- Email: [email protected]
O27. Effects of inhaled nitric oxide on human orthotopic liver transplantation
A8
Oral Sessions Papers / Nitric Oxide 14 (2006) A1–A16
lines used here the methylation degrees differ considerably in the order: A549 HaCaT > EaHy, demonstrating that the efficacy of NOS2 overexpression via AZA + TSA works best in cells with a highly methylated genome. Moreover, with A549 cells detailed analysis shows that the initial methylation state (set as 100%) is decreased to 25% by the AZA + TSA treatment, is reverted to 75% during the additional 48 h activation period, is reduced (25%) in the presence of NIO and fully reverted (<90%) in the presence of DETA-NO. These results demonstrate existence of a regulatory feed-back cycle at work between genome methylation and NOS2 expression and that the presence of NO leads to an effective suppression of epigenetic activation processes even in the presence of methylation inhibitors. doi:10.1016/j.niox.2006.04.028
O25. Arginine availability is critical to the innate immune response to Helicobacter pylori by regulation of iNOS translation Rupesh Chaturvedi a, Mohammad Asim b, Nuruddeen D. Lewis c, Francoise I. Bussiere b, Keith T. Wilson b a Department of Medicine, Division of Gastroenterology, Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare System b Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare System c Vanderbilt University School of Medicine The bacterium Helicobacter pylori (Hp) infects half of the world’s population, and causes peptic ulcer disease and gastric cancer. It elicits a vigorous immune response, but the infection persists for the life of the host. We have reported that while iNOS is induced by Hp in vivo and in vitro and NO can kill Hp, reasons for the failure of NO-mediated defense include: (1) Hp induces ornithine decarboxylase (ODC) that generates the polyamine spermine, which inhibits iNOS at the level of translation; and (2) Hp possesses an arginase that limits host NO generation. We now determined if L-arginine availability regulates iNOS response to Hp. Hp induced mRNA and protein expression of the cationic amino acid transporter (CAT) 2B in RAW 264.7 macrophages, peritoneal macrophages, and Hp gastritis tissues. Hp induced iNOS mRNA expression in macrophages in arginine-free medium, but iNOS protein expression was dependent on L-arginine concentration. These effects were due to increased iNOS translation and not an effect on protein stability. When macrophages were co-cultured with an isogenic mutant strain of Hp lacking arginase, there was significantly more iNOS protein expression and NO production, with no difference in iNOS mRNA levels. Inhibition of arginine uptake by lysine or by CAT2B siRNA had no effect on Hp-induced iNOS mRNA, but effectively blocked iNOS protein expression. Spermine inhibited L-arginine uptake and ODC siRNA potently increased L-arginine uptake. Similar effects were observed when knockdown or overexpression of spermine oxidase was used to potentiate or deplete spermine levels, respectively. These changes correlated with effects on iNOS protein and NO levels. Killing of Hp co-cultured with macrophages was dependent on L-arginine supplementation, which was prevented by lysine. In conclusion, spermine-mediated inhibition of L-arginine uptake and resulting attenuation of iNOS translation and NO production is an important mechanism of immune escape by Hp. doi:10.1016/j.niox.2006.04.029
O26. Loss of DDAH activity is involved in eNOS dysfunction following vascular injury Arthur Pope a, Wesley Johnson b, Arturo Juan Cardounel a Pharmacology, Ohio State University b Ohio State University
b
Following vascular injury, regenerated endothelial cells have been shown to exhibit impaired NOS mediated NO generation. Among the mechanisms that have been proposed for this NOS dysfunction are (1) decreased L-arginine bioavailability and (2) accumulation of the endogenous methylarginines ADMA and NMMA. These guanidino-methylated arginines are potent competitive inhibitors of NOS and are released upon protein degradation, however it is unknown whether their intracellular concentrations can reach levels sufficient to critically regulate endothelial NO production. Therefore, studies were carried out using a model of balloon mediated vascular injury of the rat carotid. HPLC measurements of intracellular methylarginine levels at 14 days post injury demonstrated a 2.4-fold increase in ADMA levels. This increase correlated with a 54% decrease in vascular relaxation in response to Ach which could be partially restored with L-arginine (200 lM) supplementation. This arginine enhancement effect was not observed in the contralateral (uninjured) vessel. The methylarginine accumulation and subsequent loss of vascular reactivity appears to be dependent on DDAH as expression of this critical ADMA metabolizing enzyme, is decreased more than 3-fold following balloon dilatation. In order to determine whether the loss of eNOS dependent vascular relaxation following balloon injury is due to decreased DDAH expression and subsequent ADMA accumulation, studies were performed using AAV mediated DDAH transduction. Successful transduction of DDAH was verified by pcr and resulted in gain of function following agonist induced vascular relaxation of the injured vessel. DDAH overexpression resulted in a 66% increase in vascular relaxation as compared to the injured control (AAV-GFP). These results demonstrate that modulation of methylarginine metabolizing enzymes in response to vascular injury results in ADMA accumulation with concentrations reaching levels sufficient to inhibit eNOS function. These effects are mediated through DDAH and implicate this enzyme as a potential target in the treatment of endothelial dysfunction. doi:10.1016/j.niox.2006.04.030
O27. Effects of inhaled transplantation
nitric
oxide
on
human
orthotopic
liver
Rakesh P. Patel a, Xinjun Teng b, Jack H. Crawford b, T. Scott Isbell b, Balu K. Chacko b, Yuliang Liu b, Phillip H. Chumley b, Nirag C. Jhala b, A. Blair Smith b, Luc Frenette b, Diana W. Wilhite b, J.S. Bynon b, Devin E. Eckhoff b, John D. Lang Jr b a Pathology, Anesthesiology, Surgery, University of Alabama at Birmingham b University of Alabama at Birmingham Hepatic ischemia-reperfusion (I–R) injury contributes significantly to organ dysfunction associated with liver transplantation. A key element in I–R injury is the acute inflammatory response that results after exposure of the reperfused tissue to leukocyte-derived reactive oxygen and nitrogen species leading to toxicity. Nitric oxide (NO) protects against hepatic I–R injury by inhibiting the inflammatory response. Inhaled NO (iNO) has been shown to replenish NO-mediated function in extra-pulmonary vascular beds during conditions characterized by loss of NO-bioavailability including I–R injury. We hypothesized that iNO will decrease hepatic inflammation and thereby minimize ischemia/reperfsion injury during liver transplantation. In this study, we report preliminary data from 14 patients undergoing liver transplantation who were randomized in an ongoing doubled-blinded placebo controlled clinical trial in which patients were administered either nitrogen (placebo) or iNO (80 ppm) during the transplantation procedure. The inhaled gas was administered after the induction of anesthesia and maintained until 1 h post arterial/portal reperfusion. Arterial and venous blood samples were collected for determination of plasma and red cell NOx profiles (nitrate, nitrite, S-nitrosothiols an C/N-nitrosamines). Moreover, liver biopsies pre- and one hour post-reperfusion were collected to assess for histological tissue injury and to determine markers of inflammation. Interim analysis demonstrated that
Oral Sessions Papers / Nitric Oxide 14 (2006) A1–A16 iNO was devoid of toxicity and was associated with increases in whole blood and plasma nitrite and nitrate. Interestingly, significant A-V nitrite gradients were observed which were increased with iNO, consistent with nitrite consumption across the microcirculation. S-Nitrosothiol concentrations in the plasma did not change significantly, but increased slightly in the RBC, and to a lesser magnitude than increases in nitrite. There was no significant difference observed in liver ischemic times between placebo and iNO groups. As expected, evaluation of liver tissue histology indicated that reperfusion resulted in increased inflammation and leukocyte accumulation. Tissue injury however, assessed histologically based on Suzuki scoring demonstrated significant reduction in injury with the administration of iNO (scores were 2.88 ± 0.3 and 2 ± 0 (means ± SEM) for placebo and iNO, respectively). Of significant clinical and economic importance, patients receiving iNO were discharged earlier compared to placebo group (30%). In summary, these preliminary analyses suggest that iNO represents a safe and viable therapy to limit hepatic I–R injury during transplantation. We hypothesize that iNO protects against liver I–R injury via increased circulating nitrite levels. doi:10.1016/j.niox.2006.04.031
O28. NO biomarkers: Windows to NO-mediated pathology and therapeutic intervention Thomas P. Misko a, Olga V. Nemirovskiy b, Melissa R. b Radabaugh , Poonam Aggarwal b, Maureen K. Highkin b, L. Stefan Lohmander c, Marie-Pierre Hellio le Graverand-Gastineau b, Candace Bramson b, Pamela T. Manning b, W. Rodney Mathews b a Discovery Biology, Pfizer b Pfizer c Lund University Inducible nitric oxide synthase (iNOS) has been implicated as a key player in the pathophysiology of numerous human diseases. Tissue-localized enzyme expression with associated nitrotyrosine formation has been observed in diseases such as stroke, multiple sclerosis, Alzheimer’s, rheumatoid arthritis and osteoarthritis suggesting that selective inhibition of iNOS may offer a route of therapeutic intervention. NO and/ or peroxynitrite appear to act as downstream effectors of the inflammatory cascade by contributing to free radical-mediated tissue injury, exerting effects via lipid, protein or DNA damage as well as through modulation of signal transduction pathways subserving normal cellular function. As a result, measurements of NOx, SNOs, and nitrotyrosine (NT) have proven to be invaluable for monitoring the temporal expression/activity of iNOS in both animal models of inflammation and in human disease. Analysis of biological fluids for nitrotyrosine has frequently proven problematic, suffering from artifact, lack of sensitivity, and signal to noise issues. Since it is a biomarker not only of NOS activity but also nitrative damage of surrounding tissue, we have developed a sensitive assay to measure levels of both free and protein-bound nitrotyrosine in accessible biological fluids including urine, plasma, serum and synovial fluid. The assay exploits an immuno-affinity based chromatographic separation of nitrotyrosine with subsequent detection by tandem mass spectrometry. The assay has a lower limit of sensitivity of 5 pg/ml (22 pM) and has been successfully utilized to follow iNOS inhibitor-mediated reduction of NT in animal models of inflammation and arthritis. Decrease in NT levels correlated well with the attenuation of paw edema, a disease-associated readout. Importantly, in both early and late stage OA we have observed a significant elevation of synovial fluid and plasma NT when compared to non-arthritis control groups. The utility of nitrotyrosine detection as a biomarker for iNOS-driven pathology in human disease states will be discussed. doi:10.1016/j.niox.2006.04.032
A9
O29. Nitric oxide is neuroprotective in a mouse model of Ab-mediated chronic neurodegeneration Carol Colton a, David Wink b, J. Brice Weinberg c, William Van Nostrand d, Hana Dawson c, Michael Vitek c a Neurology, Duke University Medical Center b NIH c Duke University Medical Center d State University of New York at Stony Brook Alzheimer’s disease is characterized by CNS insoluble amyloid deposits and accumulation of abnormally phosphorylated and aggregated forms of tau, a microtubule binding protein. Mouse models that over-express mutated forms of APP demonstrate amyloid plaque formation, but fail to demonstrate tau pathology that is fully reminiscent of AD. We now report the induction of somatodendritic tau pathology in cortical and hippocampal neurons in a well-established, common mouse model of AD that expresses the Swedish familial AD double mutation K670N-M671L in APP (Tg2576) but also lacks a functional NOS2 gene. The presence of abnormally phosphorylated tau was detected in APPswNOS2 / double transgenic mice using immunocytochemistry on brain sections and wellcharacterized antibodies (AT8, CP-13 and AT180) against specific disease-associated tau protein phosphorylation sites. To confirm the presence of aggregated tau, a filter assay that traps aggregated proteins was used to isolate and identify immunoreactive aggregates in brain lysates derived from NOS2 / , APPswNOS2 / and P301L control mice. Tau aggregation was confirmed by using scanning EM and thioflavin S histochemistry. Amyloid plaque morphology and distribution in the APPswNOS2 / brain was similar to that observed in the APPsw littermate mice. However, a significant increase in total and insoluble Ab peptides was observed. The activated form of caspase 3, a known executioner caspase involved in apoptosis, was observed in cell bodies and apical dendrites in neurons in the APPswNOS2 / mouse brain sections. Tau cleavage was detected by using an antibody directed against a C-terminal peptide of tau. In summary, the APPswNOS2 / mouse provides clear genetic data that removal of a major source of NO during a life time exposure to Ab proteins promotes pathological changes. The potential for NO to act as an inhibitory modulator of caspase activity places NO at a junction point between Ab peptides, caspase cleavage of tau and tau aggregation. doi:10.1016/j.niox.2006.04.033
O30. Unstirred layer limitation of nitric oxide depletion by oxyhemoglobin in erythrocytes Harry K. Mahtani, Charles A. Bosworth, Jack R. Lancaster Jr. Anesthesiology, University of Alabama at Birmingham (UAB) The reaction of nitric oxide (NO) with free hemoglobin in vitro is extremely rapid (kNO = 3.4 · 107 M 1 s 1) resulting in a half-life for NO (at blood hemoglobin concentrations) of only 2 ls [1]. This runs counter to the fact that NO is the primary mediator of endothelium-dependent vasodilation. There, however, actually is a dramatic decrease in the reaction rate of NO with erythrocyte-entrapped hemoglobin compared to circulating free hemoglobin. Does the composition of the red blood cell (RBC) membrane provide an intrinsic barrier to NO diffusion [2] or is the diffusion of NO in erythrocytes restricted by the existence of a surrounding unstirred layer [3]? Although erythrocytes are in constant flux, there exists a very thin layer around the cells where fluid is not being agitated by convection currents [4]. NO by necessity must diffuse through this unstirred layer before it can approach the oxyhemoglobin in the cell. The rate of NO diffusion across this unstirred layer although appreciably fast is extremely slow relative to the consumption of NO by the massively concentrated intraerythrocytic hemoglobin presence. In the vernacular of transport phenomena, the concentration of NO in the unstirred layer is strictly speaking ‘‘zero.’’ Furthermore, the exceedingly rapid depletion of any NO in the unstirred layer propagates the void (zero concentration).
Oral Sessions Papers / Nitric Oxide 14 (2006) A1–A16
lines used here the methylation degrees differ considerably in the order: A549 HaCaT > EaHy, demonstrating that the efficacy of NOS2 overexpression via AZA + TSA works best in cells with a highly methylated genome. Moreover, with A549 cells detailed analysis shows that the initial methylation state (set as 100%) is decreased to 25% by the AZA + TSA treatment, is reverted to 75% during the additional 48 h activation period, is reduced (25%) in the presence of NIO and fully reverted (<90%) in the presence of DETA-NO. These results demonstrate existence of a regulatory feed-back cycle at work between genome methylation and NOS2 expression and that the presence of NO leads to an effective suppression of epigenetic activation processes even in the presence of methylation inhibitors. doi:10.1016/j.niox.2006.04.028
O25. Arginine availability is critical to the innate immune response to Helicobacter pylori by regulation of iNOS translation Rupesh Chaturvedi a, Mohammad Asim b, Nuruddeen D. Lewis c, Francoise I. Bussiere b, Keith T. Wilson b a Department of Medicine, Division of Gastroenterology, Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare System b Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare System c Vanderbilt University School of Medicine The bacterium Helicobacter pylori (Hp) infects half of the world’s population, and causes peptic ulcer disease and gastric cancer. It elicits a vigorous immune response, but the infection persists for the life of the host. We have reported that while iNOS is induced by Hp in vivo and in vitro and NO can kill Hp, reasons for the failure of NO-mediated defense include: (1) Hp induces ornithine decarboxylase (ODC) that generates the polyamine spermine, which inhibits iNOS at the level of translation; and (2) Hp possesses an arginase that limits host NO generation. We now determined if L-arginine availability regulates iNOS response to Hp. Hp induced mRNA and protein expression of the cationic amino acid transporter (CAT) 2B in RAW 264.7 macrophages, peritoneal macrophages, and Hp gastritis tissues. Hp induced iNOS mRNA expression in macrophages in arginine-free medium, but iNOS protein expression was dependent on L-arginine concentration. These effects were due to increased iNOS translation and not an effect on protein stability. When macrophages were co-cultured with an isogenic mutant strain of Hp lacking arginase, there was significantly more iNOS protein expression and NO production, with no difference in iNOS mRNA levels. Inhibition of arginine uptake by lysine or by CAT2B siRNA had no effect on Hp-induced iNOS mRNA, but effectively blocked iNOS protein expression. Spermine inhibited L-arginine uptake and ODC siRNA potently increased L-arginine uptake. Similar effects were observed when knockdown or overexpression of spermine oxidase was used to potentiate or deplete spermine levels, respectively. These changes correlated with effects on iNOS protein and NO levels. Killing of Hp co-cultured with macrophages was dependent on L-arginine supplementation, which was prevented by lysine. In conclusion, spermine-mediated inhibition of L-arginine uptake and resulting attenuation of iNOS translation and NO production is an important mechanism of immune escape by Hp. doi:10.1016/j.niox.2006.04.029
O26. Loss of DDAH activity is involved in eNOS dysfunction following vascular injury Arthur Pope a, Wesley Johnson b, Arturo Juan Cardounel a Pharmacology, Ohio State University b Ohio State University
b
Following vascular injury, regenerated endothelial cells have been shown to exhibit impaired NOS mediated NO generation. Among the mechanisms that have been proposed for this NOS dysfunction are (1) decreased L-arginine bioavailability and (2) accumulation of the endogenous methylarginines ADMA and NMMA. These guanidino-methylated arginines are potent competitive inhibitors of NOS and are released upon protein degradation, however it is unknown whether their intracellular concentrations can reach levels sufficient to critically regulate endothelial NO production. Therefore, studies were carried out using a model of balloon mediated vascular injury of the rat carotid. HPLC measurements of intracellular methylarginine levels at 14 days post injury demonstrated a 2.4-fold increase in ADMA levels. This increase correlated with a 54% decrease in vascular relaxation in response to Ach which could be partially restored with L-arginine (200 lM) supplementation. This arginine enhancement effect was not observed in the contralateral (uninjured) vessel. The methylarginine accumulation and subsequent loss of vascular reactivity appears to be dependent on DDAH as expression of this critical ADMA metabolizing enzyme, is decreased more than 3-fold following balloon dilatation. In order to determine whether the loss of eNOS dependent vascular relaxation following balloon injury is due to decreased DDAH expression and subsequent ADMA accumulation, studies were performed using AAV mediated DDAH transduction. Successful transduction of DDAH was verified by pcr and resulted in gain of function following agonist induced vascular relaxation of the injured vessel. DDAH overexpression resulted in a 66% increase in vascular relaxation as compared to the injured control (AAV-GFP). These results demonstrate that modulation of methylarginine metabolizing enzymes in response to vascular injury results in ADMA accumulation with concentrations reaching levels sufficient to inhibit eNOS function. These effects are mediated through DDAH and implicate this enzyme as a potential target in the treatment of endothelial dysfunction. doi:10.1016/j.niox.2006.04.030
O27. Effects of inhaled transplantation
nitric
oxide
on
human
orthotopic
liver
Rakesh P. Patel a, Xinjun Teng b, Jack H. Crawford b, T. Scott Isbell b, Balu K. Chacko b, Yuliang Liu b, Phillip H. Chumley b, Nirag C. Jhala b, A. Blair Smith b, Luc Frenette b, Diana W. Wilhite b, J.S. Bynon b, Devin E. Eckhoff b, John D. Lang Jr b a Pathology, Anesthesiology, Surgery, University of Alabama at Birmingham b University of Alabama at Birmingham Hepatic ischemia-reperfusion (I–R) injury contributes significantly to organ dysfunction associated with liver transplantation. A key element in I–R injury is the acute inflammatory response that results after exposure of the reperfused tissue to leukocyte-derived reactive oxygen and nitrogen species leading to toxicity. Nitric oxide (NO) protects against hepatic I–R injury by inhibiting the inflammatory response. Inhaled NO (iNO) has been shown to replenish NO-mediated function in extra-pulmonary vascular beds during conditions characterized by loss of NO-bioavailability including I–R injury. We hypothesized that iNO will decrease hepatic inflammation and thereby minimize ischemia/reperfsion injury during liver transplantation. In this study, we report preliminary data from 14 patients undergoing liver transplantation who were randomized in an ongoing doubled-blinded placebo controlled clinical trial in which patients were administered either nitrogen (placebo) or iNO (80 ppm) during the transplantation procedure. The inhaled gas was administered after the induction of anesthesia and maintained until 1 h post arterial/portal reperfusion. Arterial and venous blood samples were collected for determination of plasma and red cell NOx profiles (nitrate, nitrite, S-nitrosothiols an C/N-nitrosamines). Moreover, liver biopsies pre- and one hour post-reperfusion were collected to assess for histological tissue injury and to determine markers of inflammation. Interim analysis demonstrated that
Oral Sessions Papers / Nitric Oxide 14 (2006) A1–A16 iNO was devoid of toxicity and was associated with increases in whole blood and plasma nitrite and nitrate. Interestingly, significant A-V nitrite gradients were observed which were increased with iNO, consistent with nitrite consumption across the microcirculation. S-Nitrosothiol concentrations in the plasma did not change significantly, but increased slightly in the RBC, and to a lesser magnitude than increases in nitrite. There was no significant difference observed in liver ischemic times between placebo and iNO groups. As expected, evaluation of liver tissue histology indicated that reperfusion resulted in increased inflammation and leukocyte accumulation. Tissue injury however, assessed histologically based on Suzuki scoring demonstrated significant reduction in injury with the administration of iNO (scores were 2.88 ± 0.3 and 2 ± 0 (means ± SEM) for placebo and iNO, respectively). Of significant clinical and economic importance, patients receiving iNO were discharged earlier compared to placebo group (30%). In summary, these preliminary analyses suggest that iNO represents a safe and viable therapy to limit hepatic I–R injury during transplantation. We hypothesize that iNO protects against liver I–R injury via increased circulating nitrite levels. doi:10.1016/j.niox.2006.04.031
O28. NO biomarkers: Windows to NO-mediated pathology and therapeutic intervention Thomas P. Misko a, Olga V. Nemirovskiy b, Melissa R. b Radabaugh , Poonam Aggarwal b, Maureen K. Highkin b, L. Stefan Lohmander c, Marie-Pierre Hellio le Graverand-Gastineau b, Candace Bramson b, Pamela T. Manning b, W. Rodney Mathews b a Discovery Biology, Pfizer b Pfizer c Lund University Inducible nitric oxide synthase (iNOS) has been implicated as a key player in the pathophysiology of numerous human diseases. Tissue-localized enzyme expression with associated nitrotyrosine formation has been observed in diseases such as stroke, multiple sclerosis, Alzheimer’s, rheumatoid arthritis and osteoarthritis suggesting that selective inhibition of iNOS may offer a route of therapeutic intervention. NO and/ or peroxynitrite appear to act as downstream effectors of the inflammatory cascade by contributing to free radical-mediated tissue injury, exerting effects via lipid, protein or DNA damage as well as through modulation of signal transduction pathways subserving normal cellular function. As a result, measurements of NOx, SNOs, and nitrotyrosine (NT) have proven to be invaluable for monitoring the temporal expression/activity of iNOS in both animal models of inflammation and in human disease. Analysis of biological fluids for nitrotyrosine has frequently proven problematic, suffering from artifact, lack of sensitivity, and signal to noise issues. Since it is a biomarker not only of NOS activity but also nitrative damage of surrounding tissue, we have developed a sensitive assay to measure levels of both free and protein-bound nitrotyrosine in accessible biological fluids including urine, plasma, serum and synovial fluid. The assay exploits an immuno-affinity based chromatographic separation of nitrotyrosine with subsequent detection by tandem mass spectrometry. The assay has a lower limit of sensitivity of 5 pg/ml (22 pM) and has been successfully utilized to follow iNOS inhibitor-mediated reduction of NT in animal models of inflammation and arthritis. Decrease in NT levels correlated well with the attenuation of paw edema, a disease-associated readout. Importantly, in both early and late stage OA we have observed a significant elevation of synovial fluid and plasma NT when compared to non-arthritis control groups. The utility of nitrotyrosine detection as a biomarker for iNOS-driven pathology in human disease states will be discussed. doi:10.1016/j.niox.2006.04.032
A9
O29. Nitric oxide is neuroprotective in a mouse model of Ab-mediated chronic neurodegeneration Carol Colton a, David Wink b, J. Brice Weinberg c, William Van Nostrand d, Hana Dawson c, Michael Vitek c a Neurology, Duke University Medical Center b NIH c Duke University Medical Center d State University of New York at Stony Brook Alzheimer’s disease is characterized by CNS insoluble amyloid deposits and accumulation of abnormally phosphorylated and aggregated forms of tau, a microtubule binding protein. Mouse models that over-express mutated forms of APP demonstrate amyloid plaque formation, but fail to demonstrate tau pathology that is fully reminiscent of AD. We now report the induction of somatodendritic tau pathology in cortical and hippocampal neurons in a well-established, common mouse model of AD that expresses the Swedish familial AD double mutation K670N-M671L in APP (Tg2576) but also lacks a functional NOS2 gene. The presence of abnormally phosphorylated tau was detected in APPswNOS2 / double transgenic mice using immunocytochemistry on brain sections and wellcharacterized antibodies (AT8, CP-13 and AT180) against specific disease-associated tau protein phosphorylation sites. To confirm the presence of aggregated tau, a filter assay that traps aggregated proteins was used to isolate and identify immunoreactive aggregates in brain lysates derived from NOS2 / , APPswNOS2 / and P301L control mice. Tau aggregation was confirmed by using scanning EM and thioflavin S histochemistry. Amyloid plaque morphology and distribution in the APPswNOS2 / brain was similar to that observed in the APPsw littermate mice. However, a significant increase in total and insoluble Ab peptides was observed. The activated form of caspase 3, a known executioner caspase involved in apoptosis, was observed in cell bodies and apical dendrites in neurons in the APPswNOS2 / mouse brain sections. Tau cleavage was detected by using an antibody directed against a C-terminal peptide of tau. In summary, the APPswNOS2 / mouse provides clear genetic data that removal of a major source of NO during a life time exposure to Ab proteins promotes pathological changes. The potential for NO to act as an inhibitory modulator of caspase activity places NO at a junction point between Ab peptides, caspase cleavage of tau and tau aggregation. doi:10.1016/j.niox.2006.04.033
O30. Unstirred layer limitation of nitric oxide depletion by oxyhemoglobin in erythrocytes Harry K. Mahtani, Charles A. Bosworth, Jack R. Lancaster Jr. Anesthesiology, University of Alabama at Birmingham (UAB) The reaction of nitric oxide (NO) with free hemoglobin in vitro is extremely rapid (kNO = 3.4 · 107 M 1 s 1) resulting in a half-life for NO (at blood hemoglobin concentrations) of only 2 ls [1]. This runs counter to the fact that NO is the primary mediator of endothelium-dependent vasodilation. There, however, actually is a dramatic decrease in the reaction rate of NO with erythrocyte-entrapped hemoglobin compared to circulating free hemoglobin. Does the composition of the red blood cell (RBC) membrane provide an intrinsic barrier to NO diffusion [2] or is the diffusion of NO in erythrocytes restricted by the existence of a surrounding unstirred layer [3]? Although erythrocytes are in constant flux, there exists a very thin layer around the cells where fluid is not being agitated by convection currents [4]. NO by necessity must diffuse through this unstirred layer before it can approach the oxyhemoglobin in the cell. The rate of NO diffusion across this unstirred layer although appreciably fast is extremely slow relative to the consumption of NO by the massively concentrated intraerythrocytic hemoglobin presence. In the vernacular of transport phenomena, the concentration of NO in the unstirred layer is strictly speaking ‘‘zero.’’ Furthermore, the exceedingly rapid depletion of any NO in the unstirred layer propagates the void (zero concentration).