EFFECTS OF COLLAGENASE ON THE RELEASE OF [H-3]-LABELED NORADRENALINE FROM BOVINE CULTURED ADRENAL CHROMAFFIN CELLS

Bovine isolated adrenal chromaffin cells maintained in culture at 37.degree. C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine .beta.-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 [norepinephrine] (3.91-125 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 1/3 of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in 3H uptake. The enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. 3H release 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 3H evoked by ACh or nicotine. The responses to high K+, veratridine or ionophore X537A [lasalocid] were not affected. The nicotinic response was recovered 2 days after collagenase treatment. The use of collagenase to disperse the adrenomedullary tissue during the isolation procedure may 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.