Effects of collagenase on the release of [³H]‐noradrenaline from bovine cultured adrenal chromaffin cells

Abstract

Bovine isolated adrenal chromaffin cells maintained in culture at 37°C for 1–7 days become polygonal and bipolar, with typical varicosity‐like extensions. Catecholamine levels and dopamine β‐hydroxylase activity decreased after 24–48 h of culture, but recovered to normal levels 3–7 days later. Incubation of 1–7 day‐old cells in the presence of increasing concentrations of [3H]‐noradrenaline (3.91 to 125 nm) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day‐old cells took up and retained only one third of the radioactivity found in 2–7 day‐old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mm K+ was very poor in 24 h‐old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2–7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatmen. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor‐mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage‐sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.

A simple and rapid HPLC–MS method for the simultaneous determination of epinephrine, norepinephrine, dopamine and 5-hydroxytryptamine: Application to the secretion of bovine chromaffin cell cultures

“…Chromaffin cells were isolated from these adrenal glands following standard protocols [16,17]. In short, adrenal glands were digested through perfusion with a solution of 0.25% collagenase, 0.01% DNAase and 0.5% bovine serum albumin.…”

Human nicotinic receptors in chromaffin cells: characterization and pharmacology

“…Adrenal glands were then placed into a preservation liquid and transported to our laboratory, where we performed the isolation and culture of the chromaffin cells (29, 19). Experiments to record nAChR currents were started 24–48 h after platting the cells to allow recovery from enzyme digestion (2). …”

Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells

“…Isolation and culture of bovine chromaffin cells Chromaffin cells were prepared from bovine adrenal glands by collagenase digestion and further separated from debris and erythrocytes by centrifugation on Percoll gradients as described (Almazan et al, 1984;Gil et al, 1998). Cells were maintained in monolayer cultures using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 10 µM cytosine arabinoside, 10 µM 5-fluoro-2′-deoxyuridine, 50 IU/ml penicillin and 50 µg/ml streptomycin and were harvested in 35 mm Petri dishes (500,000 cells/dish, Corning, NY, USA).…”