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P031. Effects of hemolysis on nitric oxide bioavailability
A28
Posters / Nitric Oxide 14 (2006) A27–A38
transduction of the NO signal. These drugs also blocked immune responses in Drosophila larvae. These findings define a new pathway contributing to immune responses and suggest that its activity might make central contributions to immune responses in different systems. Consistent with an established pathway of response to bacteria, LPS treatment of S2 cells activated GFP-Relish by inducing a cleavage that released an N-terminal anchorage domain so that the GFP-tagged C-terminal fragment could move into the nucleus. In contrast, NO-induced GFP-Relish movement into the nucleus was not associated with cleavage of GFP-Relish. In fact, NO donors block LPS-induced cleavage of GFPRelish, indicating that an alternative mechanism of GFP-Relish activation is used. Our analysis revealed that NO stimulates the production of cyclic GMP, which acts to promote release of calcium and activation of the calcium-dependent phosphatase calcineurin, which dephosphorylates GFPRelish. The dephosphorylation of the full-length GFP-Relish triggers its nuclear localization. Analyses in larvae show that steps of this pathway are required for innate immune responses.
sation of cell-free Hb into the endothelium. Our modeling was performed using FEMLAB software. Our model considered individual RBCs within the lumen of the blood vessel, taking into account the particulate nature of blood. We examined the effect of cell-free Hb and extravasation of cellfree Hb, which can occur during hemolytic conditions such as sickle cell disease. We have found that concentrations as low as 1 lM of cell-free Hb reduces the availability of NO. Furthermore, the effect of RBC membrane permeability diminishes as cell-free Hb reaches concentrations as low as 5 lM. Based on these calculations, we hypothesize that the scavenging of NO in the lumen by cell-free Hb during hemolysis results in drastically reduced NO bioavailability. Additionally, at low hematocrit values, cell-free Hb scavenging of NO was more efficient than at high hematocrit values. Examination of the effects of extravasation, indicate that concentrations of cell-free Hb in the endothelium as low as 1 lM further reduce the bioavailability of NO. These results support experimental ones demonstrating a major role of cell-free Hb in the pathology of hemolytic conditions. Supported by NHLBI of the NIH.
doi:10.1016/j.niox.2006.04.091
doi:10.1016/j.niox.2006.04.092
P031. Effects of hemolysis on nitric oxide bioavailability
P032. The NO-releasing mechanism of 4-aryl-1,3,2-oxathiazolylium-5olates Dongning Lu a, Periannan Kuppusamy b, Peng George Wang Chemistry, The Ohio State University, USA b The Ohio State University, USA
Anne B. Jeffers a, Daniel B. Kim-Shapiro b a Biomedical Engineering, Wake Forest University School of Medicine b Wake Forest University Nitric oxide (NO) has been identified as the endothelium-derived relaxation factor (EDRF), which is responsible for smooth muscle relaxation. However, hemoglobin (Hb) is an effective scavenger of NO, which can lead to endothelial dysfunction during hemolytic conditions. There are various mechanisms that limit consumption of NO by Hb in vivo including (1) a physical barrier to NO diffusion across the red blood cell (RBC) membrane, (2) an unstirred layer creating a concentration gradient in NO outside the RBC, and (3) and a cell-free zone between the endothelium where NO is made and the location of the RBCs. However, during hemolysis, these mechanisms could be compromised, reducing levels of NO reaching the smooth muscle cells. Although the importance of hemolysis in disease has been gaining attention, some believe that small amounts of hemolysis would not effect NO bioavailability in the presence of 10 mM RBC encapsulated Hb normally found in vivo. We have performed computational modeling of NO bioavailability within blood vessels, specifically looking at the effects of hemolysis and extrava-
O
O N
Ar
H+ O
S
Ar
H2O
O
O
N S
C
Ar
S
N
Nitric oxide (NO) is a small gaseous molecule that plays a critical role in a variety of bioregulatory processes and can also inhibit metastasis, enhance cancer cell apoptosis, and assist macrophages in killing tumor cells. NO is usually generated in situ from an NO donor. S-Nitrosothiols are one type of NO donor, however, their instability toward light, heat, and metal ions limits their therapeutic applicability. Synthesizing more stable precursors to S-nitrosothiols will circumnavigate this. Recently, we noticed that 4-aryl-1,3,2-oxathiazolylium-5-olates are stable precursors and we propose based on our results that they will decompose under acidic conditions to release NO (Scheme 1). Electron Paramagnetic Resonance spectroscopy (EPR), mass spectrometry, and computational calculations were used to verify the reaction pathway and products. Herein, we would like to report the synthesis of this class of compounds (Scheme 2), their decomposition mechanism, and their therapeutic applicability. doi:10.1016/j.niox.2006.04.093
NO O
OH
C
Ar
O
O Ar
OH
~H
S
C
O Ar
O
CO2
OH S
S
Ar
HO
O
Ar
SH
Ar
SH
Ar
SH
SH
Scheme 1. NO generation from 4-aryl-1,3,2-oxathiazolylium-5-olates upon acidic activation.
O
X
O C OH Ph P, DIAD 3 OH
HSAc
O C OH
O C OH NaOMe X SAc
MeOH
b
a
X SH
O C OH
iBuONO CH2Cl2
X S NO
Scheme 2. The synthetic route of 4-aryl-1,3,2-oxathiazolylium-5-olates.
O N
DCC
S
CH2Cl2 X
Posters / Nitric Oxide 14 (2006) A27–A38
transduction of the NO signal. These drugs also blocked immune responses in Drosophila larvae. These findings define a new pathway contributing to immune responses and suggest that its activity might make central contributions to immune responses in different systems. Consistent with an established pathway of response to bacteria, LPS treatment of S2 cells activated GFP-Relish by inducing a cleavage that released an N-terminal anchorage domain so that the GFP-tagged C-terminal fragment could move into the nucleus. In contrast, NO-induced GFP-Relish movement into the nucleus was not associated with cleavage of GFP-Relish. In fact, NO donors block LPS-induced cleavage of GFPRelish, indicating that an alternative mechanism of GFP-Relish activation is used. Our analysis revealed that NO stimulates the production of cyclic GMP, which acts to promote release of calcium and activation of the calcium-dependent phosphatase calcineurin, which dephosphorylates GFPRelish. The dephosphorylation of the full-length GFP-Relish triggers its nuclear localization. Analyses in larvae show that steps of this pathway are required for innate immune responses.
sation of cell-free Hb into the endothelium. Our modeling was performed using FEMLAB software. Our model considered individual RBCs within the lumen of the blood vessel, taking into account the particulate nature of blood. We examined the effect of cell-free Hb and extravasation of cellfree Hb, which can occur during hemolytic conditions such as sickle cell disease. We have found that concentrations as low as 1 lM of cell-free Hb reduces the availability of NO. Furthermore, the effect of RBC membrane permeability diminishes as cell-free Hb reaches concentrations as low as 5 lM. Based on these calculations, we hypothesize that the scavenging of NO in the lumen by cell-free Hb during hemolysis results in drastically reduced NO bioavailability. Additionally, at low hematocrit values, cell-free Hb scavenging of NO was more efficient than at high hematocrit values. Examination of the effects of extravasation, indicate that concentrations of cell-free Hb in the endothelium as low as 1 lM further reduce the bioavailability of NO. These results support experimental ones demonstrating a major role of cell-free Hb in the pathology of hemolytic conditions. Supported by NHLBI of the NIH.
doi:10.1016/j.niox.2006.04.091
doi:10.1016/j.niox.2006.04.092
P031. Effects of hemolysis on nitric oxide bioavailability
P032. The NO-releasing mechanism of 4-aryl-1,3,2-oxathiazolylium-5olates Dongning Lu a, Periannan Kuppusamy b, Peng George Wang Chemistry, The Ohio State University, USA b The Ohio State University, USA
Anne B. Jeffers a, Daniel B. Kim-Shapiro b a Biomedical Engineering, Wake Forest University School of Medicine b Wake Forest University Nitric oxide (NO) has been identified as the endothelium-derived relaxation factor (EDRF), which is responsible for smooth muscle relaxation. However, hemoglobin (Hb) is an effective scavenger of NO, which can lead to endothelial dysfunction during hemolytic conditions. There are various mechanisms that limit consumption of NO by Hb in vivo including (1) a physical barrier to NO diffusion across the red blood cell (RBC) membrane, (2) an unstirred layer creating a concentration gradient in NO outside the RBC, and (3) and a cell-free zone between the endothelium where NO is made and the location of the RBCs. However, during hemolysis, these mechanisms could be compromised, reducing levels of NO reaching the smooth muscle cells. Although the importance of hemolysis in disease has been gaining attention, some believe that small amounts of hemolysis would not effect NO bioavailability in the presence of 10 mM RBC encapsulated Hb normally found in vivo. We have performed computational modeling of NO bioavailability within blood vessels, specifically looking at the effects of hemolysis and extrava-
O
O N
Ar
H+ O
S
Ar
H2O
O
O
N S
C
Ar
S
N
Nitric oxide (NO) is a small gaseous molecule that plays a critical role in a variety of bioregulatory processes and can also inhibit metastasis, enhance cancer cell apoptosis, and assist macrophages in killing tumor cells. NO is usually generated in situ from an NO donor. S-Nitrosothiols are one type of NO donor, however, their instability toward light, heat, and metal ions limits their therapeutic applicability. Synthesizing more stable precursors to S-nitrosothiols will circumnavigate this. Recently, we noticed that 4-aryl-1,3,2-oxathiazolylium-5-olates are stable precursors and we propose based on our results that they will decompose under acidic conditions to release NO (Scheme 1). Electron Paramagnetic Resonance spectroscopy (EPR), mass spectrometry, and computational calculations were used to verify the reaction pathway and products. Herein, we would like to report the synthesis of this class of compounds (Scheme 2), their decomposition mechanism, and their therapeutic applicability. doi:10.1016/j.niox.2006.04.093
NO O
OH
C
Ar
O
O Ar
OH
~H
S
C
O Ar
O
CO2
OH S
S
Ar
HO
O
Ar
SH
Ar
SH
Ar
SH
SH
Scheme 1. NO generation from 4-aryl-1,3,2-oxathiazolylium-5-olates upon acidic activation.
O
X
O C OH Ph P, DIAD 3 OH
HSAc
O C OH
O C OH NaOMe X SAc
MeOH
b
a
X SH
O C OH
iBuONO CH2Cl2
X S NO
Scheme 2. The synthetic route of 4-aryl-1,3,2-oxathiazolylium-5-olates.
O N
DCC
S
CH2Cl2 X