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Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets
Neuroscience Letters 362 (2004) 108–112 www.elsevier.com/locate/neulet
Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets Qazi M. Ashraf*, Syed H. Haider, Christos D. Katsetos, Maria Delivoria-Papadopoulos, Om. Mishra Department of Pediatrics, Drexel University College of Medicine and St. Christopher’s Hospital for Children, Philadelphia, PA, USA Received 22 October 2003; received in revised form 25 February 2004; accepted 3 March 2004
Abstract 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 ¼ 5), hypoxic (Hx, n ¼ 5), and 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of neuronal nitric oxide synthase (nNOS), pretreated hypoxic (7-NINA þ Hx, n ¼ 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 þ 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 þ 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. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Protein tyrosine phosphatase; Hypoxia; Brain; Piglets
Protein tyrosine phosphatases (PTPs) regulate kinasedependent signal transduction pathways and control cell proliferation, differentiation and metabolism. PTPs reverse the effects of protein tyrosine kinases by catalyzing the hydrolysis of the phosphoryl group. Protein phosphatases have been implicated in a number of pathological conditions including cancer and diabetes. Protein tyrosine phosphorylation is a post-translational modification that cells utilize to regulate signal transduction [2,16]. PTP in conjunction with protein kinase regulates many critical events including cell growth, tissue differentiation, intercellular communication, immune response, development and metabolism [20]. The PTPs can be divided into three major subfamilies: tyrosine specific, dual specific and low molecular weight phosphatases. Tyrosine specific phosphatases can be further * Corresponding author. Pediatrics/Neonatal Research, 7th Floor Heritage Building, Room 701, Drexel University College of Medicine, 3300 Henry Avenue, Philadelphia, PA 19129, USA. Tel.: þ 1-215-8424960; fax: þ 1-215-843-3505. E-mail address: [email protected] (Q.M. Ashraf).
subdivided into two groups of enzymes. The first group is receptor-like with a single trans-membrane domain and one or two intracellular catalytic domains, and a unique extracellular domain of variable length. The second group comprises of cytoplasmic enzymes, with a single catalytic domain and a variable amino- or carboxyl-terminal regulatory domain, examples of which include PTP-SH and PTP-1B. A large number of enzymes and substrates are phosphorylated during hypoxia. The phosphorylation pattern of proteins observed in synaptosomes of the guinea pig showed that the phosphorylated sites were two times higher in hypoxia than in normoxia [4]. These phosphorylation events may be regulated by free radicals. There are a number of mechanisms of free radical generation during hypoxia including the nitric oxide synthase (NOS) which results in nitric oxide (NO)-dependent peroxynitrite generation. Previous studies from our laboratory have shown that PTP activity decreases during hypoxia contributing to phosphorylation of a number of proteins including antiapoptotic protein Bcl-2 [1,6]. The purpose of the present
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.02.069
Q.M. Ashraf et al. / Neuroscience Letters 362 (2004) 108–112
study was to test the hypothesis that the hypoxia-induced changes in the activity and expression of PTPs are mediated by NO and will be prevented by blocking neuronal NOS (nNOS) by 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of nNOS. In the present study we have determined the effect of administration of 7-NINA on hypoxia-induced modification of the activity and expression, specifically (a) the activity of PTP in the membrane and cytosolic fractions, (b) the expression of the PTP-1B and PTP-SH in the cytosol, and (c) the immunohistochemical analysis of PTP-1B. Studies were performed on 2 – 4-day-old Yorkshire piglets as per standard protocol as described by Ashraf et al. [1]. The study was approved by the Institutional Animal Care and Use Committee of the Drexel University College of Medicine. ATP and phosphocreatine (PCr) concentrations were determined according to the method of Lamprecht, as mentioned in Ashraf et al. [1]. P2 membrane fractions were prepared according to the method described by Harik et al. [7]. PTP activity was determined spectrophotometrically at 405 nm in a medium containing 100 mM HEPES (pH 7.0), 1 mM EDTA, 100 mg protein [8] and 2.5 mM p-nitrophenylphosphate (pNPP) at 37 8C for 20 min in the presence or absence of 2 mM bpV(phen), a selective inhibitor of PTP. The inhibitor sensitive activity was determined and expressed as nmol pNP/mg protein per h. Immunoblotting was performed as described by Ashraf et al. [1] and cytosolic proteins incubated with primary polyclonal antibodies for PTP-1B, PTP-SH1 and PTP-SH2 (Santa Cruz Biotech, Santa Cruz, CA). Equal protein loading was confirmed by subsequent probing with mouse monoclonal Actin/GAPDH antibody (Chemicon, CA) in each experiment. The protein density was expressed as absorbance (OD £ mm2). Interleaved sections for immunohistochemistry were placed on gelatin-coated slides, and then deparaffinized and post-fixed with paraformaldehyde. Sections were then washed three times for 5 min each with PBS, and then incubated with antibody against PTP-1B (Santa Cruz) diluted 1:1000 in PBS/1% serum overnight at room temperature. Slides were then washed five times with PBS and incubated with the biotin conjugated secondary antibody (Jackson Labs) diluted 1:500 in PBS/1% serum for 2 h. The slides were washed again three times with PBS and incubated with the ABC reagent (streptavidin/biotinylated horseradish peroxidase (Vector Labs)). Antibody binding was visualized with diaminobenzidine (DAB) using a Sigma-Fast reagent kit. The data on ATP, PCr, PTP enzyme activity and expression were analyzed using one-way analysis of variance (ANOVA). A P value of , 0.05 was considered significant. Tissue hypoxia was confirmed by measuring ATP and PCr levels in the cerebral tissue. The ATP values in the normoxic (Nx), hypoxic (Hx) and 7-NINA treated hypoxic groups were 4.57 ^ 0.4, 1.29 ^ 0.2 (P , 0:05 vs. Nx) and
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Fig. 1. Effect of NOS inhibition on PTP activity during hypoxia in neuronal cell membranes and cytosol of newborn piglets.
1.5 ^ 0.14 mmol/g brain (P , 0:05 vs. Nx), respectively. PCr values in the Nx, Hx and 7-NINA treated hypoxic groups were 3.77 ^ 0.3, 0.77 ^ 0.1 (P , 0:05 vs. Nx), and 1.02 ^ 0.2 mmol/g brain (P , 0:05 vs. Nx), respectively. The data demonstrate that cerebral tissue hypoxia was achieved in the Hx and 7-NI treated hypoxic groups. The PTP activity (nmol pNP/mg protein per h) in neuronal membranes was 196.8 ^ 76.7 in the Nx group, 108 ^ 14.5 in the Hx group and 239 ^ 40.8 in the 7-NINA pretreated hypoxic group (Fig. 1a). PTP activity (Fig. 1b) in cytosol (nmol pNP/mg protein per h) was 23.88 ^ 13.7 in the Nx group, 58.18 ^ 14.5 in the Hx group (P , 0:001 vs. Nx) and 20.94 ^ 7.6 in the 7-NINA treated hypoxic group (P , 0:05 vs. Hx). Fig. 2 shows a representative immunoblot of PTP-1B, PTP-SH1 and PTP-SH2 in the Nx, Hx and 7-NINA treated hypoxic groups. The protein density (OD £ mm2) of PTP1B (Fig. 3a) was 26.42 ^ 10.1 in Nx, 110.85 ^ 39.1 in Hx (P , 0:05 vs. Nx) and 26.5 ^ 2.5 in Hx-7-NINA (P , 0:05 vs. Hx). The protein density of PTP-SH1 (Fig. 3b) was 90.16 ^ 15.5 in Nx, 225.95 ^ 18.50 in Hx (P , 0:05 vs. Nx) and 121.67 ^ 13.6 in the 7-NINA treated hypoxic group (P , 0:05 vs. Hx). PTP-SH2 density (Fig. 3c) was 75.18 ^ 11.34 in Nx, 154.66 ^ 18.05 in Hx (P , 0:05 vs. Nx) and 99.63 ^ 4.68 in 7-NINA þ Hx (P , 0:05 vs. Hx). The data are presented as mean ^ standard deviation (SD) unless otherwise noted. Furthermore, immunohistochemical results presented in Fig. 4 demonstrate an increased immunoreactivity against PTP-1B in the cortical sections from the hypoxic brain as
Fig. 2. Representative immunoblots of PTP-1B, PTP-SH1 and PTP-SH2 in the cytosol of newborn piglets. Equal protein loading was confirmed by subsequent probing with mouse monoclonal Actin/GAPDH antibody in each experiment.
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compared to normoxic brain. The data show that hypoxia results in decreased PTP activity in the cell membranes and pretreatment with 7-NINA, a selective inhibitor of nNOS, prevents the hypoxia-induced decrease in PTP activity in membranes. In addition, hypoxia resulted in increased PTP activity in cytosol and increased expression of PTP-1B and PTP-SH. The administration of nNOS inhibitor prevented the hypoxia-induced increase in PTP activity in the cytosol as well as the hypoxia-induced increased expression of PTP1B and of PTP-SH in neuronal cytosol. These results demonstrate that modifications of the activity and the expression of PTPs during hypoxia are mediated by NO. It has been suggested that hypoxic injury to the brain is free radical-mediated [9,10,12]. In the present study we have tested the hypothesis that the hypoxia-induced modifications in the activity and expression of PTPs are mediated by NO free radicals and will be prevented by blocking nNOS by 7-NINA, a selective inhibitor of nNOS. PTPs are characterized by the consensus active site sequence (I/V)HCxAGxxR(S/T)G, where x denotes any amino acid residue. The active site motif contains a nucleophilic cysteine residue that is essential for the catalytic activity and a conserved arginine residue. A single substitution of the critical cysteine by serine completely inactivates the enzyme. The structure of the consensus sequences defines the binding site for the tyrosyl phosphate substrate. Modification of this cysteine residue at the active site during hypoxia by NO free radicals or their product peroxynitrite may be the potential mechanism of modification of PTP activity. The data show that hypoxia resulted in decreased PTP activity in the cell membranes and pretreatment with an nNOS prevented the hypoxia-induced decrease in PTP activity. In addition, hypoxia resulted in increased PTP activity and expression of PTP-1B and PTP-SH in the
Fig. 3. Effect of NOS inhibition on PTP-1B, PTP-SH1 and PTP-SH2 expression during hypoxia in neuronal cytosol of newborn piglets.
Fig. 4. Representative micrograph of PTP-1B immunoreactivity in the normoxic and hypoxic sections of cerebral cortical sections of newborn piglets (also shown in £ 400 magnification in the bottom panel).
cytosol. Immunohistological data supported that hypoxia results in increased PTP-1B reactivity. The administration of nNOS inhibitor prevented the hypoxia-induced increase in PTP activity as well as the expression of PTP-1B and PTP-SH in the cytosol. These results demonstrate that modification of the activity and the expression of PTPs during hypoxia is mediated by NO. In previous studies, we have shown that hypoxia results in increased generation of NO [12]. We have also shown that administration of NOS inhibitor, N-nitro-L -arginine (NNLA), prevented the increased generation of free radicals, increased cell membrane lipid peroxidation and decreased Naþ,Kþ-ATPase activity in the membranes [12]. NO is a physiological messenger in the central nervous system. NO is a highly reactive free radical which is generated by the activity of enzyme NOS. At least three different isoforms of NOS have been characterized. nNOS is constitutively expressed in neurons and has Ca2þ-dependent activity. In comparison glial cells (astrocytes, microglia and oligodendrocytes) express Ca2þ-independent NOS after exposure to chemical and physiological stimuli. In the central nervous system NO has been proposed as a mediator with a dual role of neuroprotection and neurotoxic [3,5,12, 14]. This differential role of NO depends on the generation of NO by NOS isoforms such as the endothelial or the neuronal and the site of NO production. The toxicity of NO results when it reacts with the oxygen free radical to form peroxynitrite, a very potent oxidizing and nitrating agent [5]. In addition to its toxic effect mediated by peroxynitrite, NO can also nitrate the tyrosine residue of the N-methyl-D aspartate (NMDA) receptors [19]. We have shown that peroxynitrite treatment of the NMDA receptor modifies the
Q.M. Ashraf et al. / Neuroscience Letters 362 (2004) 108–112
receptor’s ion channel and the glutamate and spermine recognition sites [18,22]. These changes allow increased influx of Ca2þ and further NO production [5,11,12,14]. PTPs can regulate the phosphorylation status of apoptotic proteins. The phosphorylation state also determines the antias well as pro-apoptotic potential of these proteins. These proteins belong to the Bcl-2 multigene family and regulate cell death and survival. The Bcl-2 family consists of both cell death promoter (Bax) and cell death preventer (Bcl-2) proteins. Overexpression of Bax is known to promote programmed cell death and overexpression of Bcl-2 leads to cell survival [17]. When Bcl-2 heterodimerizes with the Bax, it prevents Bax-mediated activation of caspases, which execute cell death. We have shown that hypoxia results in increased tyrosine phosphorylation and serine phosphorylation of Bcl-2. Unphosphorylated Bcl-2 is anti-apoptotic, but phosphorylated Bcl-2 is pro-apoptotic [1,17]. The decrease in membrane PTP activity during hypoxia may be the mechanism of increased tyrosine phosphorylation of Bcl-2 that leads to the loss of anti-apoptotic potential of Bcl2. The present study also demonstrates that hypoxia results in increased PTP activity in the cytosol. In a separate study, we have observed that cerebral hypoxia resulted in increased phosphorylation of a number of neuronal proteins whereas a number of proteins were dephosphorylated and some of the proteins were not altered in terms of phosphorylation/dephosphorylation [4]. These results indicate that the phosphorylation/dephosphorylation equilibrium during hypoxia may further be determined by the protein substrate itself and its specificity to phosphatases. There is a possibility that the translocation of PTP from membrane into cytosol occurs during hypoxia and this translocation of PTP is NO-mediated. In previous studies we have observed that hypoxia resulted in increased expression of other proteins such as the pro-apoptotic proteins Bax and Bad [2,15,21]. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was not increased. These studies also demonstrated that the hypoxia-induced increased expression of the pro-apoptotic proteins can be blocked by the administration of NOS inhibitors [2,13,21]. Together, these studies indicate that the increased generation of NO during hypoxia mediates the increased expression of a number of proteins including the apoptotic proteins as well as phosphatases during hypoxia. In a series of studies we have observed that cerebral hypoxia resulted in increased neuronal nuclear Ca2þ influx, increased calcium calmodulin-dependent protein kinase IV (CaM kinase IV) activity [22], increased phosphorylation of cyclic AMP response element binding protein and increased expression of pro-apoptotic protein Bax [21]. Each of these steps were also blocked by the administration of NOS inhibitors during hypoxia. The hypoxia-induced increased expression of PTPs observed in the present study is potentially due to a similar mechanism that is mediated by NO during hypoxia. In summary we have shown that hypoxia results in
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decreased PTP activity in cell membranes and increased activity and expression of PTP-1B and PTP-SH in the cytosol. Pretreatment with a nNOS inhibitor prevented the hypoxia-induced modification in activity and expression of PTPs. We conclude that hypoxia-induced modification of PTP activity and expression is NO-mediated. We speculate that modification of PTP activity may lead to increased phosphorylation of the anti-apoptotic protein Bcl-2 and a decrease in its anti-apoptotic potential. The NO-mediated modification of PTPs altering the phosphorylation of apoptotic proteins may be a potential mechanism of hypoxic neuronal injury in the newborn.
Acknowledgements This research was supported by grants from the National Institutes of Health, NIH HD-20337 and NIH HD-38079.
References [1] Q.M. Ashraf, S.A. Zanelli, O.P. Mishra, M. Delivoria-Papadopoulos, Phosphorylation of Bcl-2 and Bax proteins during hypoxia in newborn piglets, Neurochem. Res. 26 (2001) 1–9. [2] D. Barford, A.J. Flint, N.K. Tonks, Crystal structure of human protein tyrosine phosphatase 1B, Science 263 (1994) 1397–1404. [3] J.P. Bolanos, A. Almeida, Roles of nitric oxide in brain hypoxiaischemia, Biochim. Biophys. Acta 1411 (1999) 415–436. [4] G. Buonocore, S. Liberatori, L. Bini, O.P. Mishra, M. DelivoriaPapadopoulos, V. Pallini, R. Bracci, Hypoxic response of synaptosomal proteins in term guinea pig fetuses, J. Neurochem. 73 (1999) 2139– 2148. [5] A. Contestabile, Roles of NMDA receptor activity and nitric oxide production in brain development, Brain Res. Brain Res. Rev. 32 (2000) 476–509. [6] S.H. Haider, O.P. Mishra, A.B. Zubrow, K.I. Fritz, M. DelivoriaPapadopoulos, Effect of nitric oxide synthase inhibition on protein tyrosine phosphatase activity during hypoxia in neuronal cell membranes of newborn piglets, Proceedings of the 5th World Congress of Perinatal Medicine, Monduzzi Editore, Bologna, 2001, pp. 780 –786. [7] S.I. Harik, G.H. Doull, A.P. Dick, Specific ouabain binding to brain microvessels and choroid plexus, J. Cereb. Blood Flow Metab. 5 (1985) 156–160. [8] O. Lowry, A. Rosenbrough, A. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265– 275. [9] O.P. Mishra, M. Delivoria-Papadopoulos, Lipid peroxidation in developing fetal guinea pig brain, Dev. Brain Res. 45 (1989) 129–135. [10] O.P. Mishra, M. Delivoria-Papadopoulos, Cellular mechanisms of hypoxic injury in the developing brain, Brain Res. Bull. 48 (1999) 233–238. [11] O.P. Mishra, M. Delivoria-Papadopoulos, Nitric oxide-mediated Ca2þ-influx in neuronal nuclei and cortical synaptosomes of normoxic and hypoxic newborn piglets, Neurosci. Lett. 318 (2002) 93– 97. [12] O.P. Mishra, S. Zanelli, S.T. Ohnishi, M. Delivoria-Papadopoulos, Hypoxia-induced generation of nitric oxide free radicals in cerebral cortex of newborn guinea pigs, Neurochem. Res. 25 (2000) 1559– 1565. [13] O.P. Mishra, Q.M. Ashraf, M. Delivoria-Papadopoulos, Phosphorylation of cAMP response element binding (CREB) protein during
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Peroxynitrite-induced modification of the N-methyl-D-aspartate receptor in the cerebral cortex of the guinea pig fetus at term, Neurosci. Lett. 296 (2000) 5–8. S.A. Zanelli, Q.M. Ashraf, O.P. Mishra, Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia, Neuroscience 112 (2002) 869–877. Z.Y. Zhang, Protein tyrosine phosphatases: structure and function, substrate specificity, and inhibitor development, Annu. Rev. Pharmacol. Toxicol. 42 (2002) 209 –234. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, J.R. Ballesteros, K.I. Fritz, O.P. Mishra, Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets, Brain Res. 954 (2002) 60–67. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, K.I. Fritz, O.P. Mishra, Nitric oxide mediated Caþþ /calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei of newborn piglets, Neurosci. Lett. 335 (2002) 5–8.
Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets Qazi M. Ashraf*, Syed H. Haider, Christos D. Katsetos, Maria Delivoria-Papadopoulos, Om. Mishra Department of Pediatrics, Drexel University College of Medicine and St. Christopher’s Hospital for Children, Philadelphia, PA, USA Received 22 October 2003; received in revised form 25 February 2004; accepted 3 March 2004
Abstract 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 ¼ 5), hypoxic (Hx, n ¼ 5), and 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of neuronal nitric oxide synthase (nNOS), pretreated hypoxic (7-NINA þ Hx, n ¼ 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 þ 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 þ 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. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Protein tyrosine phosphatase; Hypoxia; Brain; Piglets
Protein tyrosine phosphatases (PTPs) regulate kinasedependent signal transduction pathways and control cell proliferation, differentiation and metabolism. PTPs reverse the effects of protein tyrosine kinases by catalyzing the hydrolysis of the phosphoryl group. Protein phosphatases have been implicated in a number of pathological conditions including cancer and diabetes. Protein tyrosine phosphorylation is a post-translational modification that cells utilize to regulate signal transduction [2,16]. PTP in conjunction with protein kinase regulates many critical events including cell growth, tissue differentiation, intercellular communication, immune response, development and metabolism [20]. The PTPs can be divided into three major subfamilies: tyrosine specific, dual specific and low molecular weight phosphatases. Tyrosine specific phosphatases can be further * Corresponding author. Pediatrics/Neonatal Research, 7th Floor Heritage Building, Room 701, Drexel University College of Medicine, 3300 Henry Avenue, Philadelphia, PA 19129, USA. Tel.: þ 1-215-8424960; fax: þ 1-215-843-3505. E-mail address: [email protected] (Q.M. Ashraf).
subdivided into two groups of enzymes. The first group is receptor-like with a single trans-membrane domain and one or two intracellular catalytic domains, and a unique extracellular domain of variable length. The second group comprises of cytoplasmic enzymes, with a single catalytic domain and a variable amino- or carboxyl-terminal regulatory domain, examples of which include PTP-SH and PTP-1B. A large number of enzymes and substrates are phosphorylated during hypoxia. The phosphorylation pattern of proteins observed in synaptosomes of the guinea pig showed that the phosphorylated sites were two times higher in hypoxia than in normoxia [4]. These phosphorylation events may be regulated by free radicals. There are a number of mechanisms of free radical generation during hypoxia including the nitric oxide synthase (NOS) which results in nitric oxide (NO)-dependent peroxynitrite generation. Previous studies from our laboratory have shown that PTP activity decreases during hypoxia contributing to phosphorylation of a number of proteins including antiapoptotic protein Bcl-2 [1,6]. The purpose of the present
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.02.069
Q.M. Ashraf et al. / Neuroscience Letters 362 (2004) 108–112
study was to test the hypothesis that the hypoxia-induced changes in the activity and expression of PTPs are mediated by NO and will be prevented by blocking neuronal NOS (nNOS) by 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of nNOS. In the present study we have determined the effect of administration of 7-NINA on hypoxia-induced modification of the activity and expression, specifically (a) the activity of PTP in the membrane and cytosolic fractions, (b) the expression of the PTP-1B and PTP-SH in the cytosol, and (c) the immunohistochemical analysis of PTP-1B. Studies were performed on 2 – 4-day-old Yorkshire piglets as per standard protocol as described by Ashraf et al. [1]. The study was approved by the Institutional Animal Care and Use Committee of the Drexel University College of Medicine. ATP and phosphocreatine (PCr) concentrations were determined according to the method of Lamprecht, as mentioned in Ashraf et al. [1]. P2 membrane fractions were prepared according to the method described by Harik et al. [7]. PTP activity was determined spectrophotometrically at 405 nm in a medium containing 100 mM HEPES (pH 7.0), 1 mM EDTA, 100 mg protein [8] and 2.5 mM p-nitrophenylphosphate (pNPP) at 37 8C for 20 min in the presence or absence of 2 mM bpV(phen), a selective inhibitor of PTP. The inhibitor sensitive activity was determined and expressed as nmol pNP/mg protein per h. Immunoblotting was performed as described by Ashraf et al. [1] and cytosolic proteins incubated with primary polyclonal antibodies for PTP-1B, PTP-SH1 and PTP-SH2 (Santa Cruz Biotech, Santa Cruz, CA). Equal protein loading was confirmed by subsequent probing with mouse monoclonal Actin/GAPDH antibody (Chemicon, CA) in each experiment. The protein density was expressed as absorbance (OD £ mm2). Interleaved sections for immunohistochemistry were placed on gelatin-coated slides, and then deparaffinized and post-fixed with paraformaldehyde. Sections were then washed three times for 5 min each with PBS, and then incubated with antibody against PTP-1B (Santa Cruz) diluted 1:1000 in PBS/1% serum overnight at room temperature. Slides were then washed five times with PBS and incubated with the biotin conjugated secondary antibody (Jackson Labs) diluted 1:500 in PBS/1% serum for 2 h. The slides were washed again three times with PBS and incubated with the ABC reagent (streptavidin/biotinylated horseradish peroxidase (Vector Labs)). Antibody binding was visualized with diaminobenzidine (DAB) using a Sigma-Fast reagent kit. The data on ATP, PCr, PTP enzyme activity and expression were analyzed using one-way analysis of variance (ANOVA). A P value of , 0.05 was considered significant. Tissue hypoxia was confirmed by measuring ATP and PCr levels in the cerebral tissue. The ATP values in the normoxic (Nx), hypoxic (Hx) and 7-NINA treated hypoxic groups were 4.57 ^ 0.4, 1.29 ^ 0.2 (P , 0:05 vs. Nx) and
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Fig. 1. Effect of NOS inhibition on PTP activity during hypoxia in neuronal cell membranes and cytosol of newborn piglets.
1.5 ^ 0.14 mmol/g brain (P , 0:05 vs. Nx), respectively. PCr values in the Nx, Hx and 7-NINA treated hypoxic groups were 3.77 ^ 0.3, 0.77 ^ 0.1 (P , 0:05 vs. Nx), and 1.02 ^ 0.2 mmol/g brain (P , 0:05 vs. Nx), respectively. The data demonstrate that cerebral tissue hypoxia was achieved in the Hx and 7-NI treated hypoxic groups. The PTP activity (nmol pNP/mg protein per h) in neuronal membranes was 196.8 ^ 76.7 in the Nx group, 108 ^ 14.5 in the Hx group and 239 ^ 40.8 in the 7-NINA pretreated hypoxic group (Fig. 1a). PTP activity (Fig. 1b) in cytosol (nmol pNP/mg protein per h) was 23.88 ^ 13.7 in the Nx group, 58.18 ^ 14.5 in the Hx group (P , 0:001 vs. Nx) and 20.94 ^ 7.6 in the 7-NINA treated hypoxic group (P , 0:05 vs. Hx). Fig. 2 shows a representative immunoblot of PTP-1B, PTP-SH1 and PTP-SH2 in the Nx, Hx and 7-NINA treated hypoxic groups. The protein density (OD £ mm2) of PTP1B (Fig. 3a) was 26.42 ^ 10.1 in Nx, 110.85 ^ 39.1 in Hx (P , 0:05 vs. Nx) and 26.5 ^ 2.5 in Hx-7-NINA (P , 0:05 vs. Hx). The protein density of PTP-SH1 (Fig. 3b) was 90.16 ^ 15.5 in Nx, 225.95 ^ 18.50 in Hx (P , 0:05 vs. Nx) and 121.67 ^ 13.6 in the 7-NINA treated hypoxic group (P , 0:05 vs. Hx). PTP-SH2 density (Fig. 3c) was 75.18 ^ 11.34 in Nx, 154.66 ^ 18.05 in Hx (P , 0:05 vs. Nx) and 99.63 ^ 4.68 in 7-NINA þ Hx (P , 0:05 vs. Hx). The data are presented as mean ^ standard deviation (SD) unless otherwise noted. Furthermore, immunohistochemical results presented in Fig. 4 demonstrate an increased immunoreactivity against PTP-1B in the cortical sections from the hypoxic brain as
Fig. 2. Representative immunoblots of PTP-1B, PTP-SH1 and PTP-SH2 in the cytosol of newborn piglets. Equal protein loading was confirmed by subsequent probing with mouse monoclonal Actin/GAPDH antibody in each experiment.
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compared to normoxic brain. The data show that hypoxia results in decreased PTP activity in the cell membranes and pretreatment with 7-NINA, a selective inhibitor of nNOS, prevents the hypoxia-induced decrease in PTP activity in membranes. In addition, hypoxia resulted in increased PTP activity in cytosol and increased expression of PTP-1B and PTP-SH. The administration of nNOS inhibitor prevented the hypoxia-induced increase in PTP activity in the cytosol as well as the hypoxia-induced increased expression of PTP1B and of PTP-SH in neuronal cytosol. These results demonstrate that modifications of the activity and the expression of PTPs during hypoxia are mediated by NO. It has been suggested that hypoxic injury to the brain is free radical-mediated [9,10,12]. In the present study we have tested the hypothesis that the hypoxia-induced modifications in the activity and expression of PTPs are mediated by NO free radicals and will be prevented by blocking nNOS by 7-NINA, a selective inhibitor of nNOS. PTPs are characterized by the consensus active site sequence (I/V)HCxAGxxR(S/T)G, where x denotes any amino acid residue. The active site motif contains a nucleophilic cysteine residue that is essential for the catalytic activity and a conserved arginine residue. A single substitution of the critical cysteine by serine completely inactivates the enzyme. The structure of the consensus sequences defines the binding site for the tyrosyl phosphate substrate. Modification of this cysteine residue at the active site during hypoxia by NO free radicals or their product peroxynitrite may be the potential mechanism of modification of PTP activity. The data show that hypoxia resulted in decreased PTP activity in the cell membranes and pretreatment with an nNOS prevented the hypoxia-induced decrease in PTP activity. In addition, hypoxia resulted in increased PTP activity and expression of PTP-1B and PTP-SH in the
Fig. 3. Effect of NOS inhibition on PTP-1B, PTP-SH1 and PTP-SH2 expression during hypoxia in neuronal cytosol of newborn piglets.
Fig. 4. Representative micrograph of PTP-1B immunoreactivity in the normoxic and hypoxic sections of cerebral cortical sections of newborn piglets (also shown in £ 400 magnification in the bottom panel).
cytosol. Immunohistological data supported that hypoxia results in increased PTP-1B reactivity. The administration of nNOS inhibitor prevented the hypoxia-induced increase in PTP activity as well as the expression of PTP-1B and PTP-SH in the cytosol. These results demonstrate that modification of the activity and the expression of PTPs during hypoxia is mediated by NO. In previous studies, we have shown that hypoxia results in increased generation of NO [12]. We have also shown that administration of NOS inhibitor, N-nitro-L -arginine (NNLA), prevented the increased generation of free radicals, increased cell membrane lipid peroxidation and decreased Naþ,Kþ-ATPase activity in the membranes [12]. NO is a physiological messenger in the central nervous system. NO is a highly reactive free radical which is generated by the activity of enzyme NOS. At least three different isoforms of NOS have been characterized. nNOS is constitutively expressed in neurons and has Ca2þ-dependent activity. In comparison glial cells (astrocytes, microglia and oligodendrocytes) express Ca2þ-independent NOS after exposure to chemical and physiological stimuli. In the central nervous system NO has been proposed as a mediator with a dual role of neuroprotection and neurotoxic [3,5,12, 14]. This differential role of NO depends on the generation of NO by NOS isoforms such as the endothelial or the neuronal and the site of NO production. The toxicity of NO results when it reacts with the oxygen free radical to form peroxynitrite, a very potent oxidizing and nitrating agent [5]. In addition to its toxic effect mediated by peroxynitrite, NO can also nitrate the tyrosine residue of the N-methyl-D aspartate (NMDA) receptors [19]. We have shown that peroxynitrite treatment of the NMDA receptor modifies the
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receptor’s ion channel and the glutamate and spermine recognition sites [18,22]. These changes allow increased influx of Ca2þ and further NO production [5,11,12,14]. PTPs can regulate the phosphorylation status of apoptotic proteins. The phosphorylation state also determines the antias well as pro-apoptotic potential of these proteins. These proteins belong to the Bcl-2 multigene family and regulate cell death and survival. The Bcl-2 family consists of both cell death promoter (Bax) and cell death preventer (Bcl-2) proteins. Overexpression of Bax is known to promote programmed cell death and overexpression of Bcl-2 leads to cell survival [17]. When Bcl-2 heterodimerizes with the Bax, it prevents Bax-mediated activation of caspases, which execute cell death. We have shown that hypoxia results in increased tyrosine phosphorylation and serine phosphorylation of Bcl-2. Unphosphorylated Bcl-2 is anti-apoptotic, but phosphorylated Bcl-2 is pro-apoptotic [1,17]. The decrease in membrane PTP activity during hypoxia may be the mechanism of increased tyrosine phosphorylation of Bcl-2 that leads to the loss of anti-apoptotic potential of Bcl2. The present study also demonstrates that hypoxia results in increased PTP activity in the cytosol. In a separate study, we have observed that cerebral hypoxia resulted in increased phosphorylation of a number of neuronal proteins whereas a number of proteins were dephosphorylated and some of the proteins were not altered in terms of phosphorylation/dephosphorylation [4]. These results indicate that the phosphorylation/dephosphorylation equilibrium during hypoxia may further be determined by the protein substrate itself and its specificity to phosphatases. There is a possibility that the translocation of PTP from membrane into cytosol occurs during hypoxia and this translocation of PTP is NO-mediated. In previous studies we have observed that hypoxia resulted in increased expression of other proteins such as the pro-apoptotic proteins Bax and Bad [2,15,21]. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was not increased. These studies also demonstrated that the hypoxia-induced increased expression of the pro-apoptotic proteins can be blocked by the administration of NOS inhibitors [2,13,21]. Together, these studies indicate that the increased generation of NO during hypoxia mediates the increased expression of a number of proteins including the apoptotic proteins as well as phosphatases during hypoxia. In a series of studies we have observed that cerebral hypoxia resulted in increased neuronal nuclear Ca2þ influx, increased calcium calmodulin-dependent protein kinase IV (CaM kinase IV) activity [22], increased phosphorylation of cyclic AMP response element binding protein and increased expression of pro-apoptotic protein Bax [21]. Each of these steps were also blocked by the administration of NOS inhibitors during hypoxia. The hypoxia-induced increased expression of PTPs observed in the present study is potentially due to a similar mechanism that is mediated by NO during hypoxia. In summary we have shown that hypoxia results in
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decreased PTP activity in cell membranes and increased activity and expression of PTP-1B and PTP-SH in the cytosol. Pretreatment with a nNOS inhibitor prevented the hypoxia-induced modification in activity and expression of PTPs. We conclude that hypoxia-induced modification of PTP activity and expression is NO-mediated. We speculate that modification of PTP activity may lead to increased phosphorylation of the anti-apoptotic protein Bcl-2 and a decrease in its anti-apoptotic potential. The NO-mediated modification of PTPs altering the phosphorylation of apoptotic proteins may be a potential mechanism of hypoxic neuronal injury in the newborn.
Acknowledgements This research was supported by grants from the National Institutes of Health, NIH HD-20337 and NIH HD-38079.
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