Influence of a calcium dependent protease inhibitor on platelet activation and secretion

Continuous proteolysis resulting in consumption of major cytoskeletal proteins may be essential for platelet activation and aggregation. In this study we evaluated the effect of a known protease inhibitor, Leupeptin, on agonist induced platelet aggregation and secretion. Platelets exposed to 10 ugs/ml of Leupeptin did not aggregate in response to the action of thrombin (0.2u/ml). However, a concentration of Leupeptin as high as 250 ugs/ml did not prevent arachidonate induced aggregation and secretion. Leupeptin (100 ugs/ml) effectively blocked thrombin (0.2 u/ml) induced elevation of cytosolic calcium, but did not affect arachidonate induced elevation of platelet intracellular calcium levels. At a concentration of 100 ug/ml, Leupeptin effectively blocked thrombin (0.5u/ml) induced clot formation of platelet poor plasma, suggesting that it can exert its effect on thrombin by preventing fibrinogen degradation. Effective K_i for the competitive inhibition of thrombin induced hydrolysis of a chromogenic substrate, S2238, by Leupeptin was 2.4 uM. Leupeptin inhibition of platelet function was reversible by washing platelets free of the polypeptide. Results of our study demonstrate that Leupeptin inhibits thrombin induced platelet activation, probably by interfering with its proteolytic activity on the platelet surface membrane. However, inhibition of platelet surface membrane associated proteases did not prevent activation of platelets by other agonists.     

L-NAME和Leupeptin对庆大霉素所致耳蜗毛细胞损伤协同保护作用的观察

目的观察一氧化氮合酶抑制剂[L-NG-硝基精氨酸甲酯(L-NG-n itroargin ine m ethylester,L-NAME)]和钙蛋白酶抑制剂[leupeptin]对庆大霉素(gentam ic in,GM)所致耳蜗毛细胞损伤的协同防护作用。方法将20只出生后3~5天的W istar大鼠(40耳)平均分成5组,1组为正常对照,其余4组在施加GM的同时单独或联合应用不同的保护因素(L-NAME和leupeptin)。通过耳蜗器官离体培养,对基底膜铺片行TR ITC-Phalloid in荧光染色,观察各组耳蜗毛细胞的生长情况并比较存活率。结果GM组毛细胞存活极少,L-NAME和leupeptin单独应用时毛细胞存活率明显高于GM组(P<0.01),二者联合应用时耳蜗毛细胞存活率显著高于L-NAME和leupeptin单独用药组(P<0.01)。结论L-NAME和leupeptin对庆大霉素耳毒性所致毛细胞损伤具有较好的协同防护作用。     

高浓度葡萄糖诱导红细胞磷脂酰丝氨酸暴露的机制研究

本研究探讨高浓度葡萄糖诱导红细胞磷脂酰丝氨酸（PS）暴露的机制。将红细胞于葡萄糖缓冲液中孵育后用流式细胞术分析PS标记率和前向角值；检测caspase-3和easpase-8的活化情况；用流式细胞术和荧光显微镜观察亮抑酶肽对葡萄糖引起的红细胞PS暴露的抑制效果。结果表明：葡萄糖可以诱导红细胞PS暴露，而且其效率和葡萄糖浓度密切相关。随着葡萄糖浓度的增加，红细胞的PS标记率呈逐步增加的趋势，当葡萄糖浓度为0．8mol／L时，红细胞的PS标记率在80％以上。然而，在葡萄糖引起的红细胞PS暴露过程中，caspase-3和caspase-8没有活化，而亮抑酶肽却可以很好地抑制红细胞PS暴露和使其体积缩小。随着亮抑酶肽浓度的增加，红细胞PS标记率呈逐步下降的趋势，而细胞体积却呈逐步增加的趋势。结论：高浓度葡萄糖可以导致严重的红细胞PS暴露，而且这种PS暴露和caspase无关，推测葡萄糖诱导的红细胞PS暴露是受一条对亮抑酶肽高度敏感的、未知的通路调控的．     

Differential Toxicity of Protease Inhibitors in Cultures of Cerebellar Granule Neurons

Involvement of proteases has been postulated in several neurodegenerative processes. Accordingly, protease inhibition has been proposed as a potential therapeutic tool to limit damage in some neuropathological states. The timed turn-over of proteins is, however, an essential biochemical process and its prolonged block may be dangerous to the cell. We report here data on toxicity consequent to 24-h exposure of cerebellar granule neurons in culture to inhibitors of different classes of proteases. Inhibition of calpains (calcium-activated cysteine proteases) resulted in dose-dependent neuronal death which largely occurred through apoptotic process. Leupeptin, an inhibitor acting on a broad spectrum of cellular serine proteases, was less toxic but resulted in definite morphological alteration of the cells. On the contrary, inhibitors of caspases, proteases belonging to the ICE (interleukin 1-β converting enzyme) family, did not apparently damage granule neurons upon exposure for 24 h to high concentrations (up to 200 μM) of two inhibitors specific for ICE (Ac-YAVD-CHO) and CPP-32 (Ac-DEVD-CHO), respectively. These results suggest that inhibition of proteases that are activated by stressful stimuli but are not essential for the normal functioning of healthy cells, as it is likely the case for caspases, may not be harmful to neurons. Instead, the potential risks and side effects of prolonged inhibition of proteases such as calpains, that regulate the disposal and the turn-over of key cellular proteins, should be carefully tested in the assessment of possible neuroprotective roles.     

Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.     

Olfactory discrimination learning is blocked by Leupeptin, a thiol protease inhibitor

Rats were trained on successive two-odor discriminations with the cues randomly located in an 8-arm radial maze. After several days of training using different odor pairs, the thiol protease inhibitor leupeptin was infused into the ventricles and testing continued. Leupeptin caused a pronounced, dose-dependent and reversible deficit in performance in this task. Previous studies have shown that these drug concentrations do not influence spontaneous activity, feeding and drinking, or the acquisition and retention of avoidance conditioning. The results are interpreted as supporting the hypothesis that a calcium-sensitive proteinase is involved in certain forms of memory that require modification of telencephalic circuitries.     

Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells

Dicoumarol, a competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), increases intracellular superoxide and affects cell growth of tumor cells. This work was set to establish a mechanistic link between dicoumarol, superoxide and cell cycle alterations in HL-60 cells. Using ES936, a mechanism-based irreversible inhibitor of NQO1, we demonstrate that NQO1 inhibition is not a major factor involved in superoxide boost. Mitochondrial Complexes II, III and IV were directly inhibited by dicoumarol. Succinate, which inhibits superoxide generation by reversed electron flow in Complex II, significantly decreased superoxide boost in dicoumarol-treated cells and in isolated mitochondria incubated with dicoumarol and decylubiquinol. Superoxide generation in cells was strongly potentiated by blocking the quinone site of Complex II with thenoyltrifluoroacetone, supporting the involvement of cytochrome b560 to drive electrons for increasing superoxide. Simultaneous inhibition of the mitochondrial chain upstream ubiquinone and displacement of succinate from the Complex II active site is proposed as a major mechanism to explain how dicoumarol increases superoxide in HL-60 cells. Dicoumarol-treated cells accumulated in S phase due to the impairment of pyrimidine biosynthesis at dihydroorotate dehydrogenase step because blockade was overcome by addition of exogenous uridine or orotate, but not by dihydroorotate. We demonstrate for the first time that dicoumarol inhibits mitochondrial electron transport, induces superoxide release by reversed electron flow in Complex II, and inhibits pyrimidines biosynthesis. These actions must be taken into account when considering dicoumarol effects on cells.     

Testing the “garbage” accumulation theory of ageing: mitotic activity protects cells from death induced by inhibition of autophagy

Imperfect autophagic degradation of oxidatively damaged macromolecules and organelles (so-called biological garbage) is considered an important contributor to ageing and consequent death of postmitotic (non-dividing) cells, such as neurons and cardiac myocytes. In contrast, proliferating cells apparently escape senescence by a continuous dilution and repair of damaged structures during division. Postmitotic ageing can be mimicked and studied in cultures of potentially dividing cells if their mitotic activity is inhibited. To test the garbage accumulation theory of ageing, we compared survival of density-dependent growth-arrested (confluent) and proliferating human fibroblasts and astrocytes following inhibition of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent fibroblast cultures to 3MA for two weeks resulted in a significantly increased proportion of dying cells compared to both untreated confluent cultures and dividing cells with 3MA-inhibited autophagy. Similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or leupeptin-exposed cultures, dying cells were overloaded with undegraded autofluorescent material. The results support a key role of biological lysosomal garbage accumulation in the triggering of ageing and death of postmitotic cells, as well as the anti-ageing role of cell division.