Original Contribution
Elevated 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

https://doi.org/10.1016/j.freeradbiomed.2006.09.020Get rights and content

Abstract

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. 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 intracellular calcium ([Ca2+]i) response in the rat hippocampal neurons. The levels of NO and [Ca2+]i in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca2+]i responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca2+]i response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca2+]i responses to hypoxia and OGD, indicating an involvement of L-type Ca2+ channels in the NO-mediated mechanisms. In addition, the [Ca2+]i responses were attenuated by ODQ or KT5823, inhibitors of the cGMP–PKG pathway, and by acivicin, an inhibitor of γ-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca2+ currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca2+ currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca2+ current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca2+ via the hippocampal L-type Ca2+ channel by S-nitrosylation during an initial phase of energy deprivation.

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.).

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