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Adaptive responses to peroxynitrite: Increased glutathione and cystine uptake in vascular cells
SYNTHESIS AND CHARACTERIZATION LINOLEIC ACID
OF NITRATED
A. Bloodsworth,
J.P. Eiserich, P.H. Chumley, V.B. O’Donnell & B.A. Freeman. Department of Anesthesiology and the UAB Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35233.
Nitrated lipids can be formed in vitro by reactive nitrogen species (RNS) such as nitric oxide (.NO), peroxynitrite (ONOO) and nitrogen dioxide (NO,). All of these RNS can be formed within the vascular compartment by endothelial cells, smooth muscle cells and phagocytes, especially during inflammatory events. To aid m the detectlon and characterization of biologically nitrated lipids, a nitrated linoleate standard (LN02) has been synthesized. The organic synthesis was accom lished by reaction of linoleic acid with phenylselenylbromine QPheSeBr), silver nitrite and mercuric chlonde in tetrahydrofuran and acetonitrile. Phenylselenyl(PheSe-) and nitro-groups are added across one of the double bonds in linoleate. Then, the PheSeremoved by h drogen peroxide (H 0,), gYo”b! is oxidatively ylel mg LNO, with both double bon d.s mtact. Nitrated linoieate was purified by reverse-phase HPLC. Negative-ion mode mass spectrometry (MS) was used to confirm product purity; LNO, has a mass/charge (m/z) [M - H’] 324. This species fragmented on tandem-mass spectrometry (MSiMS) to give a daughter ion with m/z 46, corresponding to NO;. An “N-labeled LNO, was also synthesized with m/z 325 and fragments upon MS/MS to yield a daughter ion with m/z 47. The organically synthesized standard had the same molecular weight, MS/MS fragmentation and HPLC retention time as LNO, generated by ONOO‘. Product quantified by HPLC using a yield of syntheses was chemiluminescent-based nitrogen detector and was -30%. This new standard permits more incisive identification of biologically nitrated lipids and the study of their potential bioactivity.
I 208
DETECTION OF NITROTYROSINE
IN FETAL BRAINS FOLLOWING REPETITIVE UTERINE ISCHEMIA Rakesh Bose. M.S., Tams Itlling, M.D., Michael Capian, M.D., Matthew Derrick, MD., Sidhnrthn Tan M.D. NorthwesternUniversity,Evanston,IL 60091.
The reaction of nitric oxide-derived species, such as peroxynitrite, with proteins results in the nitration of tyrosine residues to form 3-nitrotyrosine. We hypothesized that reactive nitrogen species derived from nitric oxide may be formed in premature fetal rabbit brain following acute hypoxia-ischemia. We have previously detected nitrotyrosine in hypoxic fetal brain by immunoprecipitation and Western blot analysis with antibody to nitrotyrosine. Utilizing a recently developed electrochemical HPLC assay based on the method by Shigenaga et al (Proc Nat1 Acad Sci USA 94:3211-3216, 1997), we investigated fetal brains from 29 day gestation rabbits that were randomized to either repetitive uterine ischemia for 50 or 40 min (Hypoxia) or no ischemia (Controls). Following hysterotomy, fetal brains were obtained. Brain homogenate was processed to produce a derivative of nitrotyrosine, N-acetyl-3-aminotyrosine, that was detected by a coulometric electrode array system. Nitrotyrosine was detected only in repetitive hypoxic brains compared to the absence of nitration in control brains (n=3 in each group). Brain homogenate treated with peroxynitrite had extensive nitration of protein and served as positive controls. Repetitive hypoxia-ischemia results in the increased formation of reactive nitrogen species in premature fetal brains, which may include increased formation of peroxynitrite. It is speculated that reactive nitrogen species may contribute to free radical-mediated injury following acute hypoxia-ischemia in clinical situations of acute placental insufficiency.
I
ADAPTIVE RESPONSES TO PEROXYNITRITE: INCREASED GLUTATHIONE AND CYSTINE UPTAKE IN VASCULAR CELLS
m*
ELECTROCHEMICAL
and A. Rrchnrd Whorton, Duke Uniuemfy
Medicnl Center,
We and others recently demonstrated
increased glutathione (GSH) levels, stimulated cystine uptake and induced GSH synthetic enzymes in vascular cells exposed to nitric oxide (.NO) donors. Here we report the effects of peroxynitrite (ONOO-) on GSH and cystine uptake. Bovine aortic endothelial cells (EC) and smooth muscle cells (SMC) were treated with SIN-1 which decomposes with a half-life of 40 min and generates ONOO-. Exposure of EC to 500 uM SIN-l decreased GSH by 45% at 2 hrs and increased GSH 3.5-fold at 8 hrs. Increases of Z-4-fold were observed 17 hrs following 50-500 uM SIN-l. Since these changes were similar to those observed with the .NO donor SNAP, SIN-l treatments were carried out in the presence of Cu,Zn SOD to scavenge 02: produced during SIN-l breakdown and decrease the formation of ONOO-. SOD inhibited the response by more than 50% while heat-inactivated SOD was ineffective. MnTMPyP also inhibited the increase in GSH. These results suggest that ON00 mediated the response to SIN-l. Since GSH synthesis is regulated by the availability of precursor amino acids, we also studied cystine uptake. Incubation of EC for 19 hrs with 250-500 uM SIN-1 increased cystine uptake by 29-94%. Following 500 uM SIN-l, increased cystine uptake was evident at 6 hrs and maintained for 23 hrs. Inhibitors of protein and RNA synthesis blocked the SIN-l-mediated increase in cystine uptake. Cystine uptake was also increased in SMC treated with 1 n&l SIN-l and SNAP. Basal and stimulated cystine uptake were blocked by 5 mM glutamate in EC and SMC. These studies suggest that the xc- pathway of amino acid uptake was induced in response to SIN-l. In summary, vascular cells respond to chronic ONOO- exposure with adaptive increases in cellular GSH and cystine transport pathways.
OXYGEN
REACTIONS OF NITRIC OXIDE AND PEROXYNITRITE WITH RECOMBINANT PIG HEART MITOCHONDRIAL ACONITASE. Laura Castro, Edward Suarez-Moreira. Bruce A. Freeman and Facultad de Medicina, Rafael Radi. De to. de Bioquimica. Universidad de la i”epubhca, Montevideo, Uruguay. Mitochondrial aconitase (m-ac) has been indicated to be sensitive for O;,and NO-mediated disruption of the [4Fe-4S] cluster and inactlvation. However, the concerted generation of 0,’ and NO yield peroxynitrite (ONOO-) which can outcompete both the direct reaction of 4’ and NO with m-ac as well as the enzymatic dismutation of 0;. Herein, we report that anaerobic urified recombinant m-ac to pure NO or ex osure of inhibition of the enzyme in N8NOates lea Bs to a dose-dependent the absence of substrates. When ex osures were brief (~10 min) m-ac inhibition could be reverted l!y displacement of NO with argon, whereas longer incubation times caused irreversible inactlvation of the enzyme. However, with the enzyme in catalytic turnover, NO resulted in a marginal decrease in the activity (e.g. 100 PM NO caused a 10% inhibition with 5 mM isocitrate as substrate). On the other hand, S-nitrosothiols such as S-nitroso lutathione and S-nitrosocysteine significantly inhibited m-ac, bo t?l.m the absence or in the resence of substrates, due to direct reaction with the enzyme. ! eroxynitrite reacted readily with the [4Fe-4S] cluster, yielding in the inactive [3Fe-4S] enzyme, with substrates only partially protective. Carbon dioxide ONOO--de endent inactivation of m-ac, also promoted presumably via reactions of 0 K O&O;. Our results indicate that different mechanisms account for NO-mediated m-ac inactivation. While NO reacts with the [4Fe-4S] cluster, leading to an inhibited [4Fe-4S]-NO reversible complex which can be slowly further disrupted, ONOO- and S-nitrosothiols promote a more efficient and fast inactivation.
’ 9
9
s73
OF NITRATED
A. Bloodsworth,
J.P. Eiserich, P.H. Chumley, V.B. O’Donnell & B.A. Freeman. Department of Anesthesiology and the UAB Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35233.
Nitrated lipids can be formed in vitro by reactive nitrogen species (RNS) such as nitric oxide (.NO), peroxynitrite (ONOO) and nitrogen dioxide (NO,). All of these RNS can be formed within the vascular compartment by endothelial cells, smooth muscle cells and phagocytes, especially during inflammatory events. To aid m the detectlon and characterization of biologically nitrated lipids, a nitrated linoleate standard (LN02) has been synthesized. The organic synthesis was accom lished by reaction of linoleic acid with phenylselenylbromine QPheSeBr), silver nitrite and mercuric chlonde in tetrahydrofuran and acetonitrile. Phenylselenyl(PheSe-) and nitro-groups are added across one of the double bonds in linoleate. Then, the PheSeremoved by h drogen peroxide (H 0,), gYo”b! is oxidatively ylel mg LNO, with both double bon d.s mtact. Nitrated linoieate was purified by reverse-phase HPLC. Negative-ion mode mass spectrometry (MS) was used to confirm product purity; LNO, has a mass/charge (m/z) [M - H’] 324. This species fragmented on tandem-mass spectrometry (MSiMS) to give a daughter ion with m/z 46, corresponding to NO;. An “N-labeled LNO, was also synthesized with m/z 325 and fragments upon MS/MS to yield a daughter ion with m/z 47. The organically synthesized standard had the same molecular weight, MS/MS fragmentation and HPLC retention time as LNO, generated by ONOO‘. Product quantified by HPLC using a yield of syntheses was chemiluminescent-based nitrogen detector and was -30%. This new standard permits more incisive identification of biologically nitrated lipids and the study of their potential bioactivity.
I 208
DETECTION OF NITROTYROSINE
IN FETAL BRAINS FOLLOWING REPETITIVE UTERINE ISCHEMIA Rakesh Bose. M.S., Tams Itlling, M.D., Michael Capian, M.D., Matthew Derrick, MD., Sidhnrthn Tan M.D. NorthwesternUniversity,Evanston,IL 60091.
The reaction of nitric oxide-derived species, such as peroxynitrite, with proteins results in the nitration of tyrosine residues to form 3-nitrotyrosine. We hypothesized that reactive nitrogen species derived from nitric oxide may be formed in premature fetal rabbit brain following acute hypoxia-ischemia. We have previously detected nitrotyrosine in hypoxic fetal brain by immunoprecipitation and Western blot analysis with antibody to nitrotyrosine. Utilizing a recently developed electrochemical HPLC assay based on the method by Shigenaga et al (Proc Nat1 Acad Sci USA 94:3211-3216, 1997), we investigated fetal brains from 29 day gestation rabbits that were randomized to either repetitive uterine ischemia for 50 or 40 min (Hypoxia) or no ischemia (Controls). Following hysterotomy, fetal brains were obtained. Brain homogenate was processed to produce a derivative of nitrotyrosine, N-acetyl-3-aminotyrosine, that was detected by a coulometric electrode array system. Nitrotyrosine was detected only in repetitive hypoxic brains compared to the absence of nitration in control brains (n=3 in each group). Brain homogenate treated with peroxynitrite had extensive nitration of protein and served as positive controls. Repetitive hypoxia-ischemia results in the increased formation of reactive nitrogen species in premature fetal brains, which may include increased formation of peroxynitrite. It is speculated that reactive nitrogen species may contribute to free radical-mediated injury following acute hypoxia-ischemia in clinical situations of acute placental insufficiency.
I
ADAPTIVE RESPONSES TO PEROXYNITRITE: INCREASED GLUTATHIONE AND CYSTINE UPTAKE IN VASCULAR CELLS
m*
ELECTROCHEMICAL
and A. Rrchnrd Whorton, Duke Uniuemfy
Medicnl Center,
We and others recently demonstrated
increased glutathione (GSH) levels, stimulated cystine uptake and induced GSH synthetic enzymes in vascular cells exposed to nitric oxide (.NO) donors. Here we report the effects of peroxynitrite (ONOO-) on GSH and cystine uptake. Bovine aortic endothelial cells (EC) and smooth muscle cells (SMC) were treated with SIN-1 which decomposes with a half-life of 40 min and generates ONOO-. Exposure of EC to 500 uM SIN-l decreased GSH by 45% at 2 hrs and increased GSH 3.5-fold at 8 hrs. Increases of Z-4-fold were observed 17 hrs following 50-500 uM SIN-l. Since these changes were similar to those observed with the .NO donor SNAP, SIN-l treatments were carried out in the presence of Cu,Zn SOD to scavenge 02: produced during SIN-l breakdown and decrease the formation of ONOO-. SOD inhibited the response by more than 50% while heat-inactivated SOD was ineffective. MnTMPyP also inhibited the increase in GSH. These results suggest that ON00 mediated the response to SIN-l. Since GSH synthesis is regulated by the availability of precursor amino acids, we also studied cystine uptake. Incubation of EC for 19 hrs with 250-500 uM SIN-1 increased cystine uptake by 29-94%. Following 500 uM SIN-l, increased cystine uptake was evident at 6 hrs and maintained for 23 hrs. Inhibitors of protein and RNA synthesis blocked the SIN-l-mediated increase in cystine uptake. Cystine uptake was also increased in SMC treated with 1 n&l SIN-l and SNAP. Basal and stimulated cystine uptake were blocked by 5 mM glutamate in EC and SMC. These studies suggest that the xc- pathway of amino acid uptake was induced in response to SIN-l. In summary, vascular cells respond to chronic ONOO- exposure with adaptive increases in cellular GSH and cystine transport pathways.
OXYGEN
REACTIONS OF NITRIC OXIDE AND PEROXYNITRITE WITH RECOMBINANT PIG HEART MITOCHONDRIAL ACONITASE. Laura Castro, Edward Suarez-Moreira. Bruce A. Freeman and Facultad de Medicina, Rafael Radi. De to. de Bioquimica. Universidad de la i”epubhca, Montevideo, Uruguay. Mitochondrial aconitase (m-ac) has been indicated to be sensitive for O;,and NO-mediated disruption of the [4Fe-4S] cluster and inactlvation. However, the concerted generation of 0,’ and NO yield peroxynitrite (ONOO-) which can outcompete both the direct reaction of 4’ and NO with m-ac as well as the enzymatic dismutation of 0;. Herein, we report that anaerobic urified recombinant m-ac to pure NO or ex osure of inhibition of the enzyme in N8NOates lea Bs to a dose-dependent the absence of substrates. When ex osures were brief (~10 min) m-ac inhibition could be reverted l!y displacement of NO with argon, whereas longer incubation times caused irreversible inactlvation of the enzyme. However, with the enzyme in catalytic turnover, NO resulted in a marginal decrease in the activity (e.g. 100 PM NO caused a 10% inhibition with 5 mM isocitrate as substrate). On the other hand, S-nitrosothiols such as S-nitroso lutathione and S-nitrosocysteine significantly inhibited m-ac, bo t?l.m the absence or in the resence of substrates, due to direct reaction with the enzyme. ! eroxynitrite reacted readily with the [4Fe-4S] cluster, yielding in the inactive [3Fe-4S] enzyme, with substrates only partially protective. Carbon dioxide ONOO--de endent inactivation of m-ac, also promoted presumably via reactions of 0 K O&O;. Our results indicate that different mechanisms account for NO-mediated m-ac inactivation. While NO reacts with the [4Fe-4S] cluster, leading to an inhibited [4Fe-4S]-NO reversible complex which can be slowly further disrupted, ONOO- and S-nitrosothiols promote a more efficient and fast inactivation.
’ 9
9
s73