The aim of this study is to clarify whether the activation of a Na⁺/H⁺ exchanger (NHE) is tightly concerned with neuronal and glial cell injury induced by ischemia using a selective NHE inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate). Two hours of hypoxia followed by 24 h of reoxygenation induced lactate dehydrogenase (LDH) release, a marker of cell membrane damage, in cultured neurons and glia derived from rats. SM-20220 significantly reduced LDH release in both cells in a concentration-dependent manner, and this effect was statistically significant at concentrations of more than 10^–8 mol/l for neurons and 10^–7 mol/l for glia. A standard NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride, also reduced LDH release in neurons at concentrations of more than 10^–7 mol/l. In a rat transient middle cerebral artery occlusion model, intravenous infusion of SM-20220 reduced cerebral infarction when the serum concentration of SM- 20220 was maintained at about 10^–7 mol/l. These results suggest that the activation of the NHE plays an important role in ischemic neuronal and glial cell injury, and NHE inhibitor may have good therapeutic value for the treatment of ischemic stroke.

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ABSTRACT-Endothelial cells play an important role in the physiologic homeostasis of the cerebral circulation. Previously, we showed that the Na + / H + exchanger (NHE) inhibitor SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) improved ischemic brain injury. In this study, we investigated the effect of SM-20220 on cerebrovascular dysfunction after ischemia-reperfusion, focusing on the kinds of dysfunction that involved endothelial function. In cultured bovine brain microvascular endothelial cells (BBMCs), the IC 50 value for the NHE activity of SM-20220 was 4´10-8 M. SM-20220 also reduced the cell injury induced by hypoxia/ aglycemia-reoxygenation in BBMCs, with statistical significance at 10 -7 M (P<0.05). Next, the effect of SM-20220 on disruption of the blood-brain barrier and cerebral blood flow were evaluated using transient middle cerebral artery (MCA) occlusion models. Intravenous infusion of SM-20220 (0.4 mg/ kg per hour for 1 h) attenuated the extravasation of Evans blue, a blood-brain barrier disruption indicator, into cerebral tissue on the day after transient ischemia (P<0.05). The occlusion of the MCA decreased the cerebral blood flow in the MCA territory by about 20%, and only about 45% of the preischemic value was recovered at 1-h reperfusion. A bolus injection of SM-20220 (1 mg/kg, i.v.) improved the postischemic hypoperfusion by about 75%, without causing changes in the systemic blood pressure. These results indicate that the protective effect of NHE inhibitor on ischemic brain injury may be at least partially mediated by the prevention of endothelial dysfunction.

We studied the cerebroprotective effect of SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulphonate), a newly synthesized Na+/H+ exchanger (NHE) inhibitor, in Mongolian gerbil global ischaemia. Transient cerebral ischaemia was induced by clipping both common carotid arteries for 30 min followed by 24h reperfusion. Intravenous administration of SM-20220 (0.3 or 1.0 mg kg(-1)) immediately after reperfusion significantly shortened the consciousness recovery time (P < 0.01). SM-20220 also improved the neurological outcome (McGraw's scale) after reperfusion. At the dose of 1.0 mg kg(-1), the mortality rate was significantly reduced at 24 h after reperfusion (P < 0.01). This study shows that NHE is involved in the aggravation of cerebral function, represented by consciousness recovery, and neurological outcome following transient forebrain ischaemia, and that its inhibitor may exert protective effects on post-ischaemic brain damage.

The effect of an Na+/H+ exchange inhibitor, SM-20550, on the adhesion, emigration and accumulation of leukocytes was studied in ischemia and reperfusion injury models using rat mesenteric venules or rabbit heart. Anesthetized rats underwent occlusion of the superior mesenteric artery (20 min) followed by reperfusion (60 min). After ischemia and reperfusion, the numbers of adherent and emigrated leukocytes increased significantly. Bolus intravenous administration of SM-20550 reduced the numbers of adherent and emigrated leukocytes in a dose-dependent manner, with statistical significance at dosages >0.1 mg/kg. Anesthetized rabbits underwent occlusion of the coronary artery (30 min) followed by reperfusion (5 h). Intravenous administration of SM-20550 before ischemia significantly reduced the neutrophil accumulation in the area-at-risk by 46% and reduced the infarct size by 38%. These results suggest that the inhibitory effect of SM-20550 on the neutrophil accumulation could be one of the mechanisms by which this drug limits the severity of infarctions.