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

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 versus norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were found to be predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 sec and 85% of maximum secretion within 5 min. A critical requirement for calcium in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCI) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on calcium 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 KCI-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.The term "stimulus-secretion coupling" was originally coined by Douglas and Rubin (1, 2) more than a decade ago to describe the sequence of events initiated by acetylcholine (ACh) stimulation of adrenal chromaffin cells and leading to secretion of catecholamines by exocytosis. They had in mind the close similarity to the phenomenon of "excitation-contraction coupling" in muscle (namely, the key role of calcium in mediating both secretion and contraction and a parallel set of electrical and ionic events at the plasma membrane in response to ACh). Much progress has been made during the past 2 decades in elucidating the various steps in the mechanism of secretion, with a substantial portion of the data deriving from studies on the perfused adrenal gland (2-4). There remain, however, many important aspects of cell surface receptor regulation of secretion that are still poorly understood, such as (i) the molecular nature of the calcium permeability sites that are altered by activated cholinergic receptors and potassium depolarization, (ii) the mechanism by which calcium entry into the cell induces membrane fusion and exocytosis, (iii) the possible role of cyclic nucleotides coupled with ion translocation in mediating horm...

Stimulus‐secretion Coupling in Isolated Bovine Adrenal Medullary Cells

“…The muscarinic agonist methacholine did not promote catecholamine release. These results are quantitatively consistent both with findings in the intact gland and in cells obtained by collagenase digestion (Schneider et al 1977;Greene & Rein, 1978;Mizobe et al 1979). …”

“…In recent years evidence has accumulated in support of the view that in the cells of the adrenal medulla, acetylcholine sets up a train of sodium action potentials which, by further depolarizing the cells, open channels through which Ca enters to elevate the cytosolic level of ionized Ca and trigger exocytosis (Biales, Dichter & Tischler, 1976;Brandt, Hagiwara, Kidokoro & Miyazaki, 1976; Kidokoro & Ritchie, 1980;Baker & Rink, 1975). Study of the steps that couple excitation to secretion is greatly simplified in isolated cells and much effort has been directed towards generating preparations of cells that retain the properties of the intact gland (Hochman & Perlman, 1976;Chalfie & Perlman, 1976;Brooks, 1977;Schneider, Herz &Preparation and handling ofmedullary cells The adrenal cortex, which is visually distinguishable from the medulla, was removed and discarded. Thin slices (less than 1 mm thick) were cut from the isolated medulla using stacked single edged razor blades and any obvious islands of cortical tissue removed.…”

The action of volatile anaesthetics on stimulus‐secretion coupling in bovine adrenal chromaffin cells

“…Although bovine chromaffin cells possess both nicotinic and muscarinic cholinoceptors, catecholamine secretion can only be induced by nicotinic agonists (Schneider et al, 1977;Derome et al, 1981). The muscarinic receptors may modulate catecholamine secretion induced by nicotinic agonists (Derome et al, 1981).…”

CATECHOLAMINES AND DOPAMINE‐β‐HYDROXYLASE SECRETION FROM PERFUSED GOAT ADRENAL GLANDS

“…In chicken, only the muscarinic receptor mediates CA secretion (Ledbetter & Kirshner, 1975). On the other hand, only the nicotinic receptor contributes to CA secretion in calf (Schneider, Herz & Rosenbeck, 1977) and hamster (Liang & Perlman, 1979).…”