Rev. Neurosci. 2015; 26(1): 105–117
Haiting Liu, Jiao Li, Fengyan Zhao, Huiqing Wang, Yi Qu and Dezhi Mu*
Nitric oxide synthase in hypoxic or ischemic brain injury Abstract: Hypoxic or ischemic stress causes many serious brain injuries, including stroke and neonatal hypoxia ischemia encephalopathy. During brain hypoxia ischemia processes, nitric oxide (NO) may play either a neurotoxic or a neuroprotective role, depending upon factors such as the NO synthase (NOS) isoform, the cell type by which NO is produced, and the temporal stage after the onset of the hypoxic ischemic brain injury. Excessive NO production can be neurotoxic, leading to cascade reactions of excitotoxicity, inflammation, apoptosis, and deteriorating primary brain injury. In contrast, NO produced by endothelial NOS plays a neuroprotective role by maintaining cerebral blood flow and preventing neuronal injury, as well as inhibiting platelet and leukocyte adhesion. Sometimes, NO-derived inducible NOS and neuronal NOS in special areas may also play neuroprotective roles. Therefore, this review summarizes the different roles and the regulation of the three NOS isoforms in hypoxic or ischemic brain injury as revealed in research in recent years, focusing on the neurotoxic role of the three NOS isoforms involved in mechanisms of hypoxic or ischemic brain injury. Keywords: brain injury; hypoxia; ischemia; nitric oxide synthase. DOI 10.1515/revneuro-2014-0041 Received June 19, 2014; accepted July 30, 2014; previously published online September 16, 2014
*Corresponding author: Dezhi Mu, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; Key Laboratory of Obstetric and Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; and Department of Pediatrics and Neurology, University of California, San Francisco, CA 94143, USA, Fax: +86-28-85559065, e-mail: [email protected] Haiting Liu, Jiao Li, Fengyan Zhao, Huiqing Wang and Yi Qu: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; and Key Laboratory of Obstetric and Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China
Introduction Hypoxic and ischemic stresses have been considered to be critical factors in many human central nervous system diseases, including stroke and neonatal hypoxia ischemia encephalopathy. Over the past decade, significant advancement has been made in our understanding of the mechanisms underlying these brain injuries, including the release of neurotoxic substances, inflammation, apoptosis, and excitotoxicity (Doyle et al., 2008). Nitric oxide (NO) was identified as a biological intercellular messenger just over 20 years ago; it was first identified as the primary mediator for endothelium-dependent vascular relaxation (Ignarro et al., 1987). Subsequent studies showed that NO conveys neuronal signal transmission in central nerve systems (Martinez-Ruiz et al., 2011). In addition, NO also participates in the inflammatory response. As a free radical, NO can directly disrupt protein structure, damage mitochondrion function, and induce apoptosis (Zhang et al., 2013). In particular, excessive NO production is neurotoxic. It is reported that excessive NO production can mediate excitotoxicity (Gu et al., 2010) and stimulate energy depletion-induced neuronal necrosis (Brown, 2010). Endogenous NO in mammalian species is formed from l-arginine and molecular oxygen by the action of three highly homologous NO synthases (NOSs): neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) (Qi et al., 2013). The structure of NOS involves two structural domains, the C-terminal reductase domain and the N-terminal oxygenase domain (Forstermann and Sessa, 2012). The N-terminal domain has oxygenase activity-containing sites to bind heme, (6R-)5,6,7,8-tetrahydrol-biopterin (BH4), and l-arginine (Guix et al., 2005). The C-terminal reductase domain containing binding sites for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide-adenine- dinucleotide phosphate (NADPH) is linked by a CaM-recognition site to the N-terminal domain (Alderton et al., 2001). All three isoforms of NOS utilize l-arginine and molecular oxygen as the substrate and require cofactors, i.e., NADPH, FAD, FMN, and BH4 (Forstermann and Sessa, 2012). A functional NOS is a homodimer. In nNOS and
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106 H. Liu et al.: Nitric oxide synthase in hypoxic or ischemic brain injury eNOS, calmodulin binding is induced by increased intracellular Ca2+ concentrations. In iNOS, calmodulin already binds at extremely low intracellular Ca2+ concentrations due to a different amino acid structure of the calmodulinbinding site (Forstermann and Sessa, 2012). In the healthy brain, NO is produced mainly by nNOS in a small subset of neurons and eNOS in the endothelium. It has also been reported that nNOS can be found at low levels in astrocytes and that eNOS can be found in a subset of neurons and astrocytes (Brown, 2010). Unlike the constitutively expressed nNOS and eNOS, little iNOS is detected in quiescent cells (Zhang et al., 2013). However, iNOS can be induced by inflammatory mediators and cytokines (Zhu et al., 2013), expressing primarily in macrophages, microglia, infiltrating neutrophils, and, to some extent, neurons (Gunther et al., 2012), and thus can produce high amounts of NO (Zhu et al., 2013). In this review, we summarize recent research into the different roles and the regulation mechanisms of the three NOS isoforms in hypoxic or ischemic brain injury, focusing on the neurotoxic role of the NOS involved in the genesis and development of brain ischemic or ischemic brain injury.
Different roles of NO/NOS in hypoxic or ischemic brain injury It is reported that cerebral ischemia itself or hypoxia affects activity and expression of NOS (Table 1). The effects of NO on the ischemic brain are thought to have either neurotoxic or neuroprotective effects, depending upon factors such as the NOS isoform, the cell type by which NO is produced, or the stage of the hypoxic or ischemic process (Pei et al., 2008). Therefore, different NOS isotypes and their derived NO play different roles in hypoxic or ischemic brain injury (Table 2).
nNOS In the acute stage of hypoxic or ischemic brain injury, many studies have shown that nNOS expression is increased, accompanied by the enhancement of NO production (Koh, 2008; Liu et al., 2011). Zhou et al. (2008) further found that the phosphorylation of nNOS (Ser847) reached a peak at 30 min of reperfusion and decreased to basal level at 6 h of reperfusion. Additionally, Ito et al. (2010) demonstrated that relative cerebral blood flow (CBF) was significantly higher in nNOS-/- mice than in control mice
at 70 min and 90–120 min after the early stage of reperfusion, which suggested that NO production by nNOS may enhance ischemic injury, especially in the early stage of reperfusion. Later, Zeng et al. (2011) found that the early c-Jun N-terminal kinase 1/2 (JNK1/2) activation, a signaling pathway involved in neuronal death, was due to the generation of NO from nNOS during early reperfusion, and they further confirmed that nNOS and nNOS-derived NO involved mainly the early stages of hypoxic or ischemic brain injury. However, Li et al. (2010) found that nNOS expression was upregulated only during the later phase of middle cerebral artery occlusion/reperfusion (MCAO/R) (12–24 h after ischemia). This may be because the distribution of nNOS is different at different times during hypoxic or ischemic injury. Zhang et al. (2007b) found in the striatal region that the immunoreactivity of nNOS was similar compared with the control at 1 day following ischemia; it was lower at 3 days and more intense at 14 days following ischemia. In the cortical areas, the immunoreactivity of nNOS was similar to the control at 1 day and 14 days following ischemia; it was also lower at 3 days following ischemia (Zhang et al., 2007b). In addition, ischemia or hypoxia stress may result in a change in the subcellular distribution. Brain nNOS exists in particulate and soluble forms and is distributed mainly in the cytosol (Zhou et al., 2010), while the solubility of nNOS decreases after ischemia (Takagi et al., 2000). Zhou et al. (2010) speculated that ischemia-induced nNOS translocation from cytosol to membrane exists via nNOS-postsynaptic density protein (PSD-95) interaction. Suppression of nNOS activity can reduce cellular death in cerebral ischemia (Sun et al., 2009), while inhibition of fetal brain nNOS activity can dramatically reduce mortality and the number of newborns exhibiting signs of cerebral palsy (Ji et al., 2009).
iNOS In different animal models of hypoxia or ischemia insult such as MCAO (Chang et al., 2011; Zhou et al., 2013), oxygen glucose deprivation (OGD) (Zhang et al., 2012a), acute hypobaric hypoxia (AHH) (Fujioka et al., 2008), hypoxic-ischemic (HI) (Fujioka et al., 2008), or periventricular leukomalacia (Haynes et al., 2009), the expression of iNOS is increased in the brain, which produces a large quantity of NO. The expression of iNOS lasts for a long time, 24 h after ischemia or hypoxia insult, 3 days (Zhang et al., 2012a) or even 7 days after insult, and from 60 min to 5 days after posthypoxic exposure (Udayabanu
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H. Liu et al.: Nitric oxide synthase in hypoxic or ischemic brain injury 107 Table 1 Overview of the different animal models of hypoxia/ischemia with the three NOSs. Experimental model nNOS MCAO (FCI)
Experimental animal
Observation and conclusion
References
Adult male SD rat
In the striatal region, the immunoreactivity of nNOS was similar compared with the control at 1 day following ischemia; it was less at 3 days and intense at 14 days following ischemia. In the cortical region, the immunoreactivity of nNOS was similar compared with the control at 1 day and 14 days following ischemia; it was less at 3 days following ischemia. Brain injury induced an increase in both iNOS and nNOS levels, while eNOS levels declined in MCAO-induced brain injury in the ipsilateral cortex of the brain. The enhanced NO concentration in the acute stage of MCAO/R was coincident with increased eNOS expression, while in the subacute stage, it was coincident with increased iNOS and nNOS. Pronounced eNOS elevation was detected 2 h after the beginning of ischemia (p
Haiting Liu, Jiao Li, Fengyan Zhao, Huiqing Wang, Yi Qu and Dezhi Mu*
Nitric oxide synthase in hypoxic or ischemic brain injury Abstract: Hypoxic or ischemic stress causes many serious brain injuries, including stroke and neonatal hypoxia ischemia encephalopathy. During brain hypoxia ischemia processes, nitric oxide (NO) may play either a neurotoxic or a neuroprotective role, depending upon factors such as the NO synthase (NOS) isoform, the cell type by which NO is produced, and the temporal stage after the onset of the hypoxic ischemic brain injury. Excessive NO production can be neurotoxic, leading to cascade reactions of excitotoxicity, inflammation, apoptosis, and deteriorating primary brain injury. In contrast, NO produced by endothelial NOS plays a neuroprotective role by maintaining cerebral blood flow and preventing neuronal injury, as well as inhibiting platelet and leukocyte adhesion. Sometimes, NO-derived inducible NOS and neuronal NOS in special areas may also play neuroprotective roles. Therefore, this review summarizes the different roles and the regulation of the three NOS isoforms in hypoxic or ischemic brain injury as revealed in research in recent years, focusing on the neurotoxic role of the three NOS isoforms involved in mechanisms of hypoxic or ischemic brain injury. Keywords: brain injury; hypoxia; ischemia; nitric oxide synthase. DOI 10.1515/revneuro-2014-0041 Received June 19, 2014; accepted July 30, 2014; previously published online September 16, 2014
*Corresponding author: Dezhi Mu, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; Key Laboratory of Obstetric and Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; and Department of Pediatrics and Neurology, University of California, San Francisco, CA 94143, USA, Fax: +86-28-85559065, e-mail: [email protected] Haiting Liu, Jiao Li, Fengyan Zhao, Huiqing Wang and Yi Qu: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China; and Key Laboratory of Obstetric and Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China
Introduction Hypoxic and ischemic stresses have been considered to be critical factors in many human central nervous system diseases, including stroke and neonatal hypoxia ischemia encephalopathy. Over the past decade, significant advancement has been made in our understanding of the mechanisms underlying these brain injuries, including the release of neurotoxic substances, inflammation, apoptosis, and excitotoxicity (Doyle et al., 2008). Nitric oxide (NO) was identified as a biological intercellular messenger just over 20 years ago; it was first identified as the primary mediator for endothelium-dependent vascular relaxation (Ignarro et al., 1987). Subsequent studies showed that NO conveys neuronal signal transmission in central nerve systems (Martinez-Ruiz et al., 2011). In addition, NO also participates in the inflammatory response. As a free radical, NO can directly disrupt protein structure, damage mitochondrion function, and induce apoptosis (Zhang et al., 2013). In particular, excessive NO production is neurotoxic. It is reported that excessive NO production can mediate excitotoxicity (Gu et al., 2010) and stimulate energy depletion-induced neuronal necrosis (Brown, 2010). Endogenous NO in mammalian species is formed from l-arginine and molecular oxygen by the action of three highly homologous NO synthases (NOSs): neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) (Qi et al., 2013). The structure of NOS involves two structural domains, the C-terminal reductase domain and the N-terminal oxygenase domain (Forstermann and Sessa, 2012). The N-terminal domain has oxygenase activity-containing sites to bind heme, (6R-)5,6,7,8-tetrahydrol-biopterin (BH4), and l-arginine (Guix et al., 2005). The C-terminal reductase domain containing binding sites for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide-adenine- dinucleotide phosphate (NADPH) is linked by a CaM-recognition site to the N-terminal domain (Alderton et al., 2001). All three isoforms of NOS utilize l-arginine and molecular oxygen as the substrate and require cofactors, i.e., NADPH, FAD, FMN, and BH4 (Forstermann and Sessa, 2012). A functional NOS is a homodimer. In nNOS and
Brought to you by | Rutgers University Authenticated Download Date | 6/4/15 3:57 AM
106 H. Liu et al.: Nitric oxide synthase in hypoxic or ischemic brain injury eNOS, calmodulin binding is induced by increased intracellular Ca2+ concentrations. In iNOS, calmodulin already binds at extremely low intracellular Ca2+ concentrations due to a different amino acid structure of the calmodulinbinding site (Forstermann and Sessa, 2012). In the healthy brain, NO is produced mainly by nNOS in a small subset of neurons and eNOS in the endothelium. It has also been reported that nNOS can be found at low levels in astrocytes and that eNOS can be found in a subset of neurons and astrocytes (Brown, 2010). Unlike the constitutively expressed nNOS and eNOS, little iNOS is detected in quiescent cells (Zhang et al., 2013). However, iNOS can be induced by inflammatory mediators and cytokines (Zhu et al., 2013), expressing primarily in macrophages, microglia, infiltrating neutrophils, and, to some extent, neurons (Gunther et al., 2012), and thus can produce high amounts of NO (Zhu et al., 2013). In this review, we summarize recent research into the different roles and the regulation mechanisms of the three NOS isoforms in hypoxic or ischemic brain injury, focusing on the neurotoxic role of the NOS involved in the genesis and development of brain ischemic or ischemic brain injury.
Different roles of NO/NOS in hypoxic or ischemic brain injury It is reported that cerebral ischemia itself or hypoxia affects activity and expression of NOS (Table 1). The effects of NO on the ischemic brain are thought to have either neurotoxic or neuroprotective effects, depending upon factors such as the NOS isoform, the cell type by which NO is produced, or the stage of the hypoxic or ischemic process (Pei et al., 2008). Therefore, different NOS isotypes and their derived NO play different roles in hypoxic or ischemic brain injury (Table 2).
nNOS In the acute stage of hypoxic or ischemic brain injury, many studies have shown that nNOS expression is increased, accompanied by the enhancement of NO production (Koh, 2008; Liu et al., 2011). Zhou et al. (2008) further found that the phosphorylation of nNOS (Ser847) reached a peak at 30 min of reperfusion and decreased to basal level at 6 h of reperfusion. Additionally, Ito et al. (2010) demonstrated that relative cerebral blood flow (CBF) was significantly higher in nNOS-/- mice than in control mice
at 70 min and 90–120 min after the early stage of reperfusion, which suggested that NO production by nNOS may enhance ischemic injury, especially in the early stage of reperfusion. Later, Zeng et al. (2011) found that the early c-Jun N-terminal kinase 1/2 (JNK1/2) activation, a signaling pathway involved in neuronal death, was due to the generation of NO from nNOS during early reperfusion, and they further confirmed that nNOS and nNOS-derived NO involved mainly the early stages of hypoxic or ischemic brain injury. However, Li et al. (2010) found that nNOS expression was upregulated only during the later phase of middle cerebral artery occlusion/reperfusion (MCAO/R) (12–24 h after ischemia). This may be because the distribution of nNOS is different at different times during hypoxic or ischemic injury. Zhang et al. (2007b) found in the striatal region that the immunoreactivity of nNOS was similar compared with the control at 1 day following ischemia; it was lower at 3 days and more intense at 14 days following ischemia. In the cortical areas, the immunoreactivity of nNOS was similar to the control at 1 day and 14 days following ischemia; it was also lower at 3 days following ischemia (Zhang et al., 2007b). In addition, ischemia or hypoxia stress may result in a change in the subcellular distribution. Brain nNOS exists in particulate and soluble forms and is distributed mainly in the cytosol (Zhou et al., 2010), while the solubility of nNOS decreases after ischemia (Takagi et al., 2000). Zhou et al. (2010) speculated that ischemia-induced nNOS translocation from cytosol to membrane exists via nNOS-postsynaptic density protein (PSD-95) interaction. Suppression of nNOS activity can reduce cellular death in cerebral ischemia (Sun et al., 2009), while inhibition of fetal brain nNOS activity can dramatically reduce mortality and the number of newborns exhibiting signs of cerebral palsy (Ji et al., 2009).
iNOS In different animal models of hypoxia or ischemia insult such as MCAO (Chang et al., 2011; Zhou et al., 2013), oxygen glucose deprivation (OGD) (Zhang et al., 2012a), acute hypobaric hypoxia (AHH) (Fujioka et al., 2008), hypoxic-ischemic (HI) (Fujioka et al., 2008), or periventricular leukomalacia (Haynes et al., 2009), the expression of iNOS is increased in the brain, which produces a large quantity of NO. The expression of iNOS lasts for a long time, 24 h after ischemia or hypoxia insult, 3 days (Zhang et al., 2012a) or even 7 days after insult, and from 60 min to 5 days after posthypoxic exposure (Udayabanu
Brought to you by | Rutgers University Authenticated Download Date | 6/4/15 3:57 AM
H. Liu et al.: Nitric oxide synthase in hypoxic or ischemic brain injury 107 Table 1 Overview of the different animal models of hypoxia/ischemia with the three NOSs. Experimental model nNOS MCAO (FCI)
Experimental animal
Observation and conclusion
References
Adult male SD rat
In the striatal region, the immunoreactivity of nNOS was similar compared with the control at 1 day following ischemia; it was less at 3 days and intense at 14 days following ischemia. In the cortical region, the immunoreactivity of nNOS was similar compared with the control at 1 day and 14 days following ischemia; it was less at 3 days following ischemia. Brain injury induced an increase in both iNOS and nNOS levels, while eNOS levels declined in MCAO-induced brain injury in the ipsilateral cortex of the brain. The enhanced NO concentration in the acute stage of MCAO/R was coincident with increased eNOS expression, while in the subacute stage, it was coincident with increased iNOS and nNOS. Pronounced eNOS elevation was detected 2 h after the beginning of ischemia (p
