Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets

The present study tested the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase (PTP) activity in the membranes and increased activity and expression of PTPs (PTP-1B, PTP-SH1 and 2) in the cytosol of the cerebral cortex of newborn piglets are mediated by nitric oxide (NO). To test this hypothesis, PTP activity in cell membranes and activity and expression were measured in the cytosol of normoxic (Nx, n = 5), hypoxic (Hx, n = 5), and 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of neuronal nitric oxide synthase (nNOS), pretreated hypoxic (7-NINA+Hx, n = 6) newborn piglets. PTP activity in cortical cell membranes was lower in the Hx group as compared to the Nx group and this decrease was prevented in the 7-NINA+Hx group. The density of cytosolic PTP-1B, cytosolic PTP-SH1 and PTP-SH2 was increased in the Hx group and this increase was prevented in the 7-NINA+Hx group. Immunohistochemistry results show an increased immunoreactivity to PTP-1B in the Hx as compared to Nx animals. The data show that pretreatment with 7-NINA, a selective inhibitor of nNOS, prevents the hypoxia-induced decrease in PTP activity in membranes. nNOS inhibition also prevented the hypoxia-induced increase in PTP activity and expression in cytosol, and therefore we conclude that modification of PTP during hypoxia is NO-mediated.     

Nitric oxide-mediated Ca++-influx in neuronal nuclei and cortical synaptosomes of normoxic and hypoxic newborn piglets.

Previous studies have shown that hypoxia results in the generation of nitric oxide (NO) free radicals in the cerebral cortex of newborn animals. The present study tested the hypothesis that NO increases Ca++-influx in neuronal nuclei as well as N-methyl-D aspartate (NMDA) receptor-mediated Ca++-influx in cortical synaptosomes of newborn piglets. Studies were performed in five normoxic (Nx) and 6 hypoxic (Hx) newborn piglets. Cerebral tissue hypoxia was documented by determining the levels of ATP and phosphocreatine (PCr). 45Ca++ -influx was determined in the presence of sodium nitroprusside (SNP, 10 microM), a NO donor, and peroxynitrite (10 microM). In the Hx group, ATP levels decreased to 1.40+or-0.69 vs 4.27+or-0.80 micromoles/g brain in the Nx group (P<0.05). Similarly, PCr levels decreased to 0.91+or-0.57 vs 3.40+or-0.99 micromoles/g brain (P<0.001). Nuclear 45Ca++-influx increased from 3.57+or-1.46 pmoles/mg protein in Nx nuclei to 8.64+or-3.50 in Hx nuclei (P<0.05). SNP increased neuronal nuclear Ca++ influx in the Nx from 3.57+or-1.46 to 5.47+or-2.52 pmoles/mg protein (P<0.05) but did not affect Ca++ influx in the Hx group (8.64+or-3.50 vs. 10.17+or-4.00 pmoles/mg protein). The level of Ca++ influx in the presence of SNP in Nx nuclei was similar to that seen in Hx nuclei alone. Peroxynitrite did not affect nuclear Ca++-influx in either Nx or Hx group. Synaptosomal Ca++-influx in the presence of glu + gly was 40+or-11 pmoles/mg protein in the Nx group and 80+or-16 pmoles/mg protein in the Hx group (P<0.05). Both SNP and peroxynitrite increased Ca++ influx in Nx and Hx synaptosomes. These results show that hypoxia results in increased nuclear and synaptosomal Ca++-influx. Further, the data demonstrate that NO increases intranuclear as well as intrasynaptosomal Ca++-influx and suggest that during hypoxia, the increase in intranuclear and intraynaptosomal Ca++ is NO-mediated. We propose that NO-mediated modification (by nitrosylation/nitration) of nuclear membrane high affinity Ca++-ATPase and neuronal membrane NMDA receptor, resulting in increased intranuclear and intracellular Ca++ influx, are potential NO-mediated mechanisms of Hx neuronal injury.     

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.     

Nitric oxide-mediated activation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance ODxmm(2). ATP levels were 4.57+/-0.45 micromoles/g brain in the Nx group, 1.29+/-0.23 micromoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50+/-0.14 micromoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77+/-0.36 micromoles/g brain in the Nx group, 0.77+/-0.13 micromoles/g brain in the hypoxic group (P<0.05) and 1.02+/-0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5+/-53.7 ODxmm(2) in the Nx group as compared with 419.6+/-63.9 ODxmm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0+/-28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8+/-42.8 ODxmm(2) in the normoxic group as compared with 364.6+/-60.1 ODxmm(2) in the Hx group (P<0.002) and 254.8+/-24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated     

ATP and cytochrome c-dependent inhibition of caspase-9 activity in the cerebral cortex of newborn piglets

The present study investigates the mechanism of activation of caspase-9 during hypoxia and tests the hypothesis that ATP and cytochrome c regulate the activity of caspase-9 in the cerebral cortex of newborn piglets. Cerebral tissue hypoxia was documented by decreased levels of high energy phosphates, ATP and phosphocreatine (PCr). Cytosolic fractions were prepared from cerebral cortices and passed through a G50 column, to remove endogenous ATP and cytochrome c. Caspase-9 activity was determined spectrofluorometrically using a specific fluorogenic substrate for caspase-9 at increasing concentrations of ATP (0-1.0 mM) or cytochrome c (0-3.0 microM). Caspase-9 activity (nmol/mg protein/h) was 1.26 +/- 0.15 in the normoxic and 2.13 +/- 0.14 in the hypoxic group (P < 0.05). The enzyme activity was inhibited by ATP or cytochrome c in both normoxic and hypoxic groups. The IC50 for ATP and cytochrome c increased 5-fold and 1.5-fold, respectively, following hypoxia, suggesting a hypoxia-induced modification of the ATP and cytochrome binding sites. The data demonstrate that ATP (1 mM) and cytochrome c (3.0 microM) inhibit caspase-9 activity by approximately 70%. On the basis of these observations, we propose a new and novel concept that the caspase-9 activity remains inhibited under the normoxic conditions and during hypoxia the decrease in ATP and decreases in the affinity for ATP and cytochrome c release the inhibitory block to activate the enzyme. Results of ATP- and cytochrome c-dependent inhibition of purified caspase-9 human recombinant show that the inhibitory effect by ATP and cytochrome c does not require Apaf-1. To our knowledge, this is a completely new concept and a new mechanism of regulation of caspase-9 activity that may lead to hypoxia-induced programmed cell death.     

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.     

Role of endogenous regucalcin in brain function: suppression of cytosolic nitric oxide synthase and nuclear protein tyrosine phosphatase activities in brain tissue of transgenic rats

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of brain function was investigated by using regucalcin transgenic (TG) rats. Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol and nucleus of the brain tissue of TG female rats (5-week-old) as compared with that of wild-type (wt) female rats. In TG male rats, the enhancement of regucalcin expression in the brain cytosol and nucleus was only slight. Nitric oxide (NO) synthase activity was significantly decreased in the brain cytosol of TG female rats. Protein tyrosine phosphatase activity was not significantly altered in the brain nucleus of TG female rats. The presence of calcium chloride (10 microM) or anti-regucalcin monoclonal antibody (50 ng/ml) in the enzyme reaction mixture caused a significant increase in cytosolic NO synthase and nuclear protein tyrosine phosphatase activities in the brain tissue of wt rats. The increase was significantly prevented in the brain cytosol and nucleus of TG rats. The present study supports the view that endogenous regucalcin plays a suppressive role in the regulation of brain function in rats.     

Effect of neuronal nitric oxide synthase inhibition on CA2+/calmodulin kinase kinase and CA2+/calmodulin kinase IV activity during hypoxia in cortical nuclei of newborn piglets

The present study tests the hypothesis that cerebral tissue hypoxia results in increased Ca(2+)/calmodulin (CaM) kinase kinase activity and that the administration of nitric oxide synthase inhibitors (N-nitro-l-arginine [NNLA], or 7-nitroindazole sodium [7-NINA]) prior to the onset of hypoxia will prevent the hypoxia-induced increase in the enzyme activity. To test this hypothesis, CaM kinase kinase and CaM kinase IV activities were determined in normoxic, hypoxic, NNLA-treated hypoxic, and 7-NINA-treated hypoxic piglets. Hypoxia was induced (FiO(2)=0.05-0.08x1 h) and confirmed biochemically by tissue levels of ATP and phosphocreatine. CaM kinase kinase activity was determined in a medium containing protein kinase and phosphatase inhibitors, calmodulin, and a specifically designed CaM kinase kinase target peptide. CaM kinase IV activity was determined by (33)P-incorporation into syntide-2 in a buffer containing protein kinase and phosphatase inhibitors. Compared with normoxic animals, ATP and phosphocreatine levels were significantly lower in all hypoxic piglets whether or not pretreated with nitric oxide synthase inhibitors. There was a significant difference among CaM kinase kinase activity (pmol/mg protein/min) in normoxic (76.84+/-14.1), hypoxic (138.86+/-18.2, P<0.05 vs normoxia), NNLA-pretreated hypoxic (91.34+/-19.3; P=NS vs normoxia, P<0.05 vs hypoxia) and 7-NINA-pretreated hypoxic animals (100.12+/-23.3; P=NS vs normoxia, P<0.05 vs hypoxia). There was a significant difference among CaM kinase IV activity (pmol/mg protein/min) in normoxia (1270.80+/-126.1), hypoxia (2680.80+/-136.7; P<0.05 vs normoxia), NNLA-pretreated hypoxia (1666.00+/-154.8; P<0.05 vs normoxia, P<0.05 vs hypoxia), and 7-NINA-pretreated hypoxic (1712.9+/-231.5; P=NS vs normoxia, P<0.05 vs hypoxia). We conclude that the hypoxia-induced increase in CaM kinase kinase and CaM kinase IV activity is mediated by neuronal NOS-derived NO.     

High levels of peroxynitrite are generated in the lungs of irradiated mice given cyclophosphamide and allogeneic T cells. A potential mechanism of injury after marrow transplantation.

In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (.NO) production, and by Cy, which stimulates superoxide production.NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells +/- Cy. AMs obtained on Day 7 after BMT from mice receiving T cells +/- Cy spontaneously generated between 20 and 40 microM nitrite in culture, versus < 2 microM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived.NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.     

Effect of neuronal nitric oxide synthase inhibition on caspase-9 activity during hypoxia in the cerebral cortex of newborn piglets

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death. We have also shown increased nitric oxide (NO) free radical generation during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced increase in caspase-9 activity in the cerebral cortex of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). To test this hypothesis, cytosolic caspase-9 activity was determined in 15 newborn piglets divided into three groups: normoxic (Nx, n=5), hypoxic (Hx, n=5), and Hx pretreated with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, 1mg/kg, i.p., 1h prior to hypoxia (Hx+7NI, n=5). The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. The cytosolic fraction was obtained from the cerebral cortical tissue following centrifugation at 100,000 x g for 1h and caspase-9 activity was assayed using Ac-Leu-Glu-His-Asp-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-9. Caspase-9 activity was determined spectroflourometrically at 460 nm using 380 nm as excitation wavelength. ATP levels (micromol/g brain) were 4.35+/-0.21 in the Nx 1.43+/-0.28 in the Hx (p<0.05 versus Nx), and 1.73+/-0.33 in the Hx+7-NINA group (p<0.05 versus Nx, p=NS versus Hx). PCr levels (micromol/g brain) were 3.80+/-0.26 in the Nx, 0.96+/-0.20 in the Hx (p<0.05 versus Nx), and 1.09+/-0.39 in the Hx+7 NINA group (p<0.05 versus Nx, p=NS versus Hx). Cytosolic caspase-9 activity (nmol/mg protein/h), increased from 1.27+/-0.15 in the Nx to 2.13+/-0.14 in the Hx (p<0.05 versus Nx) compared to 1.10+/-0.21 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). Caspase-3 activity (nmol/mg protein/h) also increased from 9.39+/-0.73 in Nx to 18.94+/-3.64 in Hx (p<0.05 versus Nx) compared to 8.04+/-1.05 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). The data show that administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increase in caspase-9 activity that leads to increase in caspase-3 activity. Since nNOS inhibition blocked the increase in caspase-9 activity during hypoxia, we conclude that hypoxia-induced increase in caspase-9 activity is mediated by nNOS derived NO. We propose that the NO generated during hypoxia leads to activation of caspase-9 and results in initiation of caspase-cascade-dependent hypoxic neuronal death.     

Effect of hypoxia on protein tyrosine phosphatase activity and expression of protein tyrosine phosphatases PTP-1B, PTP-SH1 and PTP-SH2 in the cerebral cortex of guinea pig fetus

The present study tested the hypothesis that the hypoxia in utero results in decreased protein tyrosine phosphatase (PTP) activity in cytosolic and membrane fractions and increased expression of PTPs (PTP-1B, PTP-SH1 and PTP-SH2) in the cytosol and the membrane fraction of the cerebral cortex of guinea pig fetus. In addition, we hypothesize that the increased expression is mediated by nitric oxide (NO). To test this hypothesis, PTP activity in cytosol and cell membrane, and expression in the cytosol and membrane fraction were measured in the cerebral cortex of normoxic, hypoxic and L-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), pretreated hypoxic (L-NAME+Hx) guinea pig fetuses. PTP activity in the cytosolic and membrane fractions was significantly lower in the Hx group as compared to the Nx group. The density of cytosolic PTP-1B, cytosolic PTP-SH1 and PTP-SH2 was increased in the Hx group and this increase was prevented in the L-NAME+Hx group. The data show that pretreatment with L-NAME, an inhibitor of NOS, prevents the hypoxia-induced increased expression of PTP-1B, PTP-SH1 and PTP-SH2 in the membrane and cytosolic fractions of the cerebral cortex of the guinea pig fetus. We conclude that the decrease in PTP activity during hypoxia is not due to protein modification of PTP and due to alteration in PTP expression.     

Phosphorylation of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets following hypoxia

We have previously shown that hypoxia leads to increased expression and increased activity of caspase-9 in the cerebral cortex of newborn piglets. Previous studies have demonstrated the importance of caspase-9 in the initiation of the apoptotic cascade, however, the mechanism of caspase-9 activation is not well understood. Experiments were conducted on newborn piglets 2-3 days of age that were anesthetized and mechanically ventilated. Hypoxia was induced by lowering the FiO(2) to 0.05-0.07 x 1h, and was confirmed biochemically by demonstrating decreased levels of ATP and PCr in the hypoxic groups in comparison with the normoxic group. The ATP level was 1.99+/-0.66 in the hypoxic group versus 4.10+/-0.19 in the normoxic group, P<0.05, and the PCr value was 0.68+/-0.14 in the hypoxic group, compared to 2.98+/-0.39 in the normoxic group, P<0.05. The cytosol of the neuronal nuclei from the cerebral cortex was probed with anti-phosphorylated Ser(196) caspase-9 antibody, using Western blot analysis. Protein bands were analyzed using image densitometry. In both the hypoxic and normoxic samples, protein bands were demonstrated just above the 50 kDa marker. Phosphorylated caspase-9 expression in OD x mm(2) was 43.85+/-8.4 in the normoxic group and 67.6+/-9.88 in the hypoxic group, P<0.05. The results of this study demonstrate that caspase-9, a key protein in hypoxia induced apoptosis, is phosphorylated at the Ser(196) site during hypoxia. The results demonstrate that hypoxia results in a post-translational modification of caspase-9 at Ser(196), which may alter the activity of caspase-9 in the hypoxic newborn brain.     

Inducible nitric oxide synthase mediates prostaglandin h2 synthase nitration and suppresses eicosanoid production

Nitric oxide (NO) modulates the biological levels of arachidonate-derived cell signaling molecules by either enhancing or suppressing the activity of prostaglandin H(2) isoforms (PGHS-1 and PGHS-2). Whether NO activates or suppresses PGHS activity is determined by alternative protein modifications mediated by NO and NO-derived species. Here, we show that inducible NO synthase (iNOS) and PGHS-1 co-localize in atherosclerotic lesions of ApoE(-/-) mouse aortae. Immunoblotting and immunohistochemistry revealed Tyr nitration in PGHS-1 in aortic lesions but markedly less in adjacent nonlesion tissue. PGHS-2 was also found in lesions, but 3-nitrotyrosine incorporation was not detected. 3-Nitrotyrosine formation in proteins is considered a hallmark reaction of peroxynitrite, which can form via NO-superoxide reactions in an inflammatory setting. That iNOS-derived NO is essential for 3-nitrotyrosine modification of PGHS-1 was confirmed by the absence of 3-nitrotyrosine in lesions from ApoE(-/-)iNOS(-/-) mice. Mass spectrometric studies specifically identified the active site residue Tyr385 as a 3-nitrotyrosine modification site in purified PGHS-1 exposed to peroxynitrite. PGHS-mediated eicosanoid (PGE(2)) synthesis was more than fivefold accelerated in cultured iNOS(-/-) versus iNOS-expressing mouse aortic smooth muscle cells, suggesting that iNOS-derived NO markedly suppresses PGHS activity in vascular cells. These results further suggest a regulatory role of iNOS in eicosanoid biosynthesis in human atherosclerotic lesions.     

Nitration of N-methyl-D-aspartate receptor subunits following in vitro dephosphorylation of cerebral cortical membranes of newborn piglets.

Previous studies have shown that activity of the cerebral N-methyl-D-aspartate (NMDA) receptor is modified by receptor nitration and phosphorylation. Since the sites for tyrosine phosphorylation and nitration are on adjacent carbon atoms, the present study tested the hypothesis that in vitro dephosphorylation of the NR1, NR2A and NR2B subunits of the NMDA receptor increases receptor nitration by peroxynitrite in cortical membranes of newborn piglets. To test this hypothesis, cerebral cortical P(2) membranes were prepared from normoxic and hypoxic newborn piglets and divided into dephosphorylated and non-dephosphorylated control groups. Dephosphorylation was performed in vitro by incubation with protein tyrosine phosphatase 1B and confirmed by immunoprecipitation with antiphosphotyrosine antibody. Dephosphorylated and non-dephosphorylated samples were nitrated with 0.5 mM peroxynitrite. Nitration was measured by immunoprecipitating with agarose-conjugated anti-nitrotyrosine antibody followed by Western blot analysis using specific anti -NR1, -NR2A and -NR2B primary antibodies. The data demonstrate that nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increases following dephosphorylation in both normoxic and hypoxic animals, however increase is much higher in hypoxic animals. We conclude that dephosphorylation at the site adjacent to the nitration site (ortho-position) on tyrosine residues of the NMDA receptor enhances nitration. Since in vitro nitration of the NMDA receptor increases the affinity of the glutamate recognition site and the receptor ion channel, we speculate that tyrosine dephosphorylation of the NMDA receptor will remove steric hindrance and facilitate nitration of tyrosine residues resulting in increased ion-channel activation in the hypoxic newborn brain.     

Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: attenuation with increasing age

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 micro M) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 micro M), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10(-4)-10(-8) M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 micro M). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10(-6) M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.     

Effect of hypoxia on the expression and activity of mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and MKP-3 in neuronal nuclei of newborn piglets: the role of nitric oxide

Mitogen-activated protein kinase-1 (MAPK-1) and MAPK-3 regulate survival and programmed cell death of neurons under stress conditions. The activity of MAPK-1 and MAPK-3 is regulated by dual specificity phosphatases: MKP-1 and MKP-3. In previous studies, we have shown that cerebral hypoxia results in increased activation of MAPK-1 and MAPK-3. Furthermore, we have shown that the hypoxia-induced activation of MAPK is nitric oxide (NO)-mediated. The present study tested the hypothesis that hypoxia results in altered expression and activity of MKP-1 and MKP-3 in neuronal nuclei and the administration of 7-nitro-indazole (7-NINA; 1 mg/kg, 60 min prior to hypoxia), a selective nNOS inhibitor, will prevent the hypoxia-induced alteration in the expression and activity of MKP-1 and MKP-3. To test this hypothesis expression and activity of MKP-1 and MKP-3 were determined in neuronal nuclei of normoxic (Nx; n=5), hypoxic (Hx; n=5) and 7-NINA-pretreated-hypoxic (7-NINA-Hx; n=5). Hypoxia was achieved by exposing the animals to an FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were analyzed by Western blot using specific antibodies for MKP-1 and MKP-3 (Santa Cruz, CA, USA). The protein band density was determined by imaging densitometry and expressed as OD x mm2. The density of MKP-1 was 61.57+/-5.68, 155.86+/-44.02 and 69.88+/-25.54 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly, the density of MKP-3 was 66.46+/-5.88, 172.04+/-33.10 and 116.88+/-14.66 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). The data show an increased expression of MKP-1 and MKP-3 during hypoxia in neuronal nuclei of newborn piglets and the administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increased expression of MKP-1 and MKP-3. The activity of MKP-1 (pmol/min) was 176.17+/-16.95 in Nx, 97.56+/-10.64 in Hx and 130+/-14.42 in the 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly the activity of MKP-3 was 104.11+/-12.17 in Nx, 36.29+/-16.88 in Hx and 77.89+/-20.18 in the 7-NINA groups, respectively (P<0.05, ANOVA). The results demonstrate that cerebral hypoxia results in increased expression of MKP-1 and MKP-3 expression that was prevented by the administration of 7-NINA. In contrast, hypoxia resulted in decreased activity of MKP-1 and MKP-3 that was prevented by the administration of a nNOS inhibitor. We conclude that hypoxia-induced decrease in MKP-1 and MKP-3 activity is not due to altered expression but due to NO-mediated modification of the cysteine residue at the active site of these dual specificity phosphatases, a mechanism of their inactivation that leads to activation of MAP kinases.     

Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.     

Protein carbonyl formation and tyrosine nitration as markers of oxidative damage during ischaemia-reperfusion injury to rat sciatic nerve

We have investigated the role of oxidative damage in peripheral nerve ischaemia-reperfusion injury using a rat sciatic nerve model. After 5 h ischaemia blood flow to the sciatic nerve was restarted and markers of oxidative damage measured after various times of reperfusion. As a marker of protein oxidative damage, protein carbonyl formation was measured using a sensitive enzyme-linked immunosorbent assay. Protein carbonyl content was unaffected by ischaemia alone, but increased by 55% after 12-18 h reperfusion, correlating with the onset of nerve pathology. Pretreatment with the xanthine oxidase inhibitor allopurinol prevented these abnormalities, suggesting that xanthine oxidase activity is proximal to oxidative damage during reperfusion injury. To determine whether formation of the potent oxidant peroxynitrite from nitric oxide and superoxide contributed to ischaemia-reperfusion injury, we measured the accumulation of 3-nitrotyrosine residues in proteins. Only one protein of 49,000 mol. wt contained significant amounts of 3-nitrotyrosine residues which was shown to be glial fibrillary acidic protein, an abundant cytoskeletal protein in Schwann cells. However glial fibrillary acidic protein contained 3-nitrotyrosine residues prior to ischaemia-reperfusion, and the amount of nitrated tyrosine residues in total glial fibrillary acidic protein did not increase significantly during reperfusion, therefore it was not possible to draw conclusions about the role of peroxynitrite in nerve reperfusion injury.     

Characterization of protein tyrosine phosphatase activity in rat liver microsomes: suppressive effect of endogenous regucalcin in transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of protein phosphatase activity in rat liver microsomes was investigated. Protein phosphatase activity torward phosphotyrosine, phosphoserine, and phosphothreonine was assayed in a reaction mixture containing the microsomal protein. Protein phosphatase activity toward phosphotyrosine was strong as compared with that of the enzyme activity toward phosphoserine and phosphothreonine, indicating the existence of protein tyrosine phosphatase. Protein phosphatase activity toward three phosphoaminoacids was significantly enhanced by the addition of both calcium chloride (10 micro M) and calmodulin (2.5 or 5 micro g/ml) in the reaction mixture. The presence of ethylene glycol bis (2-amino-ethylether) N, N, N', N'-tetracetic acid (EGTA; 0.1, 1 or 2 mM) or trifluoperazine (TFP; 10, 20 or 50 micro M), an antagonist of calmodulin, did not have a significant effect on protein phosphatase activity toward phosphotyrosine without calcium addition. Microsomal protein tyrosine phosphatase activity was not changed by okadaic acid (10(-6)-10(-4) M). The enzyme activity was significantly decreased by vanadate (10, 50 or 100 micro M). The addition of regucalcin (0.25 or 0.5 micro M) in the reaction mixture caused a significant inhibition of protein tyrosine phosphatase activity in liver microsomes. Western blot analysis showed a remarkable increase in regucalcin protein level in the liver microsomes of regucalcin transgenic (TG) rats. Protein tyrosine phosphatase activity was significantly suppressed in the liver microsomes of TG rats. This study demonstrates that protein tyrosine phosphatase activity is found in the liver microsomes, and that the enzyme activity is suppressed by regucalcin.     

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.