Evoked transient intracellular free Ca 2+ changes and secretion in isolated bovine adrenal medullary cells

Abstract

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolyzed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 .mu.M range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 .+-. 4 nM (no. = 69). This level seems independent of extracellular Ca in the range 0.5-2 mM. When the extracellular Ca concentration is lowered to approximately 10-7 M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high K medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil and the absence of extracelluar Ca block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the [Ca2+]i transient is also blocked when cells are suspended in a depolarizing medium. Extracelluar Mg inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to Mg inhibition than is Ca entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with Ca, as a result of an acetylcholine or K challenge, suggest that the increase in the apparent Ca content of the cytosol might arise from 2 contributing sources of Ca entry.