Complete dissociation of rat pancreas into acini by a perfusion- incubation method

Measurement of protein synthesis and the degradation of endogenous proteins

Agnes Telbisz, A. Kovács

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 Ca2+-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.

Original languageEnglish
Pages (from-to)342-348
Number of pages7
JournalPancreas.
Volume18
Issue number4
Publication statusPublished - May 1999

Fingerprint

Proteolysis
Pancreas
Perfusion
Suspensions
Desmosomes
Proteins
Tight Junctions
Valine
Collagenases
Lysosomes
Basement Membrane
Digestion
Buffers
Electrons
Insulin
Amino Acids
In Vitro Techniques
leupeptin

Keywords

  • Exocrine cells
  • Insulin
  • Isolation technique
  • Ultrastructure
  • Uniform suspension

ASJC Scopus subject areas

  • Gastroenterology
  • Endocrinology

Cite this

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abstract = "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 Ca2+-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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