Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures

Abstract

Chromaffin cells isolated from adult bovine adrenal medullae were plated on collagen-coated dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 8–16 h in culture, the chromaffin cells had started to develop one, two or, more rarely, three neurites. The neurites grew at a rate of7.25 ± 0.07μm/day showing a linear correlation between process length and age of the culture. The neurites displayed one or more varicosity-like structures. Some neurites made long lasting contacts with other cell bodies and processes. Cytochemical tests indicated positive catecholamine fluorescence in boti cell bodies and processes. Fluorometric determinations indicated that by day 7 the catecholamine content was75.7 ± 1% of that found on day 1 and that noradrenaline and adrenaline represented24.6 ±1% and75.3 ± 1% of the total amine content respectively. Catecholamines were released from these cultured chromaffin cells in response to stimulation by either 56 m m KCl or acetylcholine. The stimulation-induced release of amines was Ca 2+-dependent and was blocked by increasing the extracellular c oncentration of Mg 2+. Substitution of (a) extracellular Na 2+ by either choline or sucrose or (b) Ca 2+ by Ba 2+ produced a sharp increase in catecholamine output. Acetylcholine produced a doserelated increase in catecholamine release; half maximal release was produced by3.5 × 10 −5 m acetylcholine and maximal release by 10 −4 m acetylcholine. The cholinergic receptor of cultured chromaffin cells appears to be nicotinic since catecholamine release was stimulated by nicotine and carbamylcholine but not by pilocarpine. Acetylcholine-evoked catecholamine release was blocked by d-tubocurarine (ID 50 = 5.5 × 10 −7 m), and by hexamethonium (ID 50 = 8 × 10 −6 m). However, the acetylcholine-evoked amine release was not blocked by α-bungarotoxin at a concentration (1.25 × 10 −7 m) which blocked frog rectus abdominus responses to acetylcholine (10 −6–10 −3 m). Tetrodotoxin (5 × 10 −6 m) produced47 ± 2% inhibition of the secretory response to acetylcholine (10 −4 m) but it did not modify the amine release in response to 56 m m KCl. Adult bovine chromaffin cells in culture display some of the morphological and functional characteristics of sympathetic neurones. Consequently, the bovine chromaffin cell in culture appears to be a promising model for studies on development and secretion.