| Abstrakt: |
Dispersed acini have proven to be particularly valuable in the study of pancreatic enzyme secretion. Complex time course studies or experiments requiring large numbers of replicates have proven difficult, however, with currently available techniques. Using a custom-designed incubation chamber, a miniaturized incubation method has been devised, which allows for continuous oxygenation of acini in 96-well microtiter plates and rapid separation of medium from acini by vacuum filtration. The filtrates from individual wells are collected into the wells of a second microtiter plate for pancreatic enzyme measurement. Using the above method, the dose response and time course of cholecystokinin (CCK-8)-stimulated amylase secretion was investigated. During a 1-h incubation, unstimulated amylase secretion was 4.1 ±0.3% of total acini content. Response to CCK was very sensitive, being detected at 10 M (p<0.05), half-maximal at 10 M CCK (14.0±0.6%, p<0.001) and maximal at 10M CCK (24.8 ± 1.0%, p<0.001). In the time course experiments, an increase in amylase secretion was detected by 2.5 min and continued to increase steadily to a plateau at 40 min, with both submaximal (10M) and maximal (10M) CCK concentrations. The potent and specific CCK-receptor antagonist, l-364,718, caused a dose-dependent decrease in CCK-stimulated amylase secretion, with a half-maximal effect at 10M. The receptor antagonist, l-364,718, at 10M completely abolished CCK-stimulated amylase secretion. This microtechnique provides a simple, reliable, and reproducible method for the study of dispersed pancreatic acini. With this new methodology, the simultaneous study of 400-500 replicates is possible from a single guinea pig pancreas. Cells can be readily harvested at the end of the incubation period for measurement of intracellular messengers. With this technique, it is possible to carry out complex time course or dose response studies that should prove useful in the study of acinar cell function. [ABSTRACT FROM AUTHOR] |
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