phalloidine and Hypoxia

A-kinase anchoring protein 5 (AKAP5) is involved in ventricular remodeling in rats with heart failure after myocardial infarction; however, the specific mechanism is not clear. This study investigated whether AKAP5 anchors calcineurin (CaN) to regulate the remodeling of H9c2 cardiomyocytes.. H9c2 cells were subjected to hypoxia stress for 3 h and reoxygenation for 24 h to create a hypoxia-reoxygenation (H/R) model. These cells were divided into three groups: H/R (model), empty vector +H/R (NC), and siRNA-AKAP5+H/R (siRNA-AKAP5) groups. The non-H/R H9c2 cells were used as normal controls. Western blotting was used to detect cardiac hypertrophy-related protein expression in the cells, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta myosin heavy chain (β-MHC), and phosphorylated nuclear factor of activated T-cell 3 (p-NFATc3). Phalloidin staining was used to label the cytoskeleton and the cell area in different groups was measured. Immunofluorescence staining and coimmunoprecipitation were used to study the relationship between AKAP5 and CaN. H9c2 cells pretreated with the CaN inhibitor FK506 were used to further verify the relationship between AKAP5 and CaN.. In the siRNA-AKAP5+H/R group, the expression level of cardiac hypertrophy-related proteins (ANP, BNP, and β-MHC) and CaN and the area of cardiomyocytes were significantly increased, while the p-NFATc3/NFATc3 ratio was decreased in H9c2H/R cells. AKAP5 and CaN proteins were colocalized and interacted in the cells. The CaN inhibitor significantly suppressed the expression of CaN, increased the p-NFATc3/NFATc3 ratio, and reduced the expression levels of ANP, BNP, and β-MHC proteins in the cells with low AKAP5 expression.. AKAP5 downregulation aggravated the remodeling of cardiomyocytes after H/R. AKAP5 may anchor CaN to form a complex, which in turn activates NFATc3 dephosphorylation and expression of hypertrophy-related proteins.

Topics: A Kinase Anchor Proteins; Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Hypoxia; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phalloidine; Rats; RNA, Small Interfering; Tacrolimus

In the present study, the association of endothelial nitric oxide synthase (eNOS) with the actin cytoskeleton in pulmonary artery endothelial cells (PAEC) was examined. We found that the protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAEC resulted in the coprecipitation of actin, and immunoprecipitation of actin from lysates of caveolar fractions resulted in the coprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS was labeled with fluorescein, F-actin was labeled with Texas red-phalloidin, and G-actin was labeled with deoxyribonuclease I conjugated with Texas red, also demonstrated an association between eNOS and F-actin or G-actin. Incubation of purified eNOS with purified F-actin and G-actin resulted in an increase in eNOS activity. The increase in eNOS activity caused by G-actin was much higher than that caused by F-actin. Incubation of PAEC with swinholide A, an actin filament disruptor, resulted in an increase in eNOS activity, eNOS protein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activity was higher than that in eNOS protein content in swinholide A-treated cells. In contrast, exposure of PAEC to phalloidin, an actin filament stabilizer, caused decreases in eNOS activity and association of eNOS with G-actin and increases in association of eNOS with F-actin. These results suggest that eNOS is associated with actin in PAEC and that actin and its polymerization state play an important role in the regulation of eNOS activity.

Topics: Actins; Animals; Caveolin 1; Caveolins; Cell Membrane; Cells, Cultured; Cytoskeleton; Endothelium, Vascular; Hypoxia; Marine Toxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Phalloidine; Swine

Hypoxia is a pathogenetic factor in various inner ear diseases, and increasing importance is attached to the protection of the cochlea from traumatic influences. It was recently demonstrated in guinea pigs that magnesium can significantly reduce ischemia- and impulse noise-induced hearing loss. The aim of this study was to evaluate if magnesium has a protective effect on hypoxia-induced hair cell loss using an in vitro model of the new-born rat cochlea In view of the NMDA receptor-antagonistic action of magnesium, we tested MK 801, a highly potent and selective non-competitive NMDA receptor antagonist. Organotypic cochlea cultures were exposed to hypoxia (pO2 = 10-20 mm Hg at 37 degrees C) in DMEM medium containing magnesium (0.75 or 3.0 mmol/l) or MK801 (1 or 10 micromol/l) for 24 or 36 h. The cultures were phalloidin-labeled for counting the number of outer and inner hair cells (OHC/IHC). The mean damage in normoxic controls was 1-4%. IHC revealed a significantly higher susceptibility to hypoxia than OHC. In the normal magnesium group (0.75 mmol/l), 36-hour exposure to hypoxia caused a mean loss of about 25% OHC and 60% IHC. In the groups treated with either 3.0 mmol magnesium or 10 microm MK 801, the damage was significantly reduced to about 10% in OHC and 35% in IHC. This study supports previous in vivo observations in the guinea pig demonstrating the protective effects of magnesium on noise-induced impairment of inner ear oxygenation.

Topics: Animals; Animals, Newborn; Cochlea; Dizocilpine Maleate; Hair Cells, Auditory, Inner; Hair Cells, Auditory, Outer; Hypoxia; Magnesium; Neuroprotective Agents; Oxygen; Phalloidine; Rats; Receptors, N-Methyl-D-Aspartate; Time Factors

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

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.

Topics: Actins; Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cytoskeleton; DNA; Female; Fluorescent Dyes; Hypoxia; Ionomycin; Kidney Cortex; Kidney Tubules, Proximal; Phalloidine; Rabbits; Rhodamines; Time Factors

Detachment of viable renal proximal tubular cells is seen in clinical and experimental acute tubular necrosis and may contribute to the acute renal dysfunction seen in acute tubular necrosis. Mechanisms of detachment of tubular cells are unknown but must involve changes in tubular cell adhesion. To begin to define mechanisms of altered cell adhesion, cultured human proximal tubular cells were made hypoxic by nitrogen gassing. Cells were monitored (blinded) for cell retraction and rounding over 90 minutes of N2. Hypoxia caused gradual alterations in cell shape, with 37.9% +/- 5.2% retracted-rounded cells by 90 minutes; control monolayers showed no significant change. Fluorescence confocal microscope imaging revealed that hypoxia caused displacement of actin filaments to basal margins of the retracted cells and produced a perinuclear aggregation of short filaments. Phalloidin (10(-6) mol/L), which stabilizes microfilaments and is able to penetrate these hypoxic cells, decreased the percentage of cells showing morphologic changes with hypoxia to < 5% by 90 minutes (p < 0.01). Viability, as assessed by Trypan blue dye exclusion, was well maintained (90% to 98% at 90 minutes) and did not correlate with shape changes. In separate experiments, cytochalasin (10(-6) mol/L)--which depolymerizes microfilaments--but not nocodazole--which disrupts microtubules--produced cell shape change in non-hypoxic monolayers. Disruption of microfilaments appears to play a role in loss of cell-to-cell and cell-to-substrate adhesion and loss of epithelial integrity in hypoxic injury to the renal tubule. These in vitro observations may be relevant to renal proximal tubular cell detachment in in vivo renal injury.

Topics: Actin Cytoskeleton; Cell Adhesion; Cytochalasins; Humans; Hypoxia; Kidney Tubules, Proximal; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Nocodazole; Phalloidine; Rhodamines

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