Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule.

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

ATP depletion in rat cholangiocytes leads to marked internalization of membrane proteins.

Intrahepatic bile ducts (BD) are a critical target of injury in the postischemic liver. Decreased vascular perfusion causes characteristic changes in the morphology of the ductular epithelia including a loss of secondary membrane structures and a decrease in plasma membrane surface area. Using adenosine triphosphate (ATP) depletion of cultured normal rat cholangiocytes (NRC) to model ischemic ducts, the present studies examined the fate of apical membrane proteins to determine whether membrane recycling might contribute to rapid functional recovery. Apical proteins, including gamma-glutamyl transpeptidase (GGT), Na(+)-glucose cotransporter (SGLT1), and apically biotinylated proteins, were not shed into the luminal space during ATP depletion. Instead, labeling of surface proteins after ATP depletion showed a significant decrease in GGT and SGLT1, consistent with membrane internalization. Similarly, z-axis confocal microscopy of biotinylated apical proteins also showed protein internalization. During ATP recovery, SGLT1 transport activity remained profoundly depressed even after 24 hours of recovery, indicating that the function of the internalized apical proteins is not rapidly recovered. These studies suggest that the membrane internalization in ATP-depleted cholangiocytes is a unidirectional process that contributes to prolonged functional deficits after restoration of normal cellular ATP levels. This sustained decrease in transport capacity may contribute to the development of ductular injury in postischemic livers.

Glutathione status of isolated rat hepatocytes affects bile acid-induced cellular necrosis but not apoptosis.

An accumulation of hydrophobic bile acids is implicated in the pathogenesis of cholestatic liver diseases. In the present study, we determined if hydrophobic bile acid-induced cellular injury compromised hepatocyte glutathione (GSH) status, and if modulating intracellular GSH levels prevented or facilitated bile acid-induced cellular cytotoxicities. Freshly isolated rat hepatocytes incubated with >/=125 microM of the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), underwent a time- and dose-dependent decrease of intracellular GSH levels by 4-h incubation. This loss of intracellular GSH was not associated with an increase of intracellular GSH disulfide (GSSG). Rather, GCDC stimulated the dose-dependent accumulation of extracellular GSSG. The mechanism for extracellular GSSG accumulation by GCDC was through increased efflux of reduced GSH from hepatocytes into the media, where it subsequently oxidized to GSSG. Treatment of hepatocytes with GCDC (0-750 microM) did not directly alter GSH-dependent enzyme activities. The reduction of intracellular GSH with 125 microM GCDC correlated with extensive apoptosis at this concentration as determined by fluorescence microscopy of DAPI (4, 6-diamindino-2-phenylindole hydrochloride)-stained nuclei. Higher concentrations of GCDC (>/=500 microM) favored cellular necrosis and lipid peroxidation. Depleting GSH by treating hepatocytes with 1-bromoheptane increased their sensitivity toward GCDC-induced cellular necrosis, but not apoptosis. However, enhancing the hepatocyte GSH content by supplementation with GSH-ethylester (GSH-EE) failed to protect hepatocytes against either mode of cellular death. In conclusion, while GCDC-induced cytotoxicities were associated with an increased efflux of GSH from rat hepatocytes, GSH status modulated GCDC-induced necrosis, but not apoptosis.

Reorganization of cholangiocyte membrane domains represents an early event in rat liver ischemia.

Cholangiocytes contribute significantly to bile formation through the vectorial secretion of water and electrolytes and are a focal site of injury in a number of diseases including liver ischemia and post-transplantation liver failure. Using ischemia in intact liver and adenosine triphosphate (ATP) depletion in cultured cells to model cholangiocyte injury, these studies examined the effects of metabolic inhibition on cholangiocyte viability and structure. During 120 minutes of ischemia or ATP depletion, cell viability and tight junctional integrity in cholangiocytes were maintained. However, both the in vivo and in vitro models displayed striking alterations in the secondary structure of the plasma membrane. After 120 minutes, the basolateral (BL) interdigitations were diminished and the apical (Ap) microvilli were significantly decreased in number. The BL and Ap membrane surface areas decreased by 42 +/- 8% and 63 +/- 2%, respectively. Despite these changes, F-actin remained predominantly localized to the membrane domains. In contrast, in a time course that paralleled the loss of microvilli, the actin-membrane linking protein ezrin progressively dissociated from the cytoskeleton. These studies indicate that cholangiocyte ATP depletion induces characteristic, domain-specific changes in the plasma membrane and implicate alterations in the membrane-cytoskeletal interactions in the initiation of the changes. Pending the re-establishment of the differentiated domains, the loss of specific secondary structures may contribute to impaired vectorial bile duct secretion and postischemic cholestasis.

Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities.

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were > 99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

Apically and basolaterally internalized aminoglycosides colocalize in LLC-PK1 lysosomes and alter cell function.

Aminoglycosides bind to apical and basolateral (BL) membranes of renal epithelial cells. However, little is known regarding differential uptake and intracellular processing after internalization across these distinct surface membrane domains. To examine these processes independently, LLC-PK1 cells were grown on porous filters, which allow selective access to both domains. Apical and BL membrane uptakes of gentamicin (0.5 mM), quantified using [3H]gentamicin, were linear from 2 to 24 h (r = 0.99). The 4-h apical gentamicin uptake was 667 +/- 59 pmol/mg protein, the BL 748 +/- 26 pmol/mg protein, and concurrent apical and BL uptake 1,389 +/- 22 pmol/mg protein. Aminoglycoside uptake, documented using indirect immunogold techniques, occurred via the apical and BL endocytic systems and colocalized with cationic ferritin. Aminoglycosides internalized via the apical (gentamicin) and BL (tobramycin) membrane converged at the lysosomal level. Gentamicin incorporated via either domain significantly decreased lysosomal N-acetylglucosaminidase below control values (P < 0.05). We conclude that, after binding, aminoglycosides are internalized equally across apical and BL membranes of LLC-PK1 cells via receptor-mediated endocytosis, colocalize within the lysosomal compartment, and alter cellular function similarly.

Ischemia-induced loss of epithelial polarity. Role of the tight junction.

In proximal tubular cells ischemia is known to result in the redistribution of apical and basolateral domain-specific lipids and proteins into the alternate surface membrane domain. Since tight junctions are required for the maintenance of surface membrane polarity, the effect of ischemia on tight junction functional integrity was investigated. In vivo microperfusion of early loops of proximal tubules with ruthenium red (0.2%) in glutaraldehyde (2%) was used to gain selective access to and outline the apical surface membrane. Under control situations ruthenium red penetrated less than 10% of the tight junctions. After 5, 15, and 30 min of ischemia, however, there was a successive stepwise increase in tight junction penetration by ruthenium red to 29, 50, and 62%, respectively. This was associated with the rapid duration-dependent redistribution of basolateral membrane domain-specific lipids and NaK-ATPase into the apical membrane domain. Taken together, these data indicate that during ischemia proximal tubule tight junctions open, which in turn leads to the lateral intramembranous diffusion of membrane components into the alternate surface membrane domain.

The effects of magnesium on nucleoside phosphatase activity in frog skin.

Histochemical tests, employing the Wachstein-Meisel medium, indicate that nucleoside triphosphatase activity is found predominantly in two areas of the frog skin epidermis: (1) in mitochondria, where activity is enhanced by dinitrophenol, Mg(2+) dependent, but inhibited by fixation; and (2) apparently associated with cell membranes of the middle and outer portions of the epidermis, where activity is inhibited by Mg(2+), unaffected by dinitrophenol, and only slightly reduced by fixation. Spectrophotometric analysis shows that Mg(2+) in the medium does not increase spontaneous hydrolysis of ATP, thus obviating the possible explanation that changes in substrate concentrations in the medium lead to alterations in the "staining" distributions. It is postulated that perhaps the two enzymes differ in their requirements for substrate-one requiring the polyphosphate to be in complexed form with Mg(2+), the other uncomplexed. Concentrations of Mg(2+) required to inhibit cell membrane nucleoside triphosphatase activity also inhibit the electrical potential difference and short-circuit current of the frog skin. Although these observations might be taken as presumptive evidence of the cell membrane enzyme as a component of the ion pump system, because of certain dissimilarities with respect to the biochemists' "transport ATPase" and for other reasons discussed in the paper, any definite conclusions in this regard are premature.