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Polynitroxylation neutralizes the hypertensive effect of αα-crosslinked hemoglobin without affecting nitric oxide scavenging
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POLYNITROXYLATION NEUTRALIZES THE HYPERTENSIVE EFFECT OF aa-CROSSLINKED HEMOGLOBIN WITHOUT AFFECTING NITRIC OXIDE SCAVENGING
OXIDE SYNTHESIS DURING CUTANEOUS MURINE LEISHMANIASIS Edlaine Linares', Selma Giorgio', Renato A. Mortara and Ohara Au usto ? Instituto de quimica , Uniyersidade de SBo Paulo, UNICAMP and UNIFESP , Brazil.
Haiquan Li’, Li Ma. Periannan Kuppusamy’, Charles E. Trimble, Jay L. Zweier*, Carleton JC Hsia, *Johns Hopkins University, Baltimore, MD; SynZyme Technologies, LLC, CA. Various cross-linked hemoglobin (Hb) compounds have vasoconstrictive activity, which may potentially cause significant adverse effects in patients requiring oxygen therapeutics. The pressor effect has been attributed to nitric oxide (NO) scavenging by oxyhemoglobin, since reducing NO scavenging activity by site-directed mutagenesis of 64 kDa recombinant Hb products can almost eliminate vasoconstrictive activity. As an alternative approach, we have labeled a&b with nitroxide. an antioxidant enzyme mimic, and shown that the resulting polynitroxyl-aaHb (PN-aaHb, where the a&b supplied by U.S. Army) has antioxidant activities mimicking SOD and catalase. In the present study PN-a&b also shows no pressor effect in a 10% top-load model in the conscious rat, where aaHb causes a marked increase in mean arterial pressure. To check whether polynitroxylation (PN) of aaHb alters NO scavenging, the rate of NO-induced oxidation (k’) was measured for aaHbO~, PN-acfHbOz, and stroma-free Hb02(A& PN-aaHb02 showed a k’ value of 60*9 &s”. which is identical to that of native HbOz (&) and aaHb02. This indicates that PN reduces the pressor effect of aaHbOz by a mechanism other than reduced NO scavenging. We propose that the mechanism involves a reduction in endogenous levels of superoxide radicals, which regulate the normal steady state level of NO in the endothelium. This compensates the NO scavenging activity of aaHb02, In addition to being a non-vasoactive oxygen carrier, PN-ctaHb may also act as an antioxidant to counter oxidative stress from ischemia and reperfusion.
Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Several evidences indicated a role for NO in the resolution of disease. the Recent studies, however, have demonstrated that during the late stages of murine leishmaniasis, susceptible animals have high parasite loads and increased NO synthesis. To investigate source of the late this synthesis we analyzed the footpad lesions of BALBfc mice infected with L. amazonensis during 20 weeks for the presence of hemenitrosyl complexes (monitored by EPR), iNOS and nitrotyrosine (monitored by confocal immunofluorescence) and Of bacteria (monitored by optical microscopy). The data show that the presence of bacteria in the lesions is accompanied by polymorphonuclear cell accumulation and NO synthesis. Consequently, the results indicate that secondary infections facilitated by the cutaneous lesions are responsible for the late NO synthesis observed in mice susceptible to Leishmania.
1236 1
1 DETERMINANTS OF ELECTRON TRANSFER IN THE NITRIC OXIDESYNTHASES Pavel Mflrt.sek, R. Timothy Miller, and Bettie Sue Masters
U. Texas Health Science Center nt San Antonio.
KINETICS OF PEROXYNITRITE-MEDIATED INACTIVATION OF MNSOD rllanC ow. fohn P. Crow, Danielle Cruthirds, and lohn A Thompson.
Univ. ofA~;abamn
Birminghnm,
Birmin@am,
AL 35216.
Each nitric oxide synthase (NOS) isoform consists of a flavin-containing reductase domain linked to a heme-containing oxygenase domain through a calmodulin binding sequence. The reductase domain accepts electrons from NADPH and, following calmodulin binding to NOS, flavin-to-heme electron transfer is facilitated. The transfer of electrons from the flavins to the heme allows for the O,-dependent conversion of the substrate, L-arginine, to L-citrulline and nitric oxide (NO). In addition to catalyzing the oxidation of L-arg, the NOS isoforms can also reduce 0, either at the flavin and/or heme centers. The three NOS isoforms differ in the rates of both flavin-mediated reactions as well as in rates of heme-mediated reactions (NO and L-citrulline formation. We are currently investigating the mechanisms involved in controlling intra-domain as well as inter-domain electron transfer in the NOS isoforms to gain a better understanding of the ability of NOS isoforms to act as either superoxide anion synthases or NO synthases; a situation which occurs during conditions of limiting cofactor availability (namely BH, and L-arg). This situation can be clinically relevant especially in atherosclerotic and hypercholesterolemic states. Data suggest that flavin-to-heme electron transfer under aerobic, turnover conditions is rate-limiting for NO and L-citrulline formation (nNOS: 242 sec.’ for flavin reduction DS49 sec.’ for heme reduction; eNOS: 107 sec.’ for flavin reduction US 2.3 set I for heme reduction). Thus, product formation may be regulated by the reductase domains of the constitutively-expressed NOS isoforms. (Supported by NIH Grants GM52419, HL.30050; Welch Fnd AQ-1192 to BSSM).
We have recently shown that the major antioxidant in mitochondria, MnSOD, was tyrosine nitrated and inactivated during human chronic renal allograft rejection (PNAS, 1996). The results shown here confirm that intracellular MnSOD is a nitration target for both authentic peroxynitrite (ONOO) as well as ON00 generated by SIN-l. The kinetics of ONOO-mediated inactivation of MnSOD in viva are currently unknown. In order to determine whether MnSOD was concomitantly inactivated under these conditions, we carried out a similar SIN-1 experiment using phenol nitration as the measured endpoint. These experiments indicated that MnSOD could be inactivated within minutes, even in the presence of SODS and that MnSOD can be dynamically inactivated under conditions of enzyme turnover. At present, it is difficult to conclude anything with regard to whether a correlation exists between nitrated MnSOD and tissue pathology. Initial studies utilizing a rat model of chronic renal allograft rejection suggest that as early as 4 weeks post-transplant, MnSOD isolated from the rat kidney is nitrated
and inactivated. Interestingly,at12weeks post-transplant, renalMnSOD enzyme activity appears to increase as the amount of nitrated protein decreases, while at even later time points (34-52 weeks post-transplant), activity once again declines as nitration increases. These preliminary results suggest that MnSOD is an early target for ONOO-mediated inactivation during chronic renal rejection. We propose that nitration and inactivation of MnSOD in viva would lead to increased mitochondrialO2. levels and concomitant increases in ON00 which would result in nitration/oxidation of key mitochondrial proteins and
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1 235
1
LATE
NITRIC
POLYNITROXYLATION NEUTRALIZES THE HYPERTENSIVE EFFECT OF aa-CROSSLINKED HEMOGLOBIN WITHOUT AFFECTING NITRIC OXIDE SCAVENGING
OXIDE SYNTHESIS DURING CUTANEOUS MURINE LEISHMANIASIS Edlaine Linares', Selma Giorgio', Renato A. Mortara and Ohara Au usto ? Instituto de quimica , Uniyersidade de SBo Paulo, UNICAMP and UNIFESP , Brazil.
Haiquan Li’, Li Ma. Periannan Kuppusamy’, Charles E. Trimble, Jay L. Zweier*, Carleton JC Hsia, *Johns Hopkins University, Baltimore, MD; SynZyme Technologies, LLC, CA. Various cross-linked hemoglobin (Hb) compounds have vasoconstrictive activity, which may potentially cause significant adverse effects in patients requiring oxygen therapeutics. The pressor effect has been attributed to nitric oxide (NO) scavenging by oxyhemoglobin, since reducing NO scavenging activity by site-directed mutagenesis of 64 kDa recombinant Hb products can almost eliminate vasoconstrictive activity. As an alternative approach, we have labeled a&b with nitroxide. an antioxidant enzyme mimic, and shown that the resulting polynitroxyl-aaHb (PN-aaHb, where the a&b supplied by U.S. Army) has antioxidant activities mimicking SOD and catalase. In the present study PN-a&b also shows no pressor effect in a 10% top-load model in the conscious rat, where aaHb causes a marked increase in mean arterial pressure. To check whether polynitroxylation (PN) of aaHb alters NO scavenging, the rate of NO-induced oxidation (k’) was measured for aaHbO~, PN-acfHbOz, and stroma-free Hb02(A& PN-aaHb02 showed a k’ value of 60*9 &s”. which is identical to that of native HbOz (&) and aaHb02. This indicates that PN reduces the pressor effect of aaHbOz by a mechanism other than reduced NO scavenging. We propose that the mechanism involves a reduction in endogenous levels of superoxide radicals, which regulate the normal steady state level of NO in the endothelium. This compensates the NO scavenging activity of aaHb02, In addition to being a non-vasoactive oxygen carrier, PN-ctaHb may also act as an antioxidant to counter oxidative stress from ischemia and reperfusion.
Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Several evidences indicated a role for NO in the resolution of disease. the Recent studies, however, have demonstrated that during the late stages of murine leishmaniasis, susceptible animals have high parasite loads and increased NO synthesis. To investigate source of the late this synthesis we analyzed the footpad lesions of BALBfc mice infected with L. amazonensis during 20 weeks for the presence of hemenitrosyl complexes (monitored by EPR), iNOS and nitrotyrosine (monitored by confocal immunofluorescence) and Of bacteria (monitored by optical microscopy). The data show that the presence of bacteria in the lesions is accompanied by polymorphonuclear cell accumulation and NO synthesis. Consequently, the results indicate that secondary infections facilitated by the cutaneous lesions are responsible for the late NO synthesis observed in mice susceptible to Leishmania.
1236 1
1 DETERMINANTS OF ELECTRON TRANSFER IN THE NITRIC OXIDESYNTHASES Pavel Mflrt.sek, R. Timothy Miller, and Bettie Sue Masters
U. Texas Health Science Center nt San Antonio.
KINETICS OF PEROXYNITRITE-MEDIATED INACTIVATION OF MNSOD rllanC ow. fohn P. Crow, Danielle Cruthirds, and lohn A Thompson.
Univ. ofA~;abamn
Birminghnm,
Birmin@am,
AL 35216.
Each nitric oxide synthase (NOS) isoform consists of a flavin-containing reductase domain linked to a heme-containing oxygenase domain through a calmodulin binding sequence. The reductase domain accepts electrons from NADPH and, following calmodulin binding to NOS, flavin-to-heme electron transfer is facilitated. The transfer of electrons from the flavins to the heme allows for the O,-dependent conversion of the substrate, L-arginine, to L-citrulline and nitric oxide (NO). In addition to catalyzing the oxidation of L-arg, the NOS isoforms can also reduce 0, either at the flavin and/or heme centers. The three NOS isoforms differ in the rates of both flavin-mediated reactions as well as in rates of heme-mediated reactions (NO and L-citrulline formation. We are currently investigating the mechanisms involved in controlling intra-domain as well as inter-domain electron transfer in the NOS isoforms to gain a better understanding of the ability of NOS isoforms to act as either superoxide anion synthases or NO synthases; a situation which occurs during conditions of limiting cofactor availability (namely BH, and L-arg). This situation can be clinically relevant especially in atherosclerotic and hypercholesterolemic states. Data suggest that flavin-to-heme electron transfer under aerobic, turnover conditions is rate-limiting for NO and L-citrulline formation (nNOS: 242 sec.’ for flavin reduction DS49 sec.’ for heme reduction; eNOS: 107 sec.’ for flavin reduction US 2.3 set I for heme reduction). Thus, product formation may be regulated by the reductase domains of the constitutively-expressed NOS isoforms. (Supported by NIH Grants GM52419, HL.30050; Welch Fnd AQ-1192 to BSSM).
We have recently shown that the major antioxidant in mitochondria, MnSOD, was tyrosine nitrated and inactivated during human chronic renal allograft rejection (PNAS, 1996). The results shown here confirm that intracellular MnSOD is a nitration target for both authentic peroxynitrite (ONOO) as well as ON00 generated by SIN-l. The kinetics of ONOO-mediated inactivation of MnSOD in viva are currently unknown. In order to determine whether MnSOD was concomitantly inactivated under these conditions, we carried out a similar SIN-1 experiment using phenol nitration as the measured endpoint. These experiments indicated that MnSOD could be inactivated within minutes, even in the presence of SODS and that MnSOD can be dynamically inactivated under conditions of enzyme turnover. At present, it is difficult to conclude anything with regard to whether a correlation exists between nitrated MnSOD and tissue pathology. Initial studies utilizing a rat model of chronic renal allograft rejection suggest that as early as 4 weeks post-transplant, MnSOD isolated from the rat kidney is nitrated
and inactivated. Interestingly,at12weeks post-transplant, renalMnSOD enzyme activity appears to increase as the amount of nitrated protein decreases, while at even later time points (34-52 weeks post-transplant), activity once again declines as nitration increases. These preliminary results suggest that MnSOD is an early target for ONOO-mediated inactivation during chronic renal rejection. We propose that nitration and inactivation of MnSOD in viva would lead to increased mitochondrialO2. levels and concomitant increases in ON00 which would result in nitration/oxidation of key mitochondrial proteins and
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