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Biomedical subjects

M E Walter

Publications and source records attributed to M E Walter.

6 recordsLinked to original sources
PubMed1995

Effect of neutrophil mediators on epithelial permeability.

Inflammatory lung disease is associated with increased epithelial permeability, but it is unclear how inflammatory cells alter epithelial permeability. Neutrophils have azurophilic granules containing elastase, cathepsin G, and defensins which are released at sites of inflammation. Experiments using whole animals and cultured cells suggest that neutrophil elastase contributes to increased epithelial permeability. Using Madin-Darby canine kidney epithelial (MDCK) monolayers, a well-described epithelial model, we asked whether neutrophil elastase directly affects epithelial permeability independent of cell death or cell detachment from the substratum. We measured permeability using 3H-mannitol. We found that neutrophil elastase increased epithelial permeability in a time- and concentration-dependent fashion. Increased permeability required prolonged (> or = 6 h) exposure to elastase, but was not associated with cytolytic injury or cell detachment. These findings are potentially relevant to the lung because we found a similar time- and concentration-dependent effect when we added elastase to cultured human bronchial epithelial cells. In MDCK cells, permeability increased without alterations in cell actin at the light microscopic level. Interestingly, elastase-induced permeability was both prevented and reversed by serum, but not by serum albumin. Complete reversal occurred if serum was added up to 16 h after adding elastase. Proteolytic activity is important in HNE-induced epithelial permeability because soy bean trypsin inhibitor completely blocks the effect and alpha 1 proteinase inhibitor (alpha 1 PI) partially blocks the effect. Charge interactions also appear to be important because the polyanions heparin and sulfated dextran completely blocked increased permeability following elastase but only partially blocked elastolytic activity in isotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Thermal stress induces epithelial permeability.

The mechanisms by which heat injury results in multiorgan system failure are unknown, but the presence of endotoxemia and intestinal hemorrhage suggests that changes in gut epithelial permeability may be crucial to this process. To determine whether alterations in epithelial permeability occur at physiologically relevant temperatures, heat-induced changes on epithelial barrier integrity were studied using a high-resistance clone of Madin-Darby canine kidney epithelial cells. Transepithelial electrical conductance increased when monolayers were heated above 38.3 degrees C. Early changes in conductance were completely reversible with cooling. Increased conductance was due to increased paracellular permeability because heat also induced increased mannitol permeability across the monolayers. A conditioning heat stress (42 degrees C for 90 min) altered heat-induced permeability. When cell monolayers were exposed to this conditioning stress 48 h before measurement of conductance with increasing temperatures, the conductance increase did not occur until they were heated to 39.4 degrees C compared with 38.8 degrees C in naive control cells. This conditioning treatment also conferred thermotolerance as measured by cell survival after a lethal 45.0 degrees C heat stress. There was no difference in the temperature at which conductance increased between preheated and control cells 96 h after a preconditioning heat stress. The conditioning heat stress resulted in accumulation of heat-shock protein (HSP) 70 in cells at 48 h, but HSP 70 returned to control levels at 96 h. These studies demonstrate that small temperature elevations increase epithelial permeability and that prior heat stress which induces HSP 70 shifts the threshold temperature required to disrupt the epithelium.

Adaptation, Physiological↗
PubMed1994

Betamethasone activation of CTP:cholinephosphate cytidylyltransferase in vivo is lipid dependent.

Glucocorticoids increase surfactant phosphatidylcholine synthesis, in part, by stimulating the rate regulatory enzyme CTP:cholinephosphate cytidylyltransferase. This enzyme exists in mammalian lung cytosol as an active lipoprotein form (H-form) and an inactive apoprotein (L-form) species. We administered betamethasone to pregnant rats to examine the mechanisms for glucocorticoid stimulation of cytidylyltransferase activity in fetal lung. The hormone stimulated cytosolic activity threefold, and this effect was nearly abolished after lipid extraction. The addition of lipid extracts isolated from betamethasone-treated cytosolic preparations to L-form species increased enzyme activity to a greater extent than lipid extracts from control lungs. Further, the glucocorticoid increased the proportion of H-form activity from 34 to 55% of the total activity in the fetal lung cytosol. These changes were associated with a marked decrease in the activity of the L-form species. Analysis of the lipid composition of the H-form revealed that betamethasone increased the content of lipid activators, including phosphatidylglycerol and fatty acids. These observations provide evidence that glucocorticoid stimulation of CTP:cholinephosphate cytidylyltransferase in vivo is mediated by a conversion of the inactive form (L-form) to the active species (H-form). These studies further emphasize the critical role of lung lipids in mediating the glucocorticoid activation of this enzyme.

Animals↗
PubMed1993

Asbestos directly increases lung epithelial permeability.

Asbestos causes the fibrotic lung disease asbestosis, but the biologic basis for this is unknown. Lung epithelial dysfunction including increased permeability is hypothesized to contribute to lung scarring in other forms of pulmonary fibrosis. Lung epithelial permeability is increased in both animals and humans exposed to asbestos. It is not known whether the increased epithelial permeability results from direct effects of asbestos or occurs as a result of the inflammatory reaction to asbestos fibers. To address this question we used a cultured human lung epithelial model, and we measured the direct effect of asbestos on lung epithelial barrier integrity as measured by mannitol permeability. We exposed the monolayer to chryogenically ground, respirable-sized chrysotile asbestos particles. This chrysotile asbestos caused a dose- and time-dependent increase in mannitol permeability across the epithelial monolayer. Increased mannitol permeability occurred both in the presence and in the absence of serum, was not due to cytotoxicity as measured by lactate dehydrogenase release, and was not associated with altered actin cytoskeleton at the light microscopic level. Permeability to 70 kDa neutral dextran also increased after asbestos exposure; however, the absolute permeability to dextran was less than mannitol permeability. Neither latex beads nor tantalum caused any change in permeability, suggesting that our findings are not explained by nonspecific effects of particles. Increased permeability did not reverse in the continued presence of asbestos and persisted even after removing the asbestos. Finally, surface-bound iron did not appear to be necessary for this effect because neither chelating iron with deferoxamine nor iron-loading the asbestos altered the effect on mannitol permeability. These results show that asbestos has direct effects on lung epithelial permeability. Together with the recent observation that asbestos directly increases epithelial fibrinolytic activity, our results suggest a novel mechanism for asbestos-induced lung injury.

Asbestos↗
PubMed1992

Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells.

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.

Animals↗