Na+/H+ exchange inhibitor SM-20220 improves endothelial dysfunction induced by ischemia-reperfusion

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-(aminoimino-methyl)-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 IC50 value for the NHE activity of SM-20220 was 4 x 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.     

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

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-1H-indole-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 microm/3 min and 3.0+/-0.7 cells/100 microm/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.     

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.     

Relationship between the neuroprotective effect of Na+/H+ exchanger inhibitor SM-20220 and the timing of its administration in a transient middle cerebral artery occlusion model of rats.

The aim of this study was to determine the relationship between the neuroprotective effect of SM-20220 (N(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) and the timing of its administration in an experimental stroke model. Two hours of occlusion followed by 22 h of perfusion of the left middle cerebral artery (MCA) was performed by inserting a nylon thread into the MCA to occlude it, and pulling the thread to initiate reperfusion. Intravenous infusion of SM-20220 for 1 h reduced the infarct volume at doses of 0.2-0.8 mg/kg in a dose-dependent manner without causing changes in the systemic arterial blood pressure or blood gases, when SM-20220 administration was started 1 h after the onset of occlusion. Administration of SM-20220 at a dose of 0.4 mg/kg also reduced the edema formation induced by ischemia. In contrast, SM-20220 failed to reduce the infarction, even at 1.6 mg/kg, when administration was started 2 h after the onset of occlusion. Thus, the therapeutic time window of SM-20220 for this transient MCA occlusion model is 1 h. Daily administration of SM-20220 (0.4 mg/kg) for the 7 d following 1.5 h of middle cerebral artery occlusion reduced the infarct volume with statistical significance (p<0.05), showing that SM-20220 did not merely delay but prevented ischemic damage.     

白三烯受体拮抗剂ONO—1078对大鼠局灶性脑缺血的神经保护作用

AIM: To determine whether ONO-1078 (pranlukast), a potent leukotriene receptor antagonist, has neuroprotective effect on focal cerebral ischemia in the rat. METHODS: Focal cerebral ischemia was induced by 30 min of middle cerebral artery (MCA) occlusion and followed by 24 h reperfusion. ONO-1078 (0.003-1.0 mg/kg) or vehicle (saline 1 mL/kg) was ip injected 30 min before MCA occlusion and 2 h after reperfusion. The neurological score, infarct volume, neuron density (in cortex, hippocampus, and striatum), brain edema, and albumin exudation around the vessels were determined 24 h after reperfusion. RESULTS: ONO-1078 slightly improved the neurological deficiency, and dramatically decreased infarct volume and neuron loss which showed a bell shaped dose response effect with highest effect at doses of 0.01-0.3 mg/kg. Enlargement of the ischemic hemisphere and albumin exudation were inhibited at doses of 0.01-1.0 mg/kg. CONCLUSION: ONO-1078 has the protective effect on focal cerebral ischemia in rats, which is partially attributed to the inhibition of brain edema. This may represent a novel approach to the treatment of acute cerebral ischemia with cysteinyl leukotriene receptor antagonists.     

FR183998, a Na+/H+ exchange inhibitor, suppresses both IL-8 content and myocardial infarct size in a cardiac ischaemia-reperfusion model in rats

The aim of this study was to determine the effect of FR183998 (5-(2,5-dichlorothiophen-3-yl)-3-[(2-dimethylaminoethyl)carbamoyl]benzoylguanidine dihydrochloride), an Na+/H+ exchange inhibitor, on myocardial interleukin-8 (IL-8) content and myocardial infarct size in a rat ischaemia and reperfusion model. Rats underwent 30 min of ischaemia followed by 1 to 24 h of reperfusion. IL-8 content rapidly increased in reperfused rat hearts. The maximum increase in IL-8 was obtained after 3 h of reperfusion. Intravenous administration of FR183998 at 1 and 3.2 mg kg(-1), 5 min before ischaemia, significantly reduced the IL-8 level after 3 h of reperfusion (122 +/- 16 and 149 +/- 23 pg mg(-1) protein, respectively), compared with that of the saline-treated group (258 +/- 27 pg mg(-1) protein). Myeloperoxidase activity after 3 h of reperfusion was also reduced by FR183998 (from 0.83+0.19 unit g(-1) weight of tissue in the saline-treated group to 0.36 +/- 0.09 and 0.33 +/- 0.06 unit g(-1) weight of tissue in FR183998-treated groups at 1.0 and 3.2 mg kg(-1), respectively). Myocardial infarction induced by 30 min of ischaemia and 24 h of reperfusion was significantly suppressed by the same doses of FR183998 (14.0 +/- 1.5,13.5 +/- 1.9% at 1.0 and 3.2 mg kg(-1)), compared with 22.2+2.7% in the saline-treated group. These results suggestthat IL-8 may contribute to the generation of myocardial infarction in an ischaemia and reperfusion model in rats.     

Therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h in rats

Pervious experimental studies have shown that XQ-1h has beneficial neuroprotective effect in the cerebral ischemia reperfusion injury. However, the therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h is not clear. Under chloral hydrate anesthesia, transient focal cerebral ischemia was induced in rats by 2h of middle cerebral artery occlusion (MCAO), followed by 24h of reperfusion. Saline as vehicle or XQ-1h at the doses of 31.2, 15.6 and 7.8 mg/kg i.v. was administered at 0.5, 1, 2, 3h after induction of ischemia. Subsequently, 24h after MCAO brain edema, infarct volume, neurological deficits and cerebral blood flow were evaluated. Administrations of XQ-1h at the doses of 31.2mg/kg at 0.5, 1, and 2h after reperfusion of MCAO significantly reduced infarct rate (%) by 75.6% (5.2 ± 1.7), 66.2% (7.2 ± 1.9), and 47.9% (11.1 ± 1.2), respectively. XQ-1h (31.2mg/kg) treatment, 0.5, 1, and 2h after reperfusion produced significant improvement in neurological score compared to vehicle-treated group (P<0.01). Administrations of XQ-1h at the doses of 31.2mg/kg and 15.6 mg/kg at 0.5, 1, and 2h after reperfusion of MCAO significantly increased cerebral blood flow (mv) by 16.9 ± 1.9, 11.7 ± 1.3, 9.5 ± 1.0, respectively (P<0.01). In conclusion the therapeutic time window of XQ-1h for cerebral ischemia reperfusion injury is within 2h. Interestingly, we also discovered that the therapeutic time window of XQ-1h is deeply related with the activity of scavenging oxidative stress products. Further studies need to be conducted more drug combination therapy programs in order to assess the potential clinical application of XQ-1h.     

Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger

1. The NHE1 isoform of the Na(+)/H(+) exchanger plays an important role in the regulation of intracellular pH and in cardiac cell injury caused by ischaemia and reperfusion. SL 59.1227 is a novel imidazolypiperidine Na(+)/H(+) antiport inhibitor which is structurally unrelated to previously described acylguanidine inhibitors such as cariporide. 2. Recovery of pH(i) following an intracellular acid load was measured in CCL39-derived PS120 variant cells, selectively expressing either NHE1 or NHE2 isoforms of the Na(+)/H(+) exchanger. pH(i) recovery was potently and selectively slowed by SL 59.1227 in NHE1-expressing cells (IC(50) 3.3+/-1.3 nM) versus NHE2-expressing cells (2.3+/-1.0 microM). The respective IC(50) values for cariporide were 103+/-28 nM (NHE1) and 73+/-46 microM (NHE2). 3. In anaesthetized rats following left coronary artery occlusion (7 min) and reperfusion (10 min) SL 59.1227 (10 - 100 microg kg(-1) min(-1) i.v.) inhibited ischaemia-mediated ventricular tachycardia (71 - 100%) and reperfusion-induced ventricular fibrillation (75 - 87%) and prevented mortality. Bolus i.v. administration of SL 59.1227 (1 mg kg(-1)) produced anti-arrhythmic effects when administered either before or during ischaemia. 4. Cardiac infarct size was determined in anaesthetized rabbits following left coronary artery occlusion (30 min) and reperfusion (120 min). Infarct size measured as a percentage of the area at risk was 36.2+/-3.4% (control group) versus 15.3+/-3.9% (SL 59.1227 0.6 mg kg(-1) i.v.). 5. SL 59.1227 is the first example of a potent and NHE1-selective non-acylguanidine Na(+)/H(+) exchanger inhibitor. It possesses marked cardioprotective properties.     

Protective effect of SM-19712, a novel and potent endothelin converting enzyme inhibitor, on ischemic acute renal failure in rats

Effects of SM-19712 (4-chloro-N-[[(4-cyano-3-methyl- 1-1-phenyl- 1H-pyrazol-5-yl)amino]carbonyl] benzenesulfonamide, monosodium salt), a novel endothelin converting enzyme (ECE) inhibitor, on ischemic acute renal failure (ARF) in rats were examined in comparison with those of phosphoramidon, a conventional ECE inhibitor. ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in ARF rats markedly decreased at 24 h after reperfusion. Intravenous bolus injection of SM-19712 (3, 10, 30 mg/kg) prior to the occlusion attenuated dose-dependently the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damages such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, all of which were dose-dependently attenuated by SM-19712. Protective effects of phosphoramidon (10 mg/kg) on ARF-induced functional and tissue damages were less potent than that of the same dose of SM-19712. Endothelin-1 (ET-1) content in the kidney after the ischemia/reperfusion was significantly increased, being the maximum level at 6 h after reperfusion, and this elevation was completely suppressed by the higher dose of SM-19712. Our findings support the view that renal ET-1 plays an important role in the development of ischemia/reperfusion-induced renal injury. SM-19712 may be useful in the treatment of ischemic ARF.     

Reduction of myocardial infarct size by SM-198110, a novel Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange inhibitor,in rabbits

The effects of 3-[2-({[amino(imino)methyl]amino}carbonyl)-4-chloro-1H-indol-1-yl]-1-propanesulphonic acid monohydrate (SM-198110), a novel potent Na⁺/H⁺ exchange inhibitor, and cariporide (Hoe642), another Na⁺/H⁺ exchange inhibitor, were studied in a myocardial ischaemia and reperfusion injury model. Anaesthetized rabbits were subjected to occlusion of the coronary artery for 30 min followed by reperfusion for 5 h. SM-198110 or cariporide was administered before ischaemia and before reperfusion. We also assessed the anti-necrotic effect of SM-198110 when given before reperfusion, both alone and together with glibenclamide, a K_ATP channel blocker, 5-hydroxydecanoate (5-HD), a mitochondrial K_ATP channel-selective blocker and 8-(p-sulphophenyl)-theophylline (8-SPT), an adenosine receptor blocker. The infarct size was reduced dose-dependently by i.v. administration of SM-198110 before ischaemia, with a significant reduction in serum creatine phosphokinase activity. Infarct sizes, normalized to the size of the area-at-risk (means±SE) were: vehicle 56.6±3.7%; low-dose SM-198110 39.2±6.3%; mid-dose 32.8±7.4% (P<0.05); high-dose 22.1±6.7% (P<0.01). This anti-necrotic effect of SM-198110 was achieved without significant haemodynamic changes. Cariporide given before ischaemia also reduced infarct size significantly and dose-dependently. SM-198110 administered before reperfusion also resulted in a dose-dependent reduction in the infarct size. Infarct sizes were: vehicle 56.6±3.7%; low-dose SM-198110 44.5±5.7%; mid-dose 36.3±6.6% (P<0.01); high-dose 34.7±3.8% (P<0.01). In contrast, cariporide given before reperfusion did not reduce infarct sizes significantly. The anti-necrotic effect of SM-198110 was observed even when given 10 min after the beginning of reperfusion. Glibenclamide and 5-HD abolished the anti-necrotic effect of treatment before reperfusion with SM-198110. However, the co-administration of 8-SPT with SM-198110 did not affect infarct size. These results suggest that, in addition to Na⁺/H⁺ exchange inhibition, mitochondrial and/or sarcolemmal K_ATP channels contribute to the anti-necrotic effect of SM-198110 when the latter is given before reperfusion.     

Differences in the effects of Na+-H+ exchange inhibitors on cardiac function and apoptosis in guinea-pig ischemia-reperfused hearts

The protective effects of the Na+-H+ exchange (NHE) inhibitors SM-198110 (2-[[(aminoiminomethyl) amino] carbonyl]-4-chloro-1H-indole-1-propanesulfonic acid monohydrate) and SM-197378 (N-(aminoiminomethyl)-1-methyl-7-(sulfooxy)-4-(trifluoromethyl)-1H-indole-2-carboxamide monohydrate) were investigated in perfused Langendorff guinea-pig hearts subjected to ischemia (40 min) and reperfusion (40 min). The recovery of left ventricular developed pressure (LVDP) from ischemia by reperfusion was 39.0% in the control, while in the hearts pretreated with SM-198110 or SM-197378 (10(-7) M), it was about 100%. The ATP level, monitored simultaneously by (31)P-nuclear magnetic resonance spectrometry, was already higher than the control value at the end of the ischemic period, and the elevation in Na+ or Ca2+ fluorometric signals induced during ischemia was suppressed. In post-treated hearts, the LVDP recovery rate was significantly higher with SM-198110 than with SM-197378. By in vitro electron paramagnetic resonance spectrometry, SM-197378 was found to directly quench the active oxygen radical, whereas SM-198110 had no effect. Numbers of apoptotic cardiomyocytes after ischemia (1 h) followed by reperfusion (5 h) were significantly lower in SM-197378-treated than in SM-198110-treated hearts, consistent with the level of activity of caspase-3. These results suggest that the antioxidant effects of NHE inhibitors have an important role in apoptosis during ischemia-reperfusion, but apoptosis is not a major manifestation of cardiac function during postischemic recovery, and that NHE-sensitive mechanisms of reperfusion injury promote both necrotic and apoptotic processes death.     

Neuroprotective effect of ONO-1078, a leukotriene receptor antagonist, on transient global cerebral ischemia in rats

AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate}, a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078 (0.03-0.3 mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 min before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-Daspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1(VCAM-1) in the cerebral cortex and hippocampus were measured at 72 h after reperfusion. RESULTS: ONO-1078 (0.1, 0.3 mg/kg) and edaravone (10 mg/     

白三烯受体拮抗剂ONO-1078对大鼠短暂性全脑缺血的神经保护作用

AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate), a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078(0.03-0.3mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 rain before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-D-aspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1 (VCAM-1) in the cerebral cortex and hippocampus were measured at 72h after reperfusion. RESULTS: ONO-1078 (0.1,0.3mg/kg) and edaravone (10 mg/kg) improved ischemia-induced neurological deficiency and reduced neuron death.ONO-1078 (0.1, 0.3mg/kg) significantly inhibited the enhanced expression of NMDA receptor subunit protein NR2A in the cortex and VCAM-1 in the hippocampus of ischemic rats. CONCLUSION: ONO-1078 possesses a neuroprotective effect on global cerebral ischemia in rats, and its mechanism may be partly related to the inhibition of the upregulation of NR2A and VCAM- 1 in different regions of the brain.     

新型神经保护剂TQ0701-2对大鼠脑缺血再灌注损伤的保护作用

目的：研究新型神经保护剂TQ0701-2对大鼠脑缺血再灌注损伤的保护作用。方法：将120只雄性SD大鼠随机分为假手术组、模型组、依达拉奉组（3.0mg/kg）以及TQ0701-2高剂量组（6.0mg/kg）、中剂量组（3.0mg/kg）、低剂量组（1.5mg/kg）。假手术组仅进行手术而不造成缺血状态,其余各组均采用Longa线栓法制备大鼠MCAO模型,在缺血2h后进行再灌注。TQ0701-2三个剂量组和依达拉奉组分别在缺血前30min以及再灌注0、2h尾静脉注射TQ0701-2和依达拉奉,假手术组和模型组则给予等量的生理盐水。再灌注24h后观察大鼠神经功能损伤症状、脑组织梗死率以及病理组织学的改变。结果：模型组大鼠神经功能损伤严重,脑组织梗死率也明显增高（P〈0.01vs假手术组）。与依达拉奉的保护作用相同,TQ0701-2高中低三个剂量均能显著降低MCAO大鼠的神经功能评分和脑组织梗死率（P〈0.01vs模型组）,并且三个剂量的改善作用是随着浓度增大而增强的,具有剂量相关性。另外,TQ0701-2对大鼠脑缺血再灌注所致的神经元变性、坏死也有一定的保护作用。结论：研究表明,依达拉奉衍生物TQ0701-2对大鼠的脑缺血再灌注损伤有明显的神经保护作用。     

The Neuroprotection of puerarin against cerebral ischemia is associated with the prevention of apoptosis in rats

Previous work has shown that puerarin (Pur), extracted from the dried root of Pueraria lobata (Wild) Ohwi, increases cerebral blood flow in dogs and attenuates cerebral and spinal cord injury resulting from ischemia and reperfusion in rats and rabbits. The present study further demonstrates the neuroprotective effects of Pur on cerebral ischemic injury in rats and the mechanisms underlying the protective effects. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAo) for 50 min followed by reperfusion for 24 h. Pur (50, 100 mg/kg, i.p) was administered at the onset of MCAo. Twenty-four hours after reperfusion, neurological deficits were evaluated in Pur- and vehicle-treated rats. The infarct volume and edema ratios were assessed from stained brain slices. The results showed that Pur (100 mg/kg) markedly decreased the infarct volume by 34 % ( P < 0.01) in cerebral cortex and improved the neurological functions ( P < 0.05) after MCAo. Furthermore, flow cytometric analysis of annexin-V and PI labeling cells showed that the percentages of apoptosis and necrosis in the dorsolateral cortex were significantly reduced by 38.6 % and 28.5 % ( P < 0.01 and P < 0.05) following treatment with Pur (100 mg/kg) in MCAo rats. Caspase-3 activity, a biochemical marker of apoptosis, was significantly inhibited after treatment with Pur in the dorsolateral cortex. In agreement with this result, the expression of the X-chromosome-linked inhibitor of apoptosis protein (XIAP) was obviously up-regulated after administration of Pur (100 mg/kg), while caspase-3 gene was down-regulated in the dorsolateral cortex. These results suggest that the neuroprotection of puerarin against cerebral ischemia is associated with anti-apoptosis.     

In vitro and in vivo pharmacology of a structurally novel Na+-H+ exchange inhibitor, T-162559

1. We investigated the inhibitory effects of a non-acylguanidine Na(+)-H(+) exchange (NHE) inhibitor, T-162559 ((5E,7S)-[7-(5-fluoro-2-methylphenyl)-4-methyl-7,8-dihydro-5(6H)-quinolinylideneamino] guanidine dimethanesulphonate), on NHE-1, and its cardioprotective effect against ischaemia and reperfusion injury in rats and rabbits. 2. T-162559 inhibited human platelet NHE-1 in a concentration-dependent manner, with an IC(50) value of 13+/-3 nmol l(-1), making it 16 and three times more potent than cariporide IC(50): 209+/-75 nmol l(-1), P<0.01) and eniporide (IC(50): 40+/-11 nmol l(-1), P=0.066), respectively. T-162559 also inhibited rat NHE-1 with an IC(50) value of 14+/-2 nmol l(-1), which was five and three times lower than that of cariporide (IC(50): 75+/-7 nmol l(-1), P<0.01) and eniporide (IC(50): 44+/-2 nmol l(-1), P<0.01), respectively. 3. T-162559 inhibited, in a concentration-dependent manner, the reduction in cardiac contractility, progression of cardiac contracture, and increase in lactate dehydrogenase release after global ischaemia and reperfusion in perfused rat hearts. The inhibitory effects of T-162559 were observed at a lower concentration range (10 - 100 nmol l(-1)) than with cariporide and eniporide. T-162559 did not alter basal cardiac contractility or coronary flow after reperfusion, suggesting that it exerts direct cardioprotective effects on the heart. 4. Intravenous administration of T-162559 (0.03 and 0.1 mg kg(-1)) significantly inhibited the progression of myocardial infarction induced by left coronary artery occlusion and reperfusion in rabbits; the infarct size normalized by area at risk was 74+/-6% in the vehicle group, and 47+/-5% and 51+/-7% in the T-162559-0.03 mg kg(-1) and T-162559-0.1 mg kg(-1) groups (both P<0.05), respectively. 5. These results indicate that the new structural NHE-1 inhibitor T-162559 is more potent than cariporide and eniporide and possesses a cardioprotective effect against ischaemia and reperfusion injury in rat and rabbit models.     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

Production of leukotrienes in a model of focal cerebral ischaemia in the rat

1. The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery. 2. A significant (P < 0.01) elevation of LTC(4), LTD(4) and LTE(4) (cysteinyl-leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham-operated animals (3998 +/- 475 and 897 +/- 170 fmol g(-1) tissue, respectively, P < 0.01). 3. The NMDA receptor antagonist MK-801 and the adenosine A(2A) receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK-886. 4. MK-886 (0.3 and 2 mg kg(-1) i.v.) and MK-801 (3 mg kg(-1) i.p.) decreased cysteinyl-leukotriene levels (-78%, P < 0.05; -100%, P < 0.01; -92%, P < 0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg(-1) i.v.) had no significant effects. 5. MK-886 (2 mg kg(-1) i.v.) was also able to significantly reduce the cortical infarct size by 30% (P < 0.05). 6. We conclude that cysteinyl-leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.     

A new ergopeptide, CBM 36-733, improves ischaemic cerebral metabolism in spontaneously hypertensive rats.

The authors have examined the protective effects of CBM 36-733 (2-methyl-alpha-ergocryptine) on experimental cerebral ischaemia in spontaneously hypertensive rats (SHR). SHR aged 6 months were divided into four groups; a vehicle-treated group, and CBM 36-733 0.01, 0.1 or 1.0 mg/kg i.v. treated groups. After anaesthesia, the bilateral common carotid artery was ligated (BCL) and supratentorial cerebral ischaemia was produced for 1 h. Cerebral blood flow to the parietal cortex was repeatedly measured by the H2 clearance technique. Brain tissue lactate, adenosine triphosphate (ATP) and pyruvate were determined by enzymatic methods. By BCL, cerebral cortical blood flow decreased to 9-19% of the resting value at 30 min and further to 5-11% at 60 min. Blood flow reduction was not altered by CBM 36-733 administration. At 1 h ischaemia, brain tissue lactate greatly increased to 27.5 +/- 2.6 (mean +/- SEM) mmol/kg in the vehicle-treated SHR, while it showed less increase, to 7.5 +/- 1.4, in the CBM 36-733 0.1 mg/kg group. Brain ATP decreased to 1.31 +/- 0.05 in vehicle-treated SHR after 1 h BCL, but it changed little, retaining almost normal levels (2.60 +/- 0.19) in rats treated with 0.1 mg/kg CBM 36-733, followed by 1.0 and 0.01 mg/kg, suggesting a beneficial effect of CBM 36-733 on the ischaemic cerebral metabolism. The present results suggest that CBM 36-733 has a protective effect on the brain against ischaemia by improving cerebral metabolism while not affecting haemodynamics.     

Antagonistic effects of ultra-low-molecular-weight heparin against cerebral ischemia/reperfusion injury in rats.

Heparin and low-molecular-weight heparin have long been proposed for stroke treatment. This study was conducted to demonstrate the antagonistic effects of ultra-low-molecular-weight heparin (ULMWH) on cerebral ischemic injury in rats and the mechanisms underlying the effects. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. ULMWH (0.5, 1 mg kg(-1), i.v.) was administered after the MCAO and reperfusion. Twenty-four hours after the reperfusion, neurological deficit scores, body weight and infarct volume were assessed. Spectrophotometric assay was used to determine the activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) of the brain. Furthermore, the intracellular Ca(2+) concentration ([Ca(2+)]i) was measured. The results showed that vein injection of ULMWH at doses of 0.5 and 1.0 mg kg(-1) exerted significant neuroprotective effects on rats with focal cerebral ischemic injury via significantly decreasing neurological deficit scores, increasing body weight, reducing the infarct volume. At the same time, ULMWH significantly decreased MDA content, and increased SOD activity in ischemic brain. Compared with model group, ULMWH decreased the intracellular calcium concentration remarkably. All these findings suggest that ultra-low-molecular-weight heparin might act as a neuroprotective agent useful in the treatment in focal cerebral ischemia.