Background/Aims: The mechanisms of chemically induced injury are poorly understood. The purpose of this study was to develop a rapid method for isolation of gastric mucosal cells (GMCs) and sensitive tests for investigating cell damage and protection. Methods: GMCs were prepared from rat glandular stomach with pronase and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and kept in albumin-containing, albumin-free, or Hank's solutions. Initial cell viability and responsiveness were tested with trypan blue exclusion, and stimulation or inhibition of pepsinogen secretion were tested with carbachol, 16,16-dimethyl prostaglandin E2, or carbachol plus atropine. Plasma membrane damage was assessed by trypan blue exclusion, lactate dehydrogenase or cell protein leakage, mitochondrial integrity by succinic dehydroge nase activity, and nuclear lesions by ethidium bromide DNA fluorescence. Results: Incubation of GMC with ethanol resulted in 50% lethal dose for ethanol values of 12.3%, 7.4%, and 10.9% by trypan blue exclusion; 10.1%, 9.4%, and 9.1% by lactate dehydrogenase leakage; 12.8%, 10.0%, and 10.4% by succinic dehydrogenase; and 14.1%, 13.0%, and 11.5% by ethidium bromide DNA by binding using albumin-containing, albumin-free, and Hank's solutions, respectively. Conclusions: Rat GMC with high and long viability can be isolated by low concentrations of pronase and EGTA. The adapted biochemical and microscopic methods are suitable for parallel examination of chemical injury of plasma membrane, mitochondria, and nuclei. Albumin increased ethanol 50% effective concentration to GMC. A combination of biochemical and morphological methods with ethanol-induced injury to GMC is proposed for direct cytoprotection investigations. © 1994.
Identification of subcellular targets and sensitive tests of ethanol-induced damage in isolated rat gastric mucosal cells
PLEBANI, MARIO;
1994
Abstract
Background/Aims: The mechanisms of chemically induced injury are poorly understood. The purpose of this study was to develop a rapid method for isolation of gastric mucosal cells (GMCs) and sensitive tests for investigating cell damage and protection. Methods: GMCs were prepared from rat glandular stomach with pronase and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and kept in albumin-containing, albumin-free, or Hank's solutions. Initial cell viability and responsiveness were tested with trypan blue exclusion, and stimulation or inhibition of pepsinogen secretion were tested with carbachol, 16,16-dimethyl prostaglandin E2, or carbachol plus atropine. Plasma membrane damage was assessed by trypan blue exclusion, lactate dehydrogenase or cell protein leakage, mitochondrial integrity by succinic dehydroge nase activity, and nuclear lesions by ethidium bromide DNA fluorescence. Results: Incubation of GMC with ethanol resulted in 50% lethal dose for ethanol values of 12.3%, 7.4%, and 10.9% by trypan blue exclusion; 10.1%, 9.4%, and 9.1% by lactate dehydrogenase leakage; 12.8%, 10.0%, and 10.4% by succinic dehydrogenase; and 14.1%, 13.0%, and 11.5% by ethidium bromide DNA by binding using albumin-containing, albumin-free, and Hank's solutions, respectively. Conclusions: Rat GMC with high and long viability can be isolated by low concentrations of pronase and EGTA. The adapted biochemical and microscopic methods are suitable for parallel examination of chemical injury of plasma membrane, mitochondria, and nuclei. Albumin increased ethanol 50% effective concentration to GMC. A combination of biochemical and morphological methods with ethanol-induced injury to GMC is proposed for direct cytoprotection investigations. © 1994.Pubblicazioni consigliate
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