Abstract
Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys331, one of the zinc-tetrathiolate cysteines, was identified as the key site of nNOS S-nitrosylation. In addition, we also found that nNOS is highly S-nitrosylated in resting rat hippocampal neurons and the enzyme undergos denitrosylation during the process of rat brain ischemia/reperfusion. Intrestingly, the process of nNOS denitrosylation is coupling with the decrease of nNOS phosphorylation at Ser847, a site associated with nNOS activation. Further more, we document that nNOS denitrosylation could be suppressed by pretreatment of neurons with MK801, an antagonist of NMDAR, GSNO, EGTA, BAPTA, W-7, an inhibitor of calmodulin as well as TrxR1 antisense oligonucleotide (AS-ODN) respectively. Taken together, our data demonstrate that the denitrosylation of nNOS induced by calcium ion influx is a NMDAR-dependent process during the early stage of ischemia/reperfusion, which is majorly mediated by thioredoxin-1 (Trx1) system. nNOS dephosphorylation may be induced by the enzyme denitrosylation, which suggest that S-nitrosylation/denitrosylation of nNOS may be an important mechanism in regulating the enzyme activity.
Highlights
Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) which has three known isoforms in mammals: neuronal NOS, endothelial NOS, and inducible NOS
We demonstrate that neuronal NOS (nNOS) denitrosylation is induced by calcium ion influx via NMDAR during the early stages of reperfusion, which is a Ca2+/CaM-dependent process principally mediated by the Trx1 system, and that nNOS denitrosylation is coupled with an increase in the activity of this enzyme
Exogenous NO S-nitrosylates nNOS and Decreases its Activity in HEK293 Cells To investigate the exogenous effects of NO on nNOS, HEK293 cells were transfected with wild type nNOS plasmid for 48 hours and treated with various concentrations of the NO donor GSNO for 60 min (Fig. 1A)
Summary
Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) which has three known isoforms in mammals: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Many studies have suggested that either exogenous or endogenous NO can S-nitrosylate cysteine residues [6]. Both nNOS and iNOS are expressed in brain. The phosphorylation of nNOS at Ser847 is induced by Ca2+/ calmodulin-dependent kinase II (CaMK II) in the rat hippocampus following transient forebrain ischemia [11,12,13]. This decreases the activity of nNOS by inhibiting the binding of Ca2+/ calmodulin. The excessive activation of glutamate receptors is the principle reason for the neuronal death that occurs in ischemic stroke as NMDAR has a higher
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