(10-4 M) inhibited the uptake of 86Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10-4 M) blocked 86Rb uptake in a similar manner. 6 The results are further evidence in favour of the presence of a Na+-Ca2+ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca2+]i and in the modulation of catecholamine secretion. This system is activated by increasing [Na+]j, either directly (ionophore X537A, increased [Na+]J) or indirectly (Na+ pump inhibition). However, the simple inhibition of Na+ pumping does not always lead to a catecholamine secretory response; such is the case for NEM.
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.
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