sq-23377 and Ischemia

To investigate the dynamics of the intracellular Ca2+ concentration ([Ca2+]i) during retinal ischemia in rats with streptozotocin (STZ)-induced diabetes and the effect of gliclazide, a sulfonylurea with a potent free-radical scavenging activity on ischemia-induced [Ca2+]i dynamics.. Rats with STZ (65 mg/kg) induced diabetes were divided into three groups: the untreated diabetic group, the gliclazide-treated group, and the glibenclamide-treated group. An ischemic condition was imposed in vitro on the retinal slices by perfusion with an oxygen/glucose deprived solution. The [Ca2+]i was measured in individual layers of the rat retinal slices loaded with the Ca2+ indicator fluo-3.. As compared to that in the normal rat retina, both the amplitude and the kinetics of the [Ca2+]i increase were suppressed in the intermediate layers of the retinal slices from the diabetic rats under the ischemic condition. These changes were attenuated by the administration of gliclazide but not by that of glibenclamide.. Hyperglycemia influences ischemia-induced [Ca2+]i dynamics predominantly in the intermediate layers of the retina, and gliclazide, as compared to glibenclamide without a free radical scavenging activity, potently attenuates the ischemia-induced changes in the calcium dynamics.

Topics: Aniline Compounds; Animals; Calcium; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Gliclazide; Glyburide; Hypoglycemic Agents; Ionomycin; Ionophores; Ischemia; Microscopy, Confocal; Rats; Rats, Sprague-Dawley; Retina; Streptozocin; Xanthenes

The activity of cyclin-dependent kinase-5 (Cdk5) is tightly regulated by binding of its neuronal activators p35 and p39. Upon neurotoxic insults, p35 is cleaved to p25 by the Ca(2+)-dependent protease calpain. p25 is accumulated in ischemic brains and in brains of patients with Alzheimer's disease. p25 deregulates Cdk5 activity by causing prolonged activation and mislocalization of Cdk5. It is unknown whether p39, which is expressed throughout the adult rat brain, is cleaved by calpain, and whether this contributes to deregulation of Cdk5. Here, we show that calpain cleaved p39 in vitro, resulting in generation of a C-terminal p29 fragment. In vivo, p29 was generated in ischemic brain concomitant with increased calpain activity. In fresh brain lysates, generation of p29 was Ca(2+)-dependent, and calpain inhibitors abolished p29 production. The Ca(2+) ionophore ionomycin and the excitotoxin glutamate induced production of p29 in cultures of cortical neurons in a calpain-dependent manner. Like p25, p29 was more stable than p39 and caused redistribution of Cdk5 in cortical neurons. Our data suggest that neurotoxic insults lead to calpain-mediated conversion of p39 to p29, which might contribute to deregulation of Cdk5.

Topics: Animals; Blotting, Western; Brain; Calcium; Calpain; Carrier Proteins; COS Cells; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; DNA; Dose-Response Relationship, Drug; Glutamic Acid; Histidine; Humans; Ionomycin; Ionophores; Ischemia; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Plasmids; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Rats; Sequence Analysis, DNA; Time Factors; Transcription, Genetic; Transfection

Isolated hepatocytes, suspended in an organ preservation solution, can be preserved at 4 degrees C for up to 6 days. After preservation, normothermic-normoxic incubation causes loss of hepatocyte viability. The addition of 3 mmol/L glycine to the rewarming medium prevents the loss of viability. In this study we investigated the cytoprotective effects of glycine under many conditions known to cause hepatocellular injury to understand the mechanism of cold-induced injury in the liver. Hepatocytes were suspended in modified Krebs-Henseleit buffer with or without 3 mmol/L glycine and exposed to agents or conditions known to induce cell death. Hepatocyte viability was assessed by measuring the percentage of lactate dehydrogenase leakage from the cells and the concentration of ATP during incubation at 37 degrees C under room air for up to 90 min. Mitochondrial inhibitors (potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone); calcium ionophores (ionomycin and A23187); an oxidizing agent, tert-butyl hydroperoxide; and anoxia were all used to cause cell injury. Hepatocytes were also isolated from fasted rats and hypothermically preserved as another model of cell death. Other amino acids were also tested in the hypothermic preservation model to study the specificity of the amino acid requirement for prevention of lactate dehydrogenase leakage. Of the amino acids tested, only alanine (10 mmol/L) and the combination of alanine (3 mmol/L) and serine (3 mmol/L) were as effective as glycine in preventing lactate dehydrogenase release in the hypothermic preservation model.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cold Temperature; Glycine; Hypoxia; Ionomycin; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Mitochondria; Potassium Cyanide; Rats; Rats, Sprague-Dawley