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.

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.

Direct measurement of free Ca in organelles of gastric epithelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. G442-G451 ◽  
Author(s):  
A. M. Hofer ◽  
T. E. Machen
Keyword(s):  
Cell Types ◽  
Ruthenium Red ◽  
Acetoxymethyl Ester ◽  
Gastric Glands ◽  
Free Concentration ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mitochondrial Inhibitors ◽  
Carbonyl Cyanide ◽  
The Relationship

When loaded as the acetoxymethyl ester (AM) derivative, the fluorescent probe mag-fura 2 accumulates in both the cytoplasm and the subcellular compartments. The relatively high dissociation constant of this dye for Ca (53 microM) permits the measurement of changes in the free concentration of this ion in a variety of organelles where Ca concentration ([Ca]) is high. To characterize Ca stores in gastric cells, we used digitonin to permeabilize cells within isolated rabbit gastric glands loaded with mag-fura 2-AM. This allowed cytosolic dye to leak out, leaving only compartmentalized dye behind. It appears that mag-fura 2 monitors [Ca] changes in several ATP-dependent Ca-sequestering pools; an inositol 1,4,5-trisphosphate (IP3)-releasable and thapsigargin-sensitive store (which probably includes the endoplasmic reticulum), a pool that is released by the mitochondrial inhibitors oligomycin+azide, valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and ruthenium red (and therefore likely represents mitochondria), and a residual pool that was resistant to release by mitochondrial inhibitors and thapsigargin. Ca sequestration into all pools was sensitive to changes in [ATP], indicating that treatments that reduce cellular [ATP] will cause certain organelles to lose their Ca to the cytoplasm. Caffeine and ryanodine, which mobilize Ca from internal stores in many cell types, induced Ca sequestration into an IP3-insensitive store of gastric cells, and caffeine caused a reduction in cytoplasmic [Ca] (as measured with fura 2). We also show that the quantitation of free [Ca] in a given pool is complicated by a nonlinearity in the relationship between the mag-fura 2 ratio and [Ca]. This effect is likely a consequence of monitoring the fluorescence from multiple pools simultaneously. However, this limitation does not detract from the ability of this method to yield important qualitative information about the nature and number of Ca stores within single gastric cells.

scholarly journals Helicobacter pylori Infection of Human and Murine Primary Gastric Cells

2004 ◽  
Vol 72 (9) ◽  
pp. 5464-5469 ◽  
Author(s):  
Marguerite Clyne ◽  
Brendan Drumm
Keyword(s):  
Animal Model ◽  
Helicobacter Pylori ◽  
Helicobacter Pylori Infection ◽  
Gastric Mucosal Cells ◽  
Cell Lysates ◽  
Gastric Cells ◽  
Mucosal Cells ◽  
H Pylori

ABSTRACT The effect of Helicobacter pylori infection on human and murine primary gastric cells was determined. CagA was phosphorylated following adherence of H. pylori to primary human gastric cells. However, it did not adhere to human primary duodenal cells or murine gastric cells, and CagA could not be detected in cell lysates. Identification of an easily available animal model of infection in which the organism adheres to gastric mucosal cells would enhance studies of the virulence of H. pylori.

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.

Nitric oxide induces necrotic cell injury to cultured gastric mucosal cells and the cellular glutathione affect this injury

1998 ◽  
Vol 114 ◽  
pp. A77
Author(s):  
Y. Boku ◽  
Y. Naito ◽  
T. Yoshikawa ◽  
T. Fujii ◽  
Y. Masui ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Injury ◽  
Necrotic Cell ◽  
Gastric Mucosal Cells ◽  
Mucosal Cells

scholarly journals cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities.

1998 ◽  
Vol 9 (7) ◽  
pp. 1178-1186
Author(s):  
D Portilla ◽  
M D Crew ◽  
D Grant ◽  
G Serrero ◽  
L M Bates ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Injury ◽  
Expressed Sequence Tag ◽  
Expression System ◽  
Consensus Sequence ◽  
Cloning And Expression ◽  
Northern Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Early Event

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.

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