant effect on amounts of IL-1 secreted into the media by M@. Taurine, the precursor of TAU-Cl, had no measurable effect on these parameters. Effects of 1 mM TAU-Cl on production of superoxide anion by MP and by polymorphornuclear cells (PMN) was measured in an SOD inhibitable cytochrome c assay using phorbol myristate acetate. Superoxide production was inhibited 67% in PMN, but not consistently affected in M$J. These results suggest that under certain circumstances TAU-CI may influence localized inflammatory responses.
4:20