cytochalasin-d and Hypoxia

The goal of this study was to determine whether anoxic membrane disruption is initiated by loss of cytoskeletal support in rabbit renal proximal tubules (PT). We specifically tested 1) whether cytoskeletal perturbation affects membrane integrity under normoxia, 2) whether cytoskeletal perturbation potentiates anoxic membrane damage, and 3) whether the membrane protection by glycine depends on cytoskeletal integrity. Cytoskeletal perturbation was achieved with 10 microM cytochalasin D (CD) because it selectively disturbs F-actin organization and has similar effects as anoxia on the cytoskeleton of PT. During normoxia, CD caused decreased basal F-actin content, microvillar breakdown, and membrane-cytoskeleton dissociation, as revealed by the use of laser tweezers. However, membrane integrity was not altered by CD, as monitored by lactate dehydrogenase release. CD pretreatment of PT did not potentiate anoxic membrane damage. Finally, plasma membrane protection by glycine during anoxia remained in CD-pretreated PT despite loss of cytoskeletal support. These results demonstrate that loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption.

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cytochalasin D; Cytoskeleton; Dextrans; Female; Glycine; Hypoxia; Kidney Tubules, Proximal; Rabbits

The effects of agents modulating the cytoskeleton, taxol (microtubuli stabilizing), vinblastine (microtubuli destabilizing) and cytochalasin D (actin destabilizing) (10(-6) M each) on enzyme and ATP release as well as on irreversible cell injury were investigated in isolated perfused hypoxic and reoxygenated rat hearts. Enzyme (creatine kinase (CK)) and ATP concentration were assayed in the interstitial transudate and venous effluent. Irreversible cell injury was determined from trypan blue uptake and nuclear staining (NS) of cardiomyocytes in histologic sections. ATP release from nonneuronal cells was only detectable in the interstitial transudate and was not significantly altered by the agents. In controls total CK release (about 4% of total CK) exceeded the percentage of irreversibly injured cells by a factor of 8. Taxol and cytochalasin D abolished the hypoxia/reoxygenation induced interstitial CK release and reduced total CK release to a highly significant extent. The percentage of irreversible injured cells was even more diminished by these agents resulting in a ratio of CK/NS of 40. The effect of cytochalasin D apparently is the consequence of decreased contractile performance as shown by analogous depression by butonedione monoxine (BDM), whereas contractile activity was not altered by taxol. Vinblastine had no influence on CK release but increased the number of irreversibly injured cells significantly. In conclusion, cytoskeletal elements apparently participate in the hypoxia/reoxygenation induced process of release of cytosolic enzymes (CK) and irreversible injury in a different way and extent. Taxol exhibits a cytoprotective effect in isolated perfused rat hearts as evaluated by the extent of enzyme release and irreversible cell injury.

Topics: Adenosine Triphosphate; Animals; Creatine Kinase; Cytochalasin D; Cytoskeleton; Female; Heart Rate; Hypoxia; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Paclitaxel; Rats; Rats, Sprague-Dawley; Systole; Vinblastine

Mouse proximal tubular (MPT) cells in culture were subjected to ATP depletion by incubating them with cyanide in the absence of dextrose for 1 h. This insult resulted in marked alterations in the actin cytoskeleton. These changes were not associated with a decrease in cell viability and thus reflected sublethal injury. The effect of sublethal injury on the functional integrity of the intercellular tight junction (TJ) was then examined in MPT cell monolayers grown on permeable supports. During chemical anoxia, monolayer permeability to the paracellular marker mannitol progressively increased to 297 +/- 62% of baseline after 1 h. Chemical anoxia also caused a reversible loss in cell-substrate adhesion when MPT cells were studied as confluent monolayers or as single cells. Thus disruption of the actin cytoskeleton in nonlethally injured cells results in important reversible alterations in renal epithelial function characterized by impairment of the "gate" function of the TJ as well as impaired cell-substrate adhesion. We hypothesize that sublethal epithelial cell injury without accompanying necrosis may contribute to the decrement in renal function characteristic of ischemic renal injury.

Topics: Actins; Adenosine Triphosphate; Animals; Cell Adhesion; Cell Survival; Cells, Cultured; Cyanides; Cytochalasin D; Cytoskeleton; Hypoxia; Intercellular Junctions; Kidney Tubules, Proximal; Mannitol; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Permeability

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.

Topics: Animals; Cell Membrane; Cytochalasin D; Cytochalasins; Cytoskeleton; Exocytosis; Hypoxia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Male; Microscopy, Electron, Scanning; Phalloidine; Proteins; Rats; Time Factors