ABSTRACT
Monolayer cultures derived from neonatal hamster or rat pancreas by two different epithelioid cell-enriching gravity sedimentation procedures varied in ability to maintain uniform levels of insulin secretion with increased culture age. Rat pancreatic cultures were superior in this respect to identically derived hamster preparations, depending on the preparative procedure employed. Quantitative differences in the temporal pattern of insulin secretion by different rat pancreatic culture preparations were ascribed to plating cell density and consequent terminal cell density as a function of preparative procedure such that reduced densities favored sustained secretory levels. These findings suggest the importance of tissue species and preparative procedure in deriving pancreatic monolayer cultures capable of sustained levels of insulin secretion with age.
Subject(s)
Glucagon/metabolism , Insulin/metabolism , Islets of Langerhans/cytology , Animals , Cell Count , Cell Survival , Cells, Cultured , Cricetinae , Insulin Secretion , Islets of Langerhans/metabolism , Kinetics , RatsABSTRACT
Exposure of hamster pancreatic islets to hyaluronidase during isolation by means of collagenase inhibits the insulinotropic action of several chemically different sulfonylureas, leucine, and glucagon without affecting glucose-stimulated insulin secretion. This inhibition is reversible for tolbutamide and leucine but irreversible for glucagon. Hyaluronidase inhibits reversibly the insulinotropic action of tolbutamide without affecting that of glucose also in mouse and rat isolated pancreatic islets . These findings suggest the existence of functionally related pancreatic beta cell receptors for tolbutamide and leucine different from those for glucose and glucagon and illustrate the potential usefulness of hyaluronidase as an enzymatic probe applicable toward investigating the cellular mechanism of action of key insulinotropic agents.
Subject(s)
Chlorpropamide/pharmacology , Glucagon/pharmacology , Glyburide/pharmacology , Hyaluronoglucosaminidase/pharmacology , Insulin/metabolism , Islets of Langerhans/metabolism , Leucine/pharmacology , Sulfonylurea Compounds/pharmacology , Tolbutamide/pharmacology , Animals , Cricetinae , Female , Glucose/pharmacology , Islets of Langerhans/drug effects , Mice , Microbial Collagenase/pharmacology , Rats , Species SpecificityABSTRACT
Cells derived by trypsinization of neonatal golden hamster pancreas were cultured in modified Eagle's medium for 120 h in the presence of glucose (0.8 mg/ml) and for an additional 48 h in medium containing glucose (0.8 or 3.1 mg/ml) or tolbutamide (1,000 microg/ml) plus glucose (0.8 mg/ml). At day 7, cultures were stained differentially for light microscopy or examined by electron microscopy. Immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) in the culture medium were measured by standard immunoassay procedures. Staining properties and ultrastructural appearance of cultured cells were comparable to those of the intact neonatal hamster pancreas. Cultures consisted predominantly of cells possessing aldehyde fuchsin positive (AF(+)) cytoplasmic granules resembling ultrastructurally those of the intact neonatal pancreatic beta cells and additionally, those of fibroblastoid, acinar, acino-insular, and aldehyde fuchsin negative (AF(-)) argyrophilic cells. IRI release rate by the cultured cells was increased in the presence of elevated glucose or tolbutamide which paralleled the loss of AF(+) granulation, but IRG release rate was suppressed by elevated glucose concentration. These findings indicate that these monolayer cultures consist of most of the cell types occurring in the neonatal pancreas, including endocrinologically competent islet cells.
