Isolation of rat pancreatic acini with crude collagenase and permeabilization of these acini with streptolysin O.
Rat pancreatic acini were prepared with collagenase A and collagenase P and their secretory response to carbachol tested. The collagenase P gave the best acinar suspension, providing that the digestion was performed in the absence of added calcium and in the presence of a low concentration of albumin. More than ninety percent of the acini prepared with this crude collagenase were viable as judged by a coloration with eosine. Their basal amylase secretion was below 3% amylase released within 20 minutes. They responded to cholecystokinin (7-fold stimulation, EC50: 10 pM), to bombesin (7-fold stimulation, EC50: 1 nM), to carbachol (7-fold stimulation, EC50: 1 microM), to fluoroaluminate (4-fold stimulation, EC50: 3 mM) or to TPA (4-fold stimulation, EC50: 100 nM). Acini preloaded with fura2 and incubated in the presence of 0.5 mM extracellular calcium were able to maintain a large (5,000-fold) gradient of calcium across their plasma membrane. Carbachol, CCK-8 and bombesin increased the intracellular calcium concentration 6-fold, within 10 seconds. This level decreased for the next 20 seconds but remained higher than the basal value for the next 10 minutes. These acini could be permeabilized with a low concentration (0.1 U/ml) of streptolysin O without affecting the basal amylase secretion, an index of the integrity of zymogen granules. It is concluded that pancreatic acini which have retained their responsiveness to major pancreatic secretagogues can be prepared with crude collagenase and permeabilized with streptolysin O. They can thus be used as a model for the study of stimulus-secretion coupling in exocrine glands.
Grosfils
K
K
Laboratoire de Biochimie générale et humaine, Institut de Pharmacie Université libre de Bruxelles, Brussels, Belgium.
Métioui
M
M
Tiouli
M
M
Dehaye
J P
JP
D000681:Amylases / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D017364:Collagenases / Q000494:pharmacology*; D003584:Cytological Techniques; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010539:Permeability / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013301:Streptolysins / Q000494:pharmacology*
D001426:Bacterial Proteins; D006150:Guanine Nucleotides; D011506:Proteins; D013301:Streptolysins; C025964:streptolysin O; D002217:Carbachol; D007770:L-Lactate Dehydrogenase; D000681:Amylases; D017364:Collagenases; D002118:Calcium