sq-23377 and Reperfusion-Injury

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca(2+)](i) by Ca(2+) ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7(-/-)) than in erythrocytes from wild type mice (anxA7(+/+)). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7(-/-) and anx7(+/+)-mice following increase of [Ca(2+)](i) by Ca(2+) ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7(-/-) than in anx7(+/+)-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7(-/-) than in anx7(+/+)-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.

Topics: Acute Kidney Injury; Animals; Annexin A5; Annexin A7; Cell Adhesion; Endothelial Cells; Erythrocytes; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Ionomycin; Mice; Mice, Knockout; Osmotic Pressure; Phosphatidylserines; Reperfusion Injury

We investigated the expression of Na(+)/Ca(2+) exchanger (NCX) and the functional role of NCX in retinal damage by using NCX1-heterozygous deficient mice (NCX1(+/-)) and SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline), a selective NCX inhibitor in vivo. We also examined the role of NCX in oxygen-glucose deprivation (OGD) stress with a retinal ganglion cell line (RGC-5) cell culture in vitro. The expression of NCX1 was confirmed and entirely localized in retina by immunoblotting and immunohistochemistry, respectively. NCX1(+/-) mice possessed significant protection against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA). SEA0400 at 3 and 10 mg/kg significantly reduced NMDA- or high intraocular pressure-induced retinal cell damage in mice. Furthermore, SEA0400 reduced the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells and the expression of phosphorylated mitogen-activated protein kinases (ERK1/2, JNK, p38) induced by NMDA injection. In RGC-5, SEA0400 at 0.3 and 1 microM significantly inhibited OGD-induced cell damage. OGD-induced cell damage was aggravated by ouabain (a Na(+),K(+)-ATPase inhibitor) at 100 microM, and this increased damage was significantly reduced by SEA0400 at 1 microM. In conclusion, these results suggest that NCX1 may play a role in retinal cell death induced by NMDA and ischemia-reperfusion.

Topics: Aniline Compounds; Animals; Cell Death; Cell Line; Enzyme Inhibitors; In Situ Nick-End Labeling; Intraocular Pressure; Ionomycin; Male; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; N-Methylaspartate; Neuroprotective Agents; Neurotoxins; Ouabain; Phenyl Ethers; Rats; Reperfusion Injury; Retina; Retinal Ganglion Cells; Retinal Neurons; Sodium-Calcium Exchanger; Stress, Physiological; Thapsigargin

Moderation of calcium perturbations has been implicated in ischemic preconditioning. As mitochondria possess an effective Ca(2+)transporting system driven by the mitochondrial membrane potential, experiments were performed to study time-averaged intracellular free calcium and the mitochondrial membrane potential during preconditioning and ischemia-reperfusion. Isolated rat hearts were subjected to 5 min of preconditioning, a 9-min intervening reperfusion and 21 min of ischemia with subsequent reperfusion. The hearts were preloaded with the Ca(2+)indicator Fura-2 or the mitochondrial membrane potential probe safranine. A method was devised for correction for NADH autofluorescence in time-averaged Ca(2+)probing with Fura-2. The pH dependence of the apparent dissociation constant of the Ca(2+)complex of Fura-2 was determined. Intracellular free Ca(2+)increased during the 5-min ischemia, and this was reversed upon reperfusion. During protracted ischemia a continual Ca(2+)rise was observed when the fluorescence data were corrected for changes in pH. An initial sharp Fura-2 fluorescence spike upon final reperfusion was caused by a pH-dependent change in the dissociation constant of the Ca(2+)complex of Fura-2. In preconditioned hearts the free Ca(2+)was somewhat lower during reperfusion, but a major effect of preconditioning was observed during the prolonged ischemia. The decrease in mitochondrial membrane potential during prolonged ischemia was faster in the preconditioned heart with no difference during the final reperfusion. The effect of preconditioning on cell survival was reflected in a decrease in the post-ischemic washout of creatine kinase. The moderation of the ischemic and post-ischemic intracellular Ca(2+)increase, and the acceleration of the ischemic mitochondrial membrane potential decrease by ischemic preconditioning is in accord with the notion of the involvement of mitochondrial ATP sensitive K(+)channels in preconditioning. In studies on ischemia it is absolutely necessary to correct for the pH-sensitivity of the apparent dissociation constant of the calcium complex of Fura-2 to obtain reliable data for intracellular free calcium.

Topics: Animals; Bradykinin; Calcium; Chelating Agents; Creatine Kinase; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Membranes; Ionomycin; Ionophores; Ischemic Preconditioning, Myocardial; Kinetics; Male; Membrane Potentials; Mitochondria; Myoglobin; NAD; Pericardium; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spectrometry, Fluorescence; Time Factors

Activated neutrophils cause conversion of xanthine dehydrogenase to its oxidase form (xanthine oxidase) in endothelial cells, the mechanism of which may be related to the cytotoxic effect of activated neutrophils. The elastase inhibitors, elastatinal, alpha 1-antitrypsin, and MeO-Suc-(Ala)2-Pro-Val-CH2Cl, significantly inhibited xanthine dehydrogenase to oxidase conversion by phorbol myristate acetate-stimulated neutrophils without inhibition of neutrophil adherence to the endothelial cell monolayer. The role of elastase in this enzyme conversion process was confirmed by the ability of purified elastase to cause conversion of xanthine dehydrogenase to xanthine oxidase in intact endothelial cells (or cell extracts) without causing cytotoxicity. In contrast, cathepsin G failed to cause conversion. The kinetics of conversion induced by elastase was relatively rapid, being essentially completed by 30 min. Upon removal of elastase, the effect was slowly (greater than 12 h) reversible and could be inhibited by cycloheximide treatment. Exposure of endothelial cells to hypoxia failed to enhance the elastase-induced conversion. Treatment of endothelial cells with Ca2+ ionophores failed to cause conversion of xanthine dehydrogenase to oxidase, suggesting that intracellular Ca(2+)-activated proteases are not sufficient to induce this process. Neutrophil-induced xanthine dehydrogenase to oxidase conversion was inhibited by concomitant treatment with antibodies to CD11b. The results suggest that activated neutrophils induce conversion of xanthine dehydrogenase to oxidase by secretion of elastase in close proximity to the endothelial cells and that this intimate contact between the two cell types enables high local concentrations of elastase to be attained, which are sufficient to cause xanthine dehydrogenase to xanthine oxidase conversion.

Topics: Animals; Calcimycin; Cell Cycle; Cycloheximide; Endothelium, Vascular; In Vitro Techniques; Ionomycin; Neutrophils; Oxygen; Pancreas; Pancreatic Elastase; Rats; Reperfusion Injury; Tetradecanoylphorbol Acetate; Xanthine Dehydrogenase; Xanthine Oxidase