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Detection of nitrosyl hemoglobin in venous blood in the treatment of sickle cell anemia with hydroxyurea
I 156-l
I 157 I
CONSTITUTIVE AND ADAPTIVE DETOXIFICATION OF NITRIC OXIDE IN ESCHERICHIA COW : ROLE OF NITRIC OXIDE DIOXYGENASE IN THE PROTECTION OF ACONITASE
DETECTION OF NITROSYL HEMOGLOBIN IN VENOUS BLOOD IN THE TREATMENT OF SICKLE CELL ANEMIA WITH HYDROXYUREA Richard E. Glover,’ Edward D. Ivy, ’ Eugene P. Orringer, ’ Hiroshi Maeda,” and Ronald P. Mason.’ ‘Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, North Carolina 27709. *Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514. ‘Department of Microbiology, Kumamoto University of Medicine, Kumamoto 860, Japan.
Paul R. Gardner, Giuseppina Costantino and Andrew L. Salzman Division of Critical Care Medicine RO40, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229 Nitric oxide (NO’) is a naturally occurring toxin which some organisms adaptively resist. In aerobic or anaerobic Escherichia cob, low levels of NO’ exposure potently inactivated the NO’-sensitive citric acid cycle enzyme aconitase when the adaptive synthesis of NO’-defensive proteins was blocked with chloramphenicol. Adaptive protection of aconitase in aerobically grown E. coli was dependent upon 0,, was potently inhibited by cyanide, and correlated with an induced rate of cellular NO’ consumption. Constitutive and adaptive cellular NO’ consumption in aerobic cells was also dependent upon 0, and inhibited by cyanide. Exposure of aerobic cells to NO’ accordingly elevated the activity of the O,-dependent and cyanide-sensitive NO’ dioxygenase (NOD). Anaerobic E. coli exposed to NO’, or nitrate, induced a modest O,-independent and cyanide-resistant NO’ metabolizing activity and a more robust O,-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO’ and suggest functions for NOD and a cyanide-resistant NO’ scavenging activity in anaerobic
cells.
ALTERATIONS IN NITRIC OXIDE METABOLISM FOLLOWING EXPERIMENTAL CEREBRAL CORTICAL IMPACT
Controlled cortical impact in rats results in an immediate reduction in cerebral blood flow (CBl? in the region of impact. To examine the mechanism of this reduction, we measured tissue nitric
oxide @JOIusing a NO electrode, and mtcrodialysate nitrate/nitrite and amino acids. Data was collected for 1 hour before impact and 2.5 hours after impact in 7 adult rats and 5 control antmals with identical
manipulations other than cortical impact. values were expressed as a percentage of the bat&ii (pm-injmy) values arid as a percentage of controlvalues.
NO measurements showed a penistent 60%reduction
intlssueNOintheinjjanimalsafterimpact. Insometherewasa brief elevation of NO immediately after impact lasting several minutes followed by this reduction. Nitrate/nitrite measmed by chemiluminescensahoweda409breductionintheHrsthDurand339b reduclioninthesecondhourcomparedtobssellneintheinjured animals; these values were 62% and 44% lower than the control animals. In the first 20 minutes after injury, however, the nitrate/nitrite levels were 18% higher in the injured animals compared to controls. Miaudialysate argbdne, the amino acid precursor of NO, declkd in control animals by 30%but was elevated post-impart in injured animals by 113%. CitruUine, the amino acid
product of NO formation, increased 227%in the control animals and 1292%in the injured anbnais. The deceased tissue NO and nitrate/nitrite coupled with elevated dtrnlbne (suggest@ tncreased NO production), raise the possibibty that NO is being diverted to free radical formation following traumatic brain injury. Loss of physiologic NO vasodilatation may account for the regional CBF reduction observed after cortical impact. (Supported by NIH POl-NS27616)
560
OXYGEN
The clinical efficacy of hydroxyurea (HU) has been mainly attributed to increased levels of fetal hemoglobin (HbF), which reduces the tendency for sickle hemoglobin (HbS) to polymerize, thereby reducing the frequency of the vase-occlusive phenomena associated with the disease. However, benefits from HU treatment in patients have been reported in advance of increased HbF levels, Thus, it has been suggested that other hydroxyurea-dependent mechanisms may, in part, account for its clinical efficacy. We have previously demonstrated that HU is metabolized in rats to release nitric oxide and, therefore, postulated the same to occur in humans. However, to our knowledge, evidence of nitric oxide production from HU-metabolism in humans has yet to be demonstrated. Here we report that oral administration of HU for the treatment of sickle cell anemia produced detectable nitrosyl hemoglobin (HbNO). The HbNO complex could be detected as early as 30 minutes after administration and persisted up to four hours. Our observations support the hypothesis that the ability of HU to ease the vasoocclusive phenomena may, in part, be attributed to vasodilation and/or decreased platelet activation induced by HU-derived nitric oxide well in advance of increased HbF levels.
THE EFFECT OF U-TOCOPHEROL ON THE NITRATION OF I-TOCOPHEROL BY PERO-. S. P. A. Goss, N. Hogg and
Biophysics Research Institute, Medical B. Kalyanaraman. College of Wisconsin, Milwaukee, WI. 53226 USA. It has been proposed (Christen, S. et. al. (1997) Proc.Natl.Acaa!Sci.USA. 94, 3217-3222) that although a-tocopherol (a-TH) is an efficient antioxidant, the presence of y-tocopherol (y-TI-I) may be required to scavenge peroxynitritederived reactive nitrogen species. To investigate the reactions between a-TH, y-TH and peroxynitrite, endogenous levels of both a-TH and y-TH were monitored when LDL was oxidized in the presence of the peroxynitrite generator S-amino-3-(4morpholinyl)-1,2,3-oxadiazolium (SIN-I). SIN-l oxidized a-TH while y-TH levels remained constant. The sparing y-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing a-TH and y-TH were incubated with either SIN-l or peroxynitrite. Our data show that a-TH inhibits peroxynitrite-mediated y-TH nitration, i.e. 5-NOs-y-tocopherol formation. The rate constants for the reactions between both a-TH and y-TH with peroxynitrite suggest that the sparing of y-TH by a-TH does not occur by competitive scavenging, but may be due to the formation of a transient y-TH intermediate. This is supported by the fact that the sparing of y-TH by a-TH only occurs when both are present within the same membrane. Both a-TH and y-TH inhibited peroxynitrite-mediated tyrosine nitration. Nitration of y-TH becomes significant only after a-TH levels have been depleted. We conclude a-TH alone is sufftcient to remove any peroxynitrite-derived reactive nitrogen species, as the presence tyrosine.
’ 9
of a-TH
8
attenuates
nitration
of both y-TH
and
I 157 I
CONSTITUTIVE AND ADAPTIVE DETOXIFICATION OF NITRIC OXIDE IN ESCHERICHIA COW : ROLE OF NITRIC OXIDE DIOXYGENASE IN THE PROTECTION OF ACONITASE
DETECTION OF NITROSYL HEMOGLOBIN IN VENOUS BLOOD IN THE TREATMENT OF SICKLE CELL ANEMIA WITH HYDROXYUREA Richard E. Glover,’ Edward D. Ivy, ’ Eugene P. Orringer, ’ Hiroshi Maeda,” and Ronald P. Mason.’ ‘Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, North Carolina 27709. *Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514. ‘Department of Microbiology, Kumamoto University of Medicine, Kumamoto 860, Japan.
Paul R. Gardner, Giuseppina Costantino and Andrew L. Salzman Division of Critical Care Medicine RO40, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229 Nitric oxide (NO’) is a naturally occurring toxin which some organisms adaptively resist. In aerobic or anaerobic Escherichia cob, low levels of NO’ exposure potently inactivated the NO’-sensitive citric acid cycle enzyme aconitase when the adaptive synthesis of NO’-defensive proteins was blocked with chloramphenicol. Adaptive protection of aconitase in aerobically grown E. coli was dependent upon 0,, was potently inhibited by cyanide, and correlated with an induced rate of cellular NO’ consumption. Constitutive and adaptive cellular NO’ consumption in aerobic cells was also dependent upon 0, and inhibited by cyanide. Exposure of aerobic cells to NO’ accordingly elevated the activity of the O,-dependent and cyanide-sensitive NO’ dioxygenase (NOD). Anaerobic E. coli exposed to NO’, or nitrate, induced a modest O,-independent and cyanide-resistant NO’ metabolizing activity and a more robust O,-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO’ and suggest functions for NOD and a cyanide-resistant NO’ scavenging activity in anaerobic
cells.
ALTERATIONS IN NITRIC OXIDE METABOLISM FOLLOWING EXPERIMENTAL CEREBRAL CORTICAL IMPACT
Controlled cortical impact in rats results in an immediate reduction in cerebral blood flow (CBl? in the region of impact. To examine the mechanism of this reduction, we measured tissue nitric
oxide @JOIusing a NO electrode, and mtcrodialysate nitrate/nitrite and amino acids. Data was collected for 1 hour before impact and 2.5 hours after impact in 7 adult rats and 5 control antmals with identical
manipulations other than cortical impact. values were expressed as a percentage of the bat&ii (pm-injmy) values arid as a percentage of controlvalues.
NO measurements showed a penistent 60%reduction
intlssueNOintheinjjanimalsafterimpact. Insometherewasa brief elevation of NO immediately after impact lasting several minutes followed by this reduction. Nitrate/nitrite measmed by chemiluminescensahoweda409breductionintheHrsthDurand339b reduclioninthesecondhourcomparedtobssellneintheinjured animals; these values were 62% and 44% lower than the control animals. In the first 20 minutes after injury, however, the nitrate/nitrite levels were 18% higher in the injured animals compared to controls. Miaudialysate argbdne, the amino acid precursor of NO, declkd in control animals by 30%but was elevated post-impart in injured animals by 113%. CitruUine, the amino acid
product of NO formation, increased 227%in the control animals and 1292%in the injured anbnais. The deceased tissue NO and nitrate/nitrite coupled with elevated dtrnlbne (suggest@ tncreased NO production), raise the possibibty that NO is being diverted to free radical formation following traumatic brain injury. Loss of physiologic NO vasodilatation may account for the regional CBF reduction observed after cortical impact. (Supported by NIH POl-NS27616)
560
OXYGEN
The clinical efficacy of hydroxyurea (HU) has been mainly attributed to increased levels of fetal hemoglobin (HbF), which reduces the tendency for sickle hemoglobin (HbS) to polymerize, thereby reducing the frequency of the vase-occlusive phenomena associated with the disease. However, benefits from HU treatment in patients have been reported in advance of increased HbF levels, Thus, it has been suggested that other hydroxyurea-dependent mechanisms may, in part, account for its clinical efficacy. We have previously demonstrated that HU is metabolized in rats to release nitric oxide and, therefore, postulated the same to occur in humans. However, to our knowledge, evidence of nitric oxide production from HU-metabolism in humans has yet to be demonstrated. Here we report that oral administration of HU for the treatment of sickle cell anemia produced detectable nitrosyl hemoglobin (HbNO). The HbNO complex could be detected as early as 30 minutes after administration and persisted up to four hours. Our observations support the hypothesis that the ability of HU to ease the vasoocclusive phenomena may, in part, be attributed to vasodilation and/or decreased platelet activation induced by HU-derived nitric oxide well in advance of increased HbF levels.
THE EFFECT OF U-TOCOPHEROL ON THE NITRATION OF I-TOCOPHEROL BY PERO-. S. P. A. Goss, N. Hogg and
Biophysics Research Institute, Medical B. Kalyanaraman. College of Wisconsin, Milwaukee, WI. 53226 USA. It has been proposed (Christen, S. et. al. (1997) Proc.Natl.Acaa!Sci.USA. 94, 3217-3222) that although a-tocopherol (a-TH) is an efficient antioxidant, the presence of y-tocopherol (y-TI-I) may be required to scavenge peroxynitritederived reactive nitrogen species. To investigate the reactions between a-TH, y-TH and peroxynitrite, endogenous levels of both a-TH and y-TH were monitored when LDL was oxidized in the presence of the peroxynitrite generator S-amino-3-(4morpholinyl)-1,2,3-oxadiazolium (SIN-I). SIN-l oxidized a-TH while y-TH levels remained constant. The sparing y-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing a-TH and y-TH were incubated with either SIN-l or peroxynitrite. Our data show that a-TH inhibits peroxynitrite-mediated y-TH nitration, i.e. 5-NOs-y-tocopherol formation. The rate constants for the reactions between both a-TH and y-TH with peroxynitrite suggest that the sparing of y-TH by a-TH does not occur by competitive scavenging, but may be due to the formation of a transient y-TH intermediate. This is supported by the fact that the sparing of y-TH by a-TH only occurs when both are present within the same membrane. Both a-TH and y-TH inhibited peroxynitrite-mediated tyrosine nitration. Nitration of y-TH becomes significant only after a-TH levels have been depleted. We conclude a-TH alone is sufftcient to remove any peroxynitrite-derived reactive nitrogen species, as the presence tyrosine.
’ 9
of a-TH
8
attenuates
nitration
of both y-TH
and