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Effects of collagenase on the release of [ 3 H]‐noradrenaline from bovine cultured adrenal chromaffin cells
Author(s) -
Almazan G.,
Aunis D.,
García A.G.,
Montiel C.,
Nicolás G.P.,
SánchezGarcía P.
Publication year - 1984
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1984.tb16124.x
Subject(s) - collagenase , nicotine , endocrinology , medicine , veratridine , acetylcholine , adrenal medulla , catecholamine , chromaffin cell , chemistry , cell culture , microbial collagenase , tritium , biology , enzyme , sodium , biochemistry , genetics , physics , organic chemistry , sodium channel , nuclear physics
1 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. 2 Incubation of 1–7 day‐old cells in the presence of increasing concentrations of [ 3 H]‐noradrenaline (3.91 to 125 n m ) 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. 3 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. 4 Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 m m 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. 5 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. 6 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 Ca 2+ channels are affected by collagenase as the responses induced by high K + or veratridine were unaffected by this enzyme.