phalloidine and Reperfusion-Injury

To ascertain the role of spermidine/spermine N-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyamine catabolism) in cell injury, cultured kidney (HEK 293) cells conditionally overexpressing SSAT were generated. The SSAT expression was induced and its enzymatic activity increased 24 h after addition of tetracycline and remained elevated over the length of the experiments. Induction of SSAT upregulated the expression of polyamine oxidase and resulted in the reduction of cellular concentration of spermidine and spermine, increased concentration of putrescine, and inhibited cell growth. SSAT overexpression increased the expression of heme oxygenase-1 (HO-1) by 350% 24 h after addition of tetracycline, indicating the induction of oxidative stress. The presence of catalase significantly prevented the upregulation of HO-1 in SSAT overexpressing cells, indicating that generation of H2O2 is partially responsible for the induction of oxidative stress. Overexpression of SSAT caused rounding and loss of cell anchorage and significantly altered the morphology of actin-containing filopodia, suggesting an adhesion defect. SSAT upregulation may mediate majority of the oxidative stress in kidney ischemia-reperfusion injury (IRI) as manifested by decreased cell growth, generation of toxic metabolites (H2O2 and putrescine), upregulation of HO-1, disruption of cell anchorage, and defect in cell adhesion. These data point to the inhibition of polyamine catabolism as a therapeutic approach for the prevention of tissue injury in kidney IRI.

Topics: Acetyltransferases; Actins; Animals; Blotting, Northern; Catalase; Cell Adhesion; Cell Division; Cell Line; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Kidney; Male; Membrane Proteins; Models, Biological; Oxidative Stress; Oxidoreductases Acting on CH-NH Group Donors; Phalloidine; Phenotype; Polyamine Oxidase; Polyamines; Protein Binding; Protein Synthesis Inhibitors; Putrescine; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA; Tetracycline; Time Factors; Transfection; Up-Regulation

Cell death and survival pathways are critical determinants of epithelial cell fate after ischemia. Forkhead proteins have been implicated in the regulation of cellular survival.. We have found that none of the forkhead family of proteins, FKHR, is phosphorylated after ischemia/reperfusion in the rat kidney. The time course of phosphorylation is similar to the time course of activation of the forkhead protein kinase Akt/protein kinase B (PKB), with maximal phosphorylation at 24 to 48 hours postreperfusion when the process of regeneration peaks. Extracellular signal-regulated kinase (ERK)1/2 activation has also been implicated as prosurvival in the injured kidney. ERK1/2 were phosphorylated in postischemic kidneys at 5, 30, and 90 minutes of reperfusion, with phosphorylation decreased by 24 and 48 hours. Immunocytochemical analysis revealed increased phospho-ERK1/2 in the thick ascending limb and isolated cells of the S3 segment, which have lost apical actin staining. To understand the relationship between forkhead phosphorylation, Akt, and ERK1/2, an in vitro model of injury was employed. After 40 minutes of chemical anoxia followed by dextrose addition for 20 minutes to replete adenosine triphosphate (ATP) levels, FKHR and FKHRL1 are phosphorylated. The levels of phospho-Akt are increased for at least 120 minutes after dextrose addition with a maximum at 20 minutes. Phosphorylation of Akt, FKHR, and FKHRL1 are phosphatidylinositol 3-kinase (PI 3-kinase) dependent since phosphorylation is reduced by the PI 3-kinase inhibitors, wortmannin, or LY294002. Inhibition of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK1/2), the upstream activator of ERK1/2, has no effect on forkhead protein phosphorylation after chemical anoxia/dextrose addition.. We conclude that PI 3-kinase and Akt are activated after renal ischemia/reperfusion and that Akt phosphorylation leads to phosphorylation of FKHR and FKHRL1, which may affect epithelial cell fate in acute renal failure.

Topics: Adenosine Triphosphate; Animals; Hypoxia; Immunohistochemistry; Kidney; LLC-PK1 Cells; Mitogen-Activated Protein Kinases; Phalloidine; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Swine

We studied and quantified the effect of ischemia-reperfusion on hepatic F-actin on bile canalicular and basolateral membranes in human liver allografts by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was normal in liver hepatocytes before reperfusion but decreased significantly after reperfusion (by 25% of controls). These results indicate that hepatic F-actin alteration is produced during the reperfusion phase. This modification, probably induced by reactive oxygen species, could impair bile canalicular contraction and tight junction permeability and consequently bile secretion in the postoperative period.

Topics: Actin Cytoskeleton; Actins; Bile Canaliculi; Biopsy; Cell Membrane; Coloring Agents; Fluorescein-5-isothiocyanate; Hepatocytes; Humans; Liver Transplantation; Microscopy, Confocal; Phalloidine; Reperfusion Injury

The purpose of this study was to examine the relationship of increased capillary network resistance due to leukocyte-capillary plugging and tissue edema through macromolecular leakage to tissue injury after ischemia-reperfusion (I/R). After a 3-h complete ischemia in the dorsal skinfold chamber of the awake Syrian hamster, the following parameters were measured: vessel diameter, macromolecular leakage, erythrocyte velocity, adherent leukocytes, rolling leukocytes, freely flowing leukocytes, functional capillary density (FCD), propidium iodide (PI)-positive cell nuclei, and increase in network flow resistance due to leukocyte-capillary plugging. These measurements were made under baseline conditions and after 0.5 and 2 h of reperfusion for I/R alone, I/R with phalloidin (PL) treatment (to block leakage), and I/R with both PL and cytochalasin D (CD) (to block both leakage and plugging). Neither treatment had an effect on the leukocyte adherence or rolling. PL treatment preserved the endothelial barrier, improved FCD, and reduced the amount of PI measured tissue damage. CD treatment eliminated the increase in network resistance due to leukocyte plugging but did not improve FCD or tissue damage. Thus, in this I/R model, macromolecular leakage plays a role in tissue injury, whereas leukocyte plugging does not appear to be an important mechanism.

Topics: Animals; Capillaries; Capillary Permeability; Cell Adhesion; Cricetinae; Cytochalasin D; Edema; Ischemia; Leukocytes; Macromolecular Substances; Male; Mesocricetus; Muscle, Skeletal; Phalloidine; Reperfusion Injury

The purpose of this study was to examine the relationship between increased capillary network resistance due to leukocyte capillary plugging and tissue injury following ischemia-reperfusion (I/R). After a 30-min complete ischemia in rat spinotrapezius muscle, the frequency and duration of leukocyte capillary plugging were measured throughout capillary networks and used to estimate the increase in network flow resistance for I/R alone, I/R with phalloidin (Pl), and I/R with both Pl and cytochalasin D. Propidium iodide (PI) was used to label nonviable muscle cell nuclei within the volume of tissue supplied by the capillary network, and counts were made before ischemia, immediately after reperfusion, and 1 h postreperfusion. For I/R alone and I/R + Pl there is a linear correlation between the increase in resistance (up to 29%) and the increase in the number of PI-positive nuclei during the reperfusion period. With both Pl and cytochalasin D present in the superfusate, the resistance increase was abolished and the amount of tissue damage during reperfusion was minimized. The results indicate that the increase in resistance is linearly related to the tissue damage and that a reduction of the leukocyte stiffness reduces the injury.

Topics: Animals; Capillaries; Coloring Agents; Cytochalasin D; Female; Ischemia; Leukocytes; Muscle, Skeletal; Phalloidine; Propidium; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion Injury; Vascular Resistance