Naohito Ohashi

Cerebroprotective properties of SM-20220, a potent Na+/H+ exchange inhibitor, in transient cerebral ischemia in rats

The aim of this study was to investigate the contribution of the Na(+)/H(+) exchanger to cerebral ischemia using SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate), a newly synthesized compound. In in vitro experiments, we evaluated the inhibitory effect of SM-20220 on the Na(+)/H(+) exchanger in cultured neurons and glial cells. The IC(50) of SM-20220 in neurons and glial cells was 5 nM and 20 nM, respectively. To examine the in vivo effects of SM-20220 on brain injury, we used a transient middle cerebral artery occlusion model in rats. SM-20220 given intravenously 1 h after occlusion significantly reduced the extent of cerebral edema, Na(+) content and infarcted area in a dose-dependent manner. The results of the present study suggest that the Na(+)/H(+) exchanger is involved in the aggravation of brain edema and infarction, and its inhibitor may exert protective effects on post-ischemic brain damage.

The Na+/H+ Exchanger SM-20220 Attenuates Ischemic Injury in in vitro and in vivo Models

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.

SM-20220, a Potent Na+/H+ Exchange Inhibitor, Improves Consciousness Recovery and Neurological Outcome Following Transient Cerebral Ischaemia in Gerbils

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.

A Na+–H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion

Na+-H+ exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na+-H+ exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na+-H+ exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia (n = 11) or before reperfusion (n = 7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size (P < 0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P < 0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.

SM-20550, a new Na+/H+ exchange inhibitor and its cardioprotective effect in ischemic/reperfused isolated rat hearts by preventing Ca2+-overload

We investigated the effect of a newly synthesized compound, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid] on Na+/H+ or Na+/Ca2+ exchange activity in rat cardiomyocytes, and on radioligand binding with several channels or receptors in membrane preparations, and ischemia/reperfusion injury in isolated perfused rat hearts. In myocytes, SM-20550 concentration-dependently inhibited the recovery from acidosis induced by an NH4Cl prepulse in HCO3(-)-free solution. Its IC50 was 10(-8) M, which was 10 times lower than that of ethylisopropyl amiloride (EIPA). SM-20550 (10(-6) M) did not affect the Na+-dependent Ca2+ influx (Na+/Ca2+ exchange activity) in cardiomyocytes. In the radioligand binding assay, SM-20550 did not have affinity for K+ channel, beta-adrenoceptor, adenosine, angiotensin, or endothelin receptors, and had low affinity for Na+ and Ca2+ channels and alpha-adrenoceptors, only at the concentrations of 10(-6)-10(-5) M. In perfused hearts exposed to 40 min of global ischemia and 20 min of reperfusion, SM-20550 (10(-8)-10(-7) M) significantly reduced the elevation of left ventricular end-diastolic pressure during reperfusion, improved the postischemic recovery of developed pressure, and prevented coronary perfusion pressure increase after reperfusion. Furthermore, SM-20550 reduced creatine phosphokinase release during reperfusion and prevented the abnormal gain of tissue Na+ and Ca2+ at the end of reperfusion. These results suggest that SM-20550 is a potent, highly specific Na+/H+ exchange inhibitor, which exerts a protective effect against myocardial ischemia/reperfusion injury. In addition, our data strongly support the hypothesis that Na+/H+ exchange plays an important role in the development of postischemic cardiac dysfunction, most likely by inducing Na+ and Ca2+ overload.

Effects of SM-20550, a selective Na+-H+ exchange inhibitor, on the ion transport of myocardial mitochondria

The effect of a novel Na+-H+ exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate] (SM) on the ion transport of myocardial mitochondria was studied using ion fluorometry and superfusion techniques. Isolated mitochondria from the guinea-pig heart were pre-loaded with fluoroprobes of either BCECF AM for H+, SBFI AM for Na+ or fura-2 AM for Ca2+. Initially, the treated mitochondria were superfused with a normal medium (MOPS-buffer, pH 7.4, 24°C), subsequently fluorometric experiments on the Na+, H+, Ca2+ mobilization across the mitochondrial membrane were performed. The intramitochondrial pH (pHm) was increased by the superfusion of Na+ at physiological cytosolic concentrations of 10 mM, indicating the existence of a Na+-H+ exchange in mitochondrial membranes. The Na+ induced elevation of pHm was dose-dependently inhibited by SM 1 μM (ΔpHm; 45% as drug-free 100%), and 10 μM (ΔpHm; 70%(, as observed in our experiments with the myocardial sarcolemmal membrane. The selective Na+-H+ exchange inhibitor SM reduced such pHm elevations more markedly than that of EIPA [5-(N-ethyl-N-isopropyl) amiloride]. The Na+-H+ exchange inhibitors, SM and EIPA suppressed the intramitochondrial Ca2+ elevation ([Ca2+]m) brought on by external Ca2+ concentration changes: The pretreatment with SM 1 μM, 10 μM and EIPA 10 μM reduced the [Ca2+]m influx by 28.3, 56.5 and 63%, respectively. Additionally, the [Ca2+]m elevation induced by acidification of the perfusate was reduced by the prior infusion of SM and EIPA. Pretreatment of mitochondria with SM or EIPA which had beneficial effects on the left ventricular developed pressure (LVDP) in the ischemia-reperfusion injury of Langendorff hearts, reduced the intramitochondrial Na+ and pHm levels, indicating interplay of the inhibitory mechanism of Ca2+-uptake into mitochondria coupled with Na+-H+ exchange. These findings suggested that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due in part to the Ca2+-paradox at the mitochondria level.

Reduction of myocardial infarct size by SM-20550, a novel Na(+)/H(+) exchange inhibitor, in rabbits.

The effects of SM-197378, 2-[[[amino(imino)methyl]amino]carbonyl]-1-methyl-4-trifluoromethyl-1H-indol-7-yl=hydrogen=sulfate monohydrate, a novel potent Na+/H+exchange inhibitor, on heart injury were studied using a rabbit model involving 30 min of myocardial ischemia and 5 h of reperfusion. Intravenous administration of SM-197378 before ischemia reduced the infarct size by approximately 30-50% in a dose-dependent manner. This anti-necrotic effect was achieved without significant hemodynamic changes. Moreover, administration of SM-197378 before reperfusion also resulted in a significant, approximately 30-40%, reduction in the infarct size. The anti-necrotic effect of pre-ischemic bolus treatment with SM-197378 was compared with that of nicorandil, a K+channel opener with nitrate-like activity, and ischemic preconditioning. With 30 and 60 min of ischemia, the anti-necrotic effects of SM-197378 and ischemic preconditioning were similar and superior to that of nicorandil. With 90 min of ischemia, the anti-necrotic effect of SM-197378 was superior to that of ischemic preconditioning.

Protective effect of SM-20550, a selective Na+ - H+ exchange inhibitor, on ischemia-reperfusion-injured hearts.

The protective effects of Na+-H+ exchange inhibitors SM-20550 (SM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) against ischemia-reperfusion injury were investigated in guinea pig Langendorff hearts. The changes in intracellular pH (pHi), high-energy phosphates, and biologic intracellular active ions ([Na+]i and [Ca2+]i) were regarded using the 31P-NMR and specific fluorescent signals from the heart tissues together with simultaneous recordings of the left ventricular developed pressure (LVDP). The recovery rate of LVDP from ischemia (40 min) by reperfusion was 36.8% in the control experiments, whereas in the presence of SM 10−7 M, a gradual increase to 75.9% (55.5% with 10−8 M), in contrast to EIPA (10−7 M), 47.5% was observed. SM 10−7 M restored the ATP level by 70% in 40-min reperfusion, which was already higher than the control in the latter half (20-40 min) of the ischemic period. The recovery rate of phosphocreatine by pretreatment of the heart with SM 10−7 M was 75% in 40 min reperfusion. The pHi estimated from Pi/phosphocreatine chemical shift became highly acidic in ischemic heart so that SM 10−7 M caused slight but significant pHi reduction from control pHi of 5.89 to 5.75. The level returned to pHi at around 7.38 during 30-40 min reperfusion, and the recovery was significantly greater than the control pHi of 7.24. The fura-2 Ca2+ or SBFI-Na+ signals during Langendorff ischemia heart increased, and rapidly returned to the control level after the reperfusion. SM suppressed the [Na+]i or [Ca2+]i elevation induced in the late stage during ischemia, resulting in LVDP restoration after reperfusion; Diastolic Ca2+ in the end period of ischemia, SM 10−7 M 194% versus drug-free 220.7%, Na+: SM 10−7 M 121.6% versus drug-free 128.0%. The present results suggest that the selective Na+-H+ exchange inhibitor SM is promising as a potent and specific protective agent against ischemia-reperfusion injuries with Ca2+ overload induced via Na+-H+, Na+-Ca2+ exchange.

Na+/H+ exchange inhibitor SM-20220 attenuates leukocyte adhesion induced by ischemia-reperfusion.

&NA; Leukocytes play a key role in ischemia‐reperfusion‐induced tissue injuries. It has been suggested that blocking the Na+/H+ exchanger improves ischemic injuries such as stroke. In this study, we investigated the effect of the Na+/H+ exchanger inhibitor SM‐20220 (N‐[aminoiminomethyl]‐1‐methyl‐1Hindole‐2‐carboxamide methanesulfonate) on leukocyte‐endothelial cell interactions during ischemia‐reperfusion. SM‐20220 (0.3–1.0 mg/kg i.v.) given after ischemia significantly attenuated the leukocyte adhesion in the mesenteric postcapillary venules that was induced by transient superior mesenteric artery occlusion. At 60 min after reperfusion, the numbers of adherent leukocytes in groups treated with vehicle or SM‐20220 (0.3 mg/kg) were 15.1 ± 2.9 cells/100 &mgr;m/3 min and 3.0 ± 0.7 cells/100 &mgr;m/3 min (p < 0.01), respectively. In a transient middle cerebral artery occlusion model, i.v. infusion of SM‐20220 (0.4 mg/kg per hour) for 1 h, beginning 1 h after the start of occlusion, significantly reduced both the infarct size and the increase in brain myeloperoxidase activity, compared with the vehicle group (p < 0.01 and p < 0.05, respectively). In summary, this is the first evidence that the leukocyte adhesion to the endothelium that is induced by ischemia‐reperfusion is attenuated by the inhibition of Na+/H+ exchanger activity in vivo. Our results suggest that Na+/H+ exchanger inhibitors may prevent ischemia‐reperfusion injuries such as stroke partly through the attenuation of leukocyte‐endothelial cell interactions.

Effect of an orally active Na+/H+ exchange inhibitor, SMP-300, on experimental angina and myocardial infarction models in rats.

The effects of SMP-300, an orally active, potent, and selective Na + /H + exchange inhibitor, were evaluated and compared with those of nifedipine, propranolol, and nicorandil on three experimental angina models and on myocardial infarction in rats. SMP-300 (0.1–1 mg/kg, p.o.) reduced isoproterenol-induced ST segment depression in a dose-dependent manner. Its maximal effect was comparable to that reported for propranolol and greater than that of nifedipine. SMP-300 (0.3–1 mg/kg) reduced vasopressin-induced ST segment depression in a dose-dependent manner, and its maximal effect was comparable to those of nifedipine and nicorandil. SMP-300 (0.3–1 mg/kg, p.o.) and propranolol (100 mg/kg, p.o.) inhibited coronary artery occlusion–induced T-wave elevation, but nifedipine (3 mg/kg, p.o.) did not. SMP-300 (1 mg/kg, p.o.) reduced myocardial infarct size after 40 min of coronary artery occlusion followed by 24 h of reperfusion, but nifedipine (3 mg/kg, p.o.) or propranolol (100 mg/kg, p.o.) did not. This study provides support for the future use of a Na + /H + exchange inhibitor as an anti-anginal drug with a novel mode of action.