Stimulus-secretion coupling in chromaffin cells isolated from bovine adrenal medulla

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Abstract
Bovine adrenal chromaffin cells were isolated by removal of the cortex and sequential collagenase digestion of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimulation, cation requirements and cytoskeletal elements. The dose-response curve for stimulated release had its half-maximum value at 10-5 M acetylcholine, and maximum secretion was on the average 7 times that of control basal secretion. The differential release of epinephrine vs. norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 s and 85% of maximum secretion within 5 min. A critical requirement for Ca in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic Ca concentrations. Stimulation by excess K (65 mM KCl) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on Ca concentration, and in its response to the local anesthetic tetracaine. Tetracaine, which is thought to inhibit membrane cation permeability, was able to block acetylcholine-stimulated but not KCl-stimulated secretion. The microtubule disrupting agent vinblastine was able to block catecholamine release whereas the microfilament disrupter cytochalasin B had little effect. The results show the isolated bovine chromaffin cells to be viable, functioning, and available in large quantity. These cells now provide an excellent system for studying cell surface regulation of hormone and neurotransmitter release.