Effect of extracellular Ca2+ on indomethacin-induced injury to rabbit dispersed gastric mucosal cells

1994 ◽  
Vol 72 (1) ◽  
pp. 63-69 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Incubation Medium ◽  
Trypan Blue ◽  
Cell Injury ◽  
Cell Damage ◽  
Cellular Damage ◽  
Blue Dye ◽  
Ionophore A23187 ◽  
Dye Uptake ◽  
Gastric Mucosal Cells ◽  
Mucosal Cells

The effects of extracellular Ca2+ on indomethacin-induced disruption to rabbit dispersed gastric mucosal cells have been examined. Fundic mucosal cells were isolated, and cellular viability and disruption were assessed by the release of the lysosomal enzyme acid phosphatase and by trypan blue dye exclusion. Addition of the Ca2+ ionophore A23187 (12.5 and 25 μM) and indomethacin (0.1–10 μM) to the incubation medium induced concentration-dependent increases in enzyme marker release and trypan blue dye uptake in the cells. The resultant cellular disruption in response to incubation of cells with combinations of indomethacin and A23187 was not significantly different from that observed when the agents were administered separately. The degree of cell injury was reduced by removal of Ca2+ from the incubation medium. Similarly preincubation with the Ca2+ channel antagonist verapamil (1 μM) reduced A23187- and indomethacin-induced cell injury. Both A23187 (25 μM) and indomethacin (10 μM) treatments increased cytosolic Ca2+ concentration ([Ca2+]i). Pretreatment of cells with 16,16-dimethylprostaglandin E2 (0.1–10 μM) reduced both indomethacin (10 μM) induced cellular damage and the elevation in [Ca2+]i. These data suggest that indomethacin-induced disruption of gastric mucosal cells is dependent to some extent upon extracellular Ca2+. An inappropriate Ca2+ flux may contribute to indomethacin-induced damage to gastric mucosal cells.Key words: ionophore A23187, indomethacin, prostaglandin, calcium, cell damage.

Effects of calcium-modifying agents on integrity of rabbit isolated gastric mucosal cells

1991 ◽  
Vol 261 (1) ◽  
pp. G119-G127 ◽  
Author(s):  
B. L. Tepperman ◽  
S. Y. Tan ◽  
B. J. Whittle
Keyword(s):  
Cell Damage ◽  
Bay K 8644 ◽  
Blue Dye ◽  
Mucosal Cell ◽  
Ionophore A23187 ◽  
Enzyme Release ◽  
Cellular Viability ◽  
Cell Integrity ◽  
Gastric Mucosal Cells ◽  
Mucosal Cells

The effects of Ca(2+)-modifying agents on the disruption of gastric isolated mucosal cells from the rabbit have been examined. Fundic mucosal cells were isolated and enriched by centrifugal elutriation, with cellular viability and disruption being assessed by trypan blue dye exclusion and by the release of lysosomal enzymes. The Ca2+ ionophore A23187 (3.125-50 microM) induced a concentration-dependent increase in enzyme marker release and decreased dye exclusion from the cells. Ionophore-induced enzyme release was reduced by removal of Ca2+ or by incubation with EDTA. Cells from the medium-sized fraction were found to be more sensitive to damage by A23187 than were larger-diameter cells. Enzyme release from these cells was also induced by the Ca(2+)-channel activator BAY K 8644 (1.5 microM). The Ca(2+)-channel antagonists, nifedipine, verapamil, and diltiazem (1 microM), abolished enzyme release in response to BAY K 8644 (1.5 microM) but did not affect A23187 (25 microM)-induced responses. Ethanol (5 and 8%) also induced a concentration-dependent increase in enzyme release and decrease in dye exclusion, but this effect was not dependent on the external Ca2+ concentration. However, threshold concentrations of A23187 (1.56 microM) substantially potentiated the cell damage induced by ethanol (5 or 8%), and this synergism was dependent on external Ca2+. These data suggest that agents that produce an inappropriate Ca2+ flux can disrupt or augment disruption of gastric mucosal cells and thus Ca2+ homeostasis is essential for maintenance of mucosal cell integrity.

Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells

1995 ◽  
Vol 269 (2) ◽  
pp. G287-G296 ◽  
Author(s):  
A. J. Dziki ◽  
S. Batzri ◽  
J. W. Harmon ◽  
M. Molloy
Keyword(s):  
Cell Death ◽  
Bile Salt ◽  
Cell Injury ◽  
Early Event ◽  
Acetoxymethyl Ester ◽  
Gastric Mucosal Cells ◽  
Maximal Increase ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mucosal Cells

Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

In Vitro ◽  
1981 ◽  
Vol 17 (12) ◽  
pp. 1100-1110 ◽  
Author(s):  
Hugo O. Jauregui ◽  
Nancy T. Hayner ◽  
James L. Driscoll ◽  
Rhonda Williams-Holland ◽  
Milton H. Lipsky ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Rat Hepatocytes ◽  
Blue Dye ◽  
Dye Uptake ◽  
Isolated Cells ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Adult Rat Hepatocytes ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures

Investigation of gastroprotective compounds at subcellular level in isolated gastric mucosal cells

2000 ◽  
Vol 279 (6) ◽  
pp. G1201-G1208 ◽  
Author(s):  
Lajos Nagy ◽  
Romeo E. Morales ◽  
Martin Beinborn ◽  
Peter Vattay ◽  
Sandor Szabo
Keyword(s):  
Plasma Membrane ◽  
Cell Injury ◽  
Succinic Dehydrogenase ◽  
Gastric Mucosal Cells ◽  
Trypan Blue Exclusion ◽  
Pepsinogen Secretion ◽  
Mucosal Cells ◽  
Nuclear Damage

We tested the hypothesis that recognized gastroprotective agents exert direct protection against ethanol-induced injury in isolated rat gastric mucosal cells in vitro. If protection exists, we also wanted to identify subcellular targets in the reversible and/or irreversible stages of cell injury. Ethanol-induced cell injury was quantified by measuring plasma membrane leakage (trypan blue exclusion and lactate dehydrogenase release), mitochondrial integrity (succinic dehydrogenase), and nuclear damage (ethidium bromide-DNA fluorescence). Initial cell viability and responsiveness were estimated by the effects of carbachol, carbachol + atropine, or 16,16-dimethyl-PGE2on chief cell pepsinogen secretion. Enriched parietal cells were stimulated by histamine, carbachol, or histamine + IBMX. Preincubation of cells with PG, sucrose octasulfate, or the sulfhydryl compounds N-acetylcysteine, taurine, or cysteamine increased cell resistance ≤21% against ethanol. Similar protection was found with low histamine concentrations, but a higher concentration aggravated ethanol toxicity. Other naturally occurring or synthetic gastroprotective agents offered partial protection or aggravated ethanol-induced cell injury. Only a few in vivo gastroprotective agents demonstrated in vitro direct cytoprotection, which involved mainly the reversible stage of cell injury (e.g., plasma membrane changes) and, less often, irreversible (e.g., mitochondrial and nuclear) damage. Our findings also indicate that a major part of the beneficial effect of gastroprotective agents is expressed at the tissue level.

Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening

2004 ◽  
Vol 97 (1) ◽  
pp. 269-276 ◽  
Author(s):  
Sarina S. Kay ◽  
Anastacia M. Bilek ◽  
Kay C. Dee ◽  
Donald P. Gaver
Keyword(s):  
Epithelial Cell ◽  
Pressure Gradient ◽  
Stimulus Duration ◽  
Cell Injury ◽  
Cell Layer ◽  
Cell Damage ◽  
Flow Chamber ◽  
Cellular Damage ◽  
Airway Reopening ◽  
Epithelial Cell Layer

The reduction of tidal volume during mechanical ventilation has been shown to reduce mortality of patients with acute respiratory distress syndrome, but epithelial cell injury can still result from mechanical stresses imposed by the opening of occluded airways. To study these stresses, a fluid-filled parallel-plate flow chamber lined with epithelial cells was used as an idealized model of an occluded airway. Airway reopening was modeled by the progression of a semi-infinite bubble of air through the length of the channel, which cleared the fluid. In our laboratory’s prior study, the magnitude of the pressure gradient near the bubble tip was directly correlated to the epithelial cell layer damage (Bilek AM, Dee KC, and Gaver DP III. J Appl Physiol 94: 770–783, 2003). However, in that study, it was not possible to discriminate the stress magnitude from the stimulus duration because the bubble propagation velocity varied between experiments. In the present study, the stress magnitude is modified by varying the viscosity of the occlusion fluid while fixing the reopening velocity across experiments. This approach causes the stimulus duration to be inversely related to the magnitude of the pressure gradient. Nevertheless, cell damage remains directly correlated with the pressure gradient, not the duration of stress exposure. The present study thus provides additional evidence that the magnitude of the pressure gradient induces cellular damage in this model of airway reopening. We explore the mechanism for acute damage and also demonstrate that repeated reopening and closure is shown to damage the epithelial cell layer, even under conditions that would not lead to extensive damage from a single reopening event.

Reduced glutathione modulates Ca(2+)-mediated damage to rabbit isolated gastric mucosal cells

1994 ◽  
Vol 267 (1) ◽  
pp. G1-G9 ◽  
Author(s):  
H. M. Wong ◽  
B. L. Tepperman
Keyword(s):  
Cell Injury ◽  
Reduced Glutathione ◽  
Cyclopiazonic Acid ◽  
Blue Dye ◽  
Disruptive Effect ◽  
Oxyntic Mucosa ◽  
A 23187 ◽  
Mucosal Cells ◽  
Exogenous Addition ◽  
Mucosa Cell

In the present study, we have examined the role of reduced glutathione (GSH) in the modulation of Ca2+ ionophore A-23187-induced gastric mucosal cellular disruption. Experiments were conducted using cells isolated from rabbit oxyntic mucosa. Cell injury was assessed by release of the lysosomal enzyme acid phosphatase, by trypan blue dye uptake, and by examination of lipid peroxidation. Cellular GSH was assessed spectrophotometrically by measuring total soluble reduced thiol content. Ionophore A-23187 (3-25 microM) induced a concentration-dependent injury to mucosal cells and a concentration-dependent reduction in cellular GSH content. Removal of Ca2+ from the incubation medium abolished both the disruptive action and the GSH-lowering effect of A-23187. Gastric cellular GSH content was reduced significantly by preincubation of cells with an agent that depletes reduced sulfhydryls, diethyl maleate (DEM; 1 mM). Furthermore, DEM significantly augmented cellular disruption in response to A-23187. Similarly, incubation of cells with L-cysteine (1 mM), a stimulant of glutathione synthesis, increased mucosal cellular GSH content and decreased the cellular disruptive effect of A-23187. Exogenous addition of GSH to the cell suspension significantly reduced Ca2+ ionophore-induced cellular disruption and hastened the recovery of cellular free Ca2+ concentration ([Ca2+]) to baseline values. Similarly, exogenous GSH reduced cellular disruption produced by cyclopiazonic acid and thapsigargin, agents that increase intracellular [Ca2+] ([Ca2+]i) by inhibiting Ca2+ sequestration. These data suggest that a sustained increase in [Ca2+]i contributes to the pathogenesis of gastric mucosal cellular injury. This effect appears to be mediated by a reduction in the cellular content of GSH.

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