Complete dissociation of rat pancreas into acini by a perfusion-incubation method: measurement of protein synthesis and the degradation of endogenous proteins.
A new method has been developed for the dissociation of rat pancreas into acinar suspension by introducing an in situ flow-through perfusion with a Ca(2+)-chelating buffer before in vitro enzymatic digestion with collagenase. As a result of the perfusion step and other minor modifications, a high-quality and uniform suspension of acini and small acinar complexes is obtained that offers the possibility to work with 50 parallel samples from a single rat for protein synthesis and degradation measurements. Initial cellular viability is 95-99% and remains > 85% even after 6 h in vitro. Electron-microscopic observations show that the cells in isolated acini retain their polarity, tight junctions remain intact, and desmosomes and basement membranes disappear. Protein synthesis shows strong dependence on the extracellular supply of amino acids and 30-50% stimulation by insulin. With the help of the new system of isolated acini, data on the degradation of intracellular proteins of the exocrine pancreatic tissue is presented for the first time in the literature. After a 2-h labeling period with [14C]valine, 2-4% of the radioactive protein is degraded per hour. Of this protein breakdown, 30-40% appears to take place in lysosomes, as measured in the presence of leupeptin, an inhibitor of lysosomal degradation.
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Department of General Zoology, Eotvos Lorand University, Budapest, Hungary. kosbor@cerberus.elte.hu
Kovacs
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D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002118:Calcium; D002614:Chelating Agents; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D010477:Perfusion; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans
D000596:Amino Acids; D002021:Buffers; D002614:Chelating Agents; D007328:Insulin; D011506:Proteins; D002118:Calcium