Different effects of eNOS and nNOS inhibition on transient forebrain ischemia

Abstract

To clarify the functions of nitric oxide (NO) induced by either neuronal NO synthase (nNOS) or endothelial NO synthase (eNOS) after transient cerebral ischemia, we investigated the effects of l-N⁵-(1-iminoethyl)ornithine (l-NIO), a relatively selective eNOS inhibitor, and 7-nitroindazole (7-NI), a relatively selective nNOS inhibitor, on hippocampal dysfunction caused by cerebral ischemia. We measured mean arterial blood pressure (MABP), hippocampal blood flow, direct current (DC) potential, CA1 population spike (PS) and extracellular concentrations of glutamate from rat hippocampus after transient forebrain ischemia, which was induced by four-vessel occlusion for 10 min. l-NIO (20 mg/kg) and 7-NI (25 mg/kg) were administered intraperitoneally 20 min before ischemia. l-NIO, but not 7-NI, increased MABP before, during and after ischemia, compared with the vehicle group. 7-NI, but not l-NIO, reduced the amplitude of anoxic depolarization induced by ischemia. 7-NI recovered the PS amplitude in part 60 min after ischemia. 7-NI, but not l-NIO, reduced the ischemia-induced levels of glutamate. These results indicate that nNOS inhibition with 7-NI improves, at least in part, hippocampal dysfunction after ischemia, while eNOS inhibition with l-NIO worsens it.

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 N^G-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.