An embryonic chick pancreas organ culture model: characterization and neural control of exocrine release

An embryonic chick (Gallus domesticus) whole-organ pancreas culture system was developed for use as an in vitro model to study cholinergic regulation of exocrine pancreatic function. The culture system was examined for characteristic exocrine function and viability by measuring enzyme release, and noting histological, morphological, and anti-amylase immuno-fluorescence staining changes over a series of incubation times. This embryonic culture system exhibits loss of viability and morphological degeneration after 12 h of incubation time. Characterization and development of this exocrine model system was an important aspect of this study. Assessment of the 18-day-old embryonic chick pancreas model clearly indicated biochemical and cholinergic functionality, and morphological integrity, of the tissue after 4-h incubation. This embryonic age and incubation period were utilized for all subsequent cholinergic studies. The in vitro model was used to study parasympathetic regulation of exocrine function via the muscarinc receptors present in the embryonic chick pancreas. The effects of synthetic muscarinic agonists (bethanechol and carbachol) and subtype-specific antagonists affected amylase release to varying degrees suggesting heterogeneity of receptors. The effects of the muscarinic receptor antagonists atropine (non-specific), pirenzepine (M(1)-selective) and 4-DAMP [4-diphenylacetoxy-N-methyl-piperidine methiodide] (M(3)-selective) on bethanechol-stimulated amylase release were examined. Atropine and 4-DAMP at concentrations of 2 microM and higher significantly inhibited (p<0.05) agonist-stimulated amylase release, while pirenzipine did not at 2 microM, but did at 200 microM. The M(3) subtype selective antagonist 4-DAMP (2 pM-2 mM) significantly inhibited (p<0.05) 5 mM bethanechol-stimulated amylase release at concentrations of 2 microM and greater (amylase activity decreased from 100.61 to 49.41 U/l/mg). The data suggest the existence of a muscarinic receptor subtype for the embryonic chick pancreas exocrine cells characteristic to the mammalian M(3) glandular subtype.