Research reportDifferent effects of eNOS and nNOS inhibition on transient forebrain ischemia
Introduction
Nitric oxide (NO) is produced endogenously by the conversion of l-arginine to citrulline by NO synthase (NOS). NO is synthesized by three different types of NOS including the constitutive calcium/calmodulin-dependent neuronal and endothelial isoforms and the inducible calcium-independent isoform [14], [35]. NO plays an important role in the modulation of vascular tone and neurotransmission [23]. Moreover, there is increasing evidence that NO produced both during and after cerebral ischemia may be an important factor in the pathogenesis of neuronal ischemic injury [14], [16]. During the early stages of cerebral ischemia, endothelial NOS (eNOS) and neuronal NOS (nNOS) are continuously active and produce large amounts of NO [14], [43], [44]. eNOS-derived NO is beneficial in promoting collateral circulation and microvascular flow [14]. In contrast, nNOS-derived NO is detrimental in the early stages of transient cerebral ischemia [8], [30].
It is well known that cerebral ischemia induces a massive increase in glutamate release (for recent review, see Ref. [28]). At the same time, glutamate-induced Ca2+ overload in ischemic neurons leads to a persistent activation of nNOS, resulting in continuous NO production [14], [35]. The overproduction of nNOS-derived NO leads to neurotoxicity [10], [35]. The neurotoxic actions of NO are mediated by peroxynitrite, the reaction product of NO and superoxide anion [4].
In global forebrain ischemia, the amount of glutamate released in rats treated with NG-nitro-l-arginine methyl ester hydrochloride (l-NAME), a non-specific NOS inhibitor, has been reported to increase [17], [42], decrease [32] or not change [46]. One reason for the discrepancy may be related to the lack of specificity of the inhibitor (l-NAME): l-NAME not only inhibits nNOS in brain but also causes a sustained increase in blood pressure by inhibiting eNOS.
l-N5-(1-iminoethyl)ornithine (l-NIO) is a relatively selective inhibitor of eNOS [26], [39]. l-NIO reduces resting cyclic GMP levels and constricts pial arteries, both consistent with the idea that eNOS contributes to resting vascular tone [23]. On the other hand, 7-nitroindazole (7-NI) is relatively selective for the neuronal isoform of NOS [25]. 7-NI has no significant effects on mean arterial blood pressure (MABP) [3], [37] and cerebral blood flow [1], [15]. Yoshida et al. [40] showed that 7-NI had no effect on MABP or on the response to acetylcholine, suggesting no eNOS activity in vivo. It has been reported that 7-NI decreases focal infarct volume [40] and provides protection against ischemia-induced cell death in the CA1 region of the gerbil hippocampus [29]. Kahn et al. [18] have recently found that 7-NI reduces the glutamate levels in the striatum during ischemia and reperfusion.
Our previous study showed that 7-NI markedly inhibits NO production in the rat hippocampus during post-ischemic early reperfusion [15]. At present, we do not know the effects of relatively selective eNOS inhibitors on hippocampal neuronal dysfunction after transient forebrain ischemia. In the present study, therefore, to better understand the differential roles of eNOS- and nNOS-derived NO, we investigated the effects of 7-NI and l-NIO, which have differential inhibitory actions on eNOS and nNOS, on transient forebrain ischemia using electrophysiological and microdialysis methods.
Section snippets
Animal preparation
All procedures followed our previous studies [15], [19] with some modifications. The present study was carried out according to ‘The Guiding Principles for Care and Use of Animals in the Field of Physiological Sciences’ (The Physiological Society of Japan). Adult male Wistar rats weighing between 280 and 320 g were used. On the day before the experiment, the rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.) and the bilateral vertebral arteries were electrocauterized. On the
Physiological variables
The arterial Pao2, Paco2 and pH values were measured at the end of each experiment. However, they did not show any significant differences among groups (data not shown).
Effects of 7-NI and l-NIO on hippocampal blood flow and MABP
Fig. 2 shows representative records of MABP, hippocampal blood flow and DC potential activity before, during and after transient forebrain ischemia in the vehicle, 7-NI and l-NIO groups. Both 7-NI and l-NIO did not affect the hippocampal blood flow values 20 min after drug administration, 10 min after the onset of ischemia and
Discussion
In the present study, we have observed for the first time the effects of l-NIO on the hippocampal CA1 PS amplitudes and levels of glutamate during transient global ischemia. The main findings were as follows: (1) l-NIO, but not 7-NI, increased MABP before, during and after ischemia; (2) 7-NI, but not l-NIO, reduced the AD amplitudes during ischemia and partially recovered the PS amplitudes 60 min after the onset of reperfusion, compared with the vehicle group; (3) 7-NI, but not l-NIO, reduced
Acknowledgements
We thank Dr Iso, Department of Behavior, Hyogo College of Medicine, for the statistical analysis. This work was supported in part by Hyogo College of Medicine Research Grant for M.-H. Jiang.
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