Original ContributionElevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia
Introduction
The level of nitric oxide (NO) in brain tissues varies over a wide concentration range during and following hypoxic or ischemic insult, which is a key factor determining the biological effects of NO. Hence, deficiency of NO production is associated with an increase in brain injuries induced by oxygen deprivation or cerebral ischemia [1], [2]. Yet, excessive amounts of NO mediate pathophysiological events in hypoxia-induced brain injuries. In patients with acute ischemic stroke, the concentration of NO metabolites in the cerebrospinal fluid is positively correlated with the early neurological worsening and the infarct volume [3]. It has also been shown that blockade of the activity of nitric oxide synthases (NOSs) ameliorates neuronal injuries following severe hypoxia or ischemia in animal models [4], [5], brain slices [6], [7], [8], and cultured neurons [9], [10].
Different pathways mediate the production of NO, which are affected by energy deprivation. The constitutive synthesis of NO is Ca2+ dependent and is mediated by neuronal and endothelial NOSs that provide basal levels of NO for maintaining physiological functions, whereas the NO production is mainly contributed by inducible NOS activated under pathophysiological conditions [11]. Moreover, the enzymatic pathways for NO production require multiple substrates including L-arginine, oxygen, NADPH, and other cofactors [11]. Considerable evidence suggests that both constitutive and inducible NOSs are involved in the excessive amount of NO production following hypoxic or ischemic insults, which are responsible for brain injuries [12], [13], [14], [15]. The endogenous NO activates downstream pathways that mediate the NO effect on neurons, the cGMP signaling cascade and S-nitrosylation of which are known to have major impacts on neuronal excitability via their modulatory effects on ionic channels [16]. Specifically, the cGMP-dependent pathway is reactive to NO at physiological levels and activates protein kinase G (PKG) for modulation of ionic channel functions. In addition, many proteins are S-nitrosylated by NO in physiological and pathological conditions [17]. Nitrosylation requires a covalent attachment of NO to the thiol side chains of cysteine residues in target proteins, and the transfer of S-nitrosothiol (SNO) signal by γ-glutamyl transpeptidase in the neural tissue [18], [19], [20].
During the early onset of acute hypoxia or ischemia, changes in ionic channel activities play an important role in the membrane depolarization and redistribution of ions across the membrane. Particularly, the acute change in the Ca2+ flux could greatly affect the activities of NO synthases and thus the amount of NO production and the level of NO in neural tissue. In addition, elevation in intracellular Ca2+ ([Ca2+]i) level is a cardinal event in neuronal response to energy deprivation, leading to subsequent neuronal injuries and delayed cell death [21]. In this context, it is not clear to what extent the endogenous NO may be related to the initial changes in the ionic events causing the Ca2+ flux. In addition, by what pathways NO could play a role in the early onset of energy deprivation is not well understood. In this study, we examined the acute effects of severe hypoxia and oxygen–glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the [Ca2+]i response in the rat hippocampal neurons, and the mechanistic effect of NO on the L-type Ca2+ current. We hypothesized that S-nitrosylation of L-type Ca2+ channels with an elevated endogenous NO production is involved in the increased Ca2+ flux in rat hippocampal neurons during an early onset of severe hypoxia and in vitro ischemia. Some of the results were communicated in abstracts.
Section snippets
Animals and hippocampal slices
The experimental protocol for this study was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. Brain slices were prepared from mature Sprague-Dawley rats (P26–P37) and the procedures have been described elsewhere [22]. In brief, rats were deeply anesthetized with methoxyflurane and were then decapitated. The brain was removed and chilled in ice-cold artificial cerebrospinal fluid (ACSF) gassed with 95% O2/5% CO2. A block of tissue
Effects of acute hypoxia and OGD on the endogenous NO production in rat hippocampal CA1 neurons
The NO concentration increased in acute hypoxia and oxygen–glucose deprivation (Fig. 1). The NO level elevated rapidly within a minute of hypoxia and reached its peak in 2–3 min of hypoxia when the oxygen level in the bath was reduced to its nadir (about 1 Torr). The NO concentration was sustained at an elevated level and then decreased gradually back to the resting level during recovery from hypoxia (Fig. 1A). On average, the NO concentration was elevated by 240.4 ± 18.3 nM (n = 10) in hypoxia
Discussion
The level of NO produced by CA1 neurons at the stratum pyramidale of the hippocampal slice was measured by an electrochemical microsensor. Results are consistent with findings of studies with imaging techniques indicating that NO-producing neurons are largely concentrated in the CA1 region of the hippocampal slices [26], [27], [28]. During the early onset of acute severe hypoxia or OGD, we observed a rapid increase in NO concentration that was maintained at elevated levels during hypoxia and
Acknowledgments
The authors gratefully acknowledge Mr. W.B. Wong for his technical assistance. The study was supported by research grants (N_HKU711/02) from the Research Grants Council of Hong Kong (M.L.F.) and by Grants NSFC/RGC (30218004), NSFC(30125013, 30330240), National Basic Research Program of China (No.2006CB504100), and Cheung Kong Scholars Programme from China (T.M.G.).
References (59)
- et al.
Nitric oxide inhibitors attenuate ischemic degeneration in the CA1 region of rat hippocampal slices
Neurosci. Lett.
(1996) - et al.
NG-nitro-L-arginine protects against hypoxia/hypoglycemia-induced decrease in CA1 presynaptic spikes in rat hippocampal slices
Eur. J. Pharmacol.
(1995) - et al.
In vitro hypoxia of cortical and hippocampal CA1 neurons: glutamate, nitric oxide, and platelet activating factor participate in the mechanism of selective neural death in CA1 neurons
Brain Res.
(1996) - et al.
Increase in nitric oxide in the hypoxic-ischemic neonatal rat brain and suppression by 7-nitroindazole and aminoguanidine
Eur. J. Pharmacol.
(1998) - et al.
Nitric oxide synthase activity and inhibition after neonatal hypoxia ischemia in the mouse brain
Brain Res. Dev. Brain Res.
(2000) - et al.
cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO
Trend Neurosci.
(2002) - et al.
Sodium homeostasis in rat hippocampal slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels
Neurosci. Lett.
(1999) - et al.
Glutamate receptor signaling interplay modulates stress-sensitive mitogen-activated protein kinases and neuronal cell death
J. Biol. Chem.
(1999) - et al.
Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4, 5-diaminofluorescein
J. Neurosci. Methods
(1999) - et al.
Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia
Biochim. Biophys. Acta
(1999)
Neonatal mice lacking neuronal nitric oxide synthase are less vulnerable to hypoxic-ischemic injury
Neurobiol. Dis.
Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia
Brain Res.
Use of brain slices in the study of pathogenic role of inducible nitric oxide synthase in cerebral ischemia-reperfusion
Gen. Pharmacol.
Ischemia/reperfusion-induced death of cardiac myocytes: possible involvement of nitric oxide in the coordination of ATP supply and demand during ischemia
J. Mol. Cell. Cardiol.
Nitric oxide tonically depresses a voltage- and Ca-dependent outward current in hippocampal slices
Neurosci. Lett.
Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex
Neuron
The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions
Cell
Nitric oxide regulates cardiac Ca2+ current: involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation
J. Biol. Chem.
The protective role of nitric oxide in the brain ischemia
J. Physiol. Pharmacol.
Nitric oxide deficiency contributes to large cerebral infarct size
Hypertension
Nitric oxide-related brain damage in acute ischemic stroke
Stroke
Low dose L-NAME reduces infarct volume in the rat MCAO/reperfusion model
J. Neurosurg. Anesthesiol.
The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels
J. Cereb. Blood Flow Metab.
NOS inhibitors decrease hypoxia-induced ATP reductions in respiring cerebrocortical slices
Anesthesiology
Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation
Mol. Chem. Neuropathol.
Endogenous nitric oxide synthsis: biological functions and pathophysiology
Free Radic. Res.
A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds
Nature
Subacute but not acute generation of nitric oxide in focal cerebral ischemia
Stroke
Protein S-nitrosylation: purview and parameters
Nat. Rev. Mol. Cell. Biol.
Cited by (31)
Kinesin-1 Regulates Extrasynaptic Targeting of NMDARs and Neuronal Vulnerability Toward Excitotoxicity
2019, iScienceCitation Excerpt :It is generated preferably from extrasynaptic NMDARs under certain neurodegenerative conditions (Molokanova et al., 2014). We used an NO-sensitive electrode to measure NO emissions from acutely prepared hippocampal slices from wild-type and Kif5b+/− mice (Tjong et al., 2007). Hypoxia (N2) and L-arginine (L-Arg) induced significantly lower NO emissions in kif5b+/− slices than in wild-type (L-Arg, p < 0.0001, t = 9.53, df = 9; N2, p < 0.0001, t = 15.53, df = 8; unpaired t test; Figure S4A).
Regulation of L-type Ca<inf>V</inf>1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway
2017, Cell CalciumCitation Excerpt :Though the reason for these discrepancies are presently unknown, they may lie in the fact that some NO effects are mediated via cGMP, but some others depend on the ability NO to generate free radicals or produce peroxynitrite leading to protein S-nitrosylation or nitrotyrosination [36]. Indeed, redox-modulation of L-type channels has been reported in cardiac myocytes, hippocampal neurons and vestibular hair cells [11,37,38]. However, in our experiments this may not be the case because the inhibitory effect of the NO donor SNP was almost fully blocked by the NO scavenger CPTIO.
Possible involvement of S-nitrosylation of brain cyclooxygenase-1 in bombesin-induced central activation of adrenomedullary outflow in rats
2012, European Journal of PharmacologyCitation Excerpt :N-Ethylmaleimide is known to produce irreversible alkylation of thiol groups, thereby blocking protein S-nitrosylation. Several groups have been using these reagents as “inhibitors of protein S-nitrosylation” in various studies including cellular (Bai et al., 2004; Zhang et al., 2008), slice cultural (Kakizawa et al., 2012; Tjong et al., 2007) and animal experiments (Pei et al., 2008). In the present experiments, central administration of dithiothreitol and N-ethylmaleimide effectively reduced the bombesin-induced elevation of plasma catecholamines, respectively.
Ionic storm in hypoxic/ischemic stress: Can opioid receptors subside it?
2010, Progress in NeurobiologyNitric oxide deficit in chronic intermittent hypoxia impairs large conductance calcium-activated potassium channel activity in rat hippocampal neurons
2008, Free Radical Biology and Medicine
- 1
These authors contributed equally to this study.