Research report
Tetrahydrobiopterin and nitric oxide synthase dimer levels are not changed following hypoxia–ischemia in the newborn rat

https://doi.org/10.1016/j.devbrainres.2005.02.008Get rights and content

Abstract

The effect of hypoxia–ischemia on the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4) and changes in the enzyme dimer state have not previously been studied. Cell-based studies have demonstrated the regulation of nitric oxide (NO) synthesis by intracellular BH4 levels. Activation of NOS requires two NOS polypeptides to form a homodimer. Dimerization results in the creation of high-affinity binding sites for BH4 and l-arginine. Our previous studies have indicated that nNOS activity falls 2 h post-hypoxia–ischemia in the immature rodent model. Thus, the objective of this study was to determine whether changes in nNOS dimeric state could be responsible for the decrease in nNOS activity. Using the immature rat model of HI in conjunction with LT-PAGE and Western blot analysis, we determined the effect of HI on NOS dimer state in hippocampus and cortex and the effects of pharmacologic modulation of NO levels during HI on dimer formation. Using high-performance liquid chromatography (HPLC) and electrospray tandem mass spectrometry (MS–MS), we measured BH4 and l-arginine levels respectively after HI under the same conditions. We found minimal or no changes in either BH4 levels or NOS dimer state at 2 h, 24 h and 7 day recovery from HI on postnatal day 7. In contrast, l-arginine levels were transiently increased in the hypoxic–ischemic hemisphere. Thus, our data suggest that the previously described decrease in NOS activity after HI is not associated with depletion of the cofactor BH4, l-arginine substrate or changes in the NOS enzyme dimer state.

Introduction

Hypoxia–ischemia (HI) in the perinatal period is associated with significant infant mortality and neurologic morbidity [23], [50]. Both cerebral ischemia in the adult and HI in the newborn [9], [11] result in glutamate-mediated excitotoxic cell death. Stimulation of glutamate receptors, including the N-methyl-d-aspartate (NMDA) receptor, results in increased production in the brain of several isoforms of nitric oxide synthase (NOS) [22]. Increase in the activity of NOS and increased release of nitric oxide (NO) are pivotal events in the pathophysiology of stroke and perinatal asphyxia [47]. Multiple lines of evidence suggest that inhibition of NOS is associated with a reduction in neurologic injury following HI in the newborn and adult brain [21]. Pharmacologic inhibition of NOS [19], targeted disruption of the NOS gene [14] or lesioning of NOS neurons [13] each reduce neurologic injury in the immature rodent brain following HI.

The NOS polypeptide comprises an N-terminal oxygenase domain and a C-terminal reductase domain [31], [48]. The oxygenase domain contains an iron protoporphyrin (heme) and the pteridine cofactor (6 R)-5,6,7,8-Tetrahydo-l-biopterin (BH4). Cell-based studies have demonstrated the regulation of NO synthesis by intracellular BH4 levels [54]. The polypeptide C-terminal domain contains a flavin mononucleotide and a flavin adenine dinucleotide with an intervening calmodulin binding sequence between the two domains. Activation of the NOS enzyme requires two NOS polypeptides to form a homodimer [41], [48]. Dimerization results in the creation of high-affinity binding sites for BH4 and l-arginine in the oxygenase domains and enables electron transfer between NOS flavin and heme groups [36], [42]. After dimerization, in the presence of five enzyme cofactors, these domains are used to catalyze a five-electron oxidation of l-arginine to NO with a stoichiometric formation of citrulline [5]. If this reaction is arrested during the reductive activation of oxygen, it may become uncoupled [37], [38], resulting in the generation of superoxide (O2) instead of l-citrulline and NO. This latter condition is favored when levels of either of two cofactors l-arginine or BH4 are reduced [32], [37], [55].

Despite the fundamental role acknowledged for NOS and NO-dependent pathways in the response cerebral ischemic injury, little is known about the changes in cofactor levels or dimerization state of NOS in response to perinatal HI. The capacity of NOS to generate O2 when uncoupled and the important regulatory role played by cofactors in the dimer state of the NOS enzyme could implicate these mechanisms in the oxidative stress response to ischemic insult in the newborn. While the neonatal brain is believed to show increased susceptibility to oxidative stress [12], the contribution of NOS to O2 mediated injury during HI, the dimerization state of NOS and the effect of HI on levels of the cofactor BH4 and the substrate l-arginine are unknown. We used the well-established immature rat model of unilateral carotid ligation with transient hypoxia [40] in conjunction with LT-PAGE and Western blotting to determine the effect of HI on neuronal (n)NOS dimer state in hippocampus and cortex and the effects of modulation of NO levels during HI on dimer formation. Using high-performance liquid chromatography (HPLC), we measured BH4 levels after HI under the same conditions. To determine whether pharmacologic manipulation of NO levels was associated with changes in cofactor levels or NOS dimer state, we treated rats during HI with either the NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) [4] or the NO donor (Z)-1-[N-(2aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETA/NONOate) [3], [56]. To determine the effects of HI on the NOS substrate, l-arginine, we used electrospray tandem mass spectrometry (MS–MS) [6].

Section snippets

Animals

All experiments were performed in accordance with the relevant National Institutes of Health Guide for Care and Use of Laboratory Animals. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Northwestern University, Chicago. Male and female Wistar rats (Charles River) at day 7 after birth were used for the studies. Pups were housed with their dam in cages in the animal facility with a 12-h light/dark cycle. All reagents were obtained from Sigma

Time course of cortex BH4 levels after HI and effect of NOS inhibition and NO donor

Rats were sacrificed at either 2 or 24 h or 7 days after HI. BH4 levels in the cortex (Figs. 1A–C) and hippocampus (Figs. 2A–C) were determined by HPLC. Values are expressed as mean ± SEM (n = at least 5 per group) in fmol/μg protein.

There were no significant differences between non-ischemic (left) and ischemic (right) cortex of vehicle-treated animals at either 2 or 24 h or 7 day recovery (Fig. 1A). After 2 h recovery from HI, there were no statistically significant differences between BH4

Discussion

There are three principal findings in this study. First, the dimer state of the nNOS enzyme remains unchanged during HI in the newborn period at any of the recovery periods studied. Levels of nNOS dimer are not affected by treatment with the NOS inhibitor l-NAME or administration of an exogenous NO donor, DETA/NONOate. Second, the apparent stability of the active dimer state of the enzyme is associated with similar stability in levels of the cofactor BH4 which are not significantly affected in

Acknowledgments

This work was supported by NINDS KO8 NS044998 (MSW), NICHD R01 HD39110 (SMB), NHLBI R01HL070061 (SMB) and NHLBI R01HL07212 (SMB). SMB also gratefully acknowledges the support of NIH P20 RR15583.

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