Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.

Abstract

Abstract. The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaflin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-1oaded NIH-3T3' cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca 2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells.In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2÷]i was followed by a secretory response which originated from the entire surface of the cell.In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaftin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (>200 nM), transient elevation in [Ca2÷]i and no detectable secretory response. The rise in [Ca2÷]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, ~25 % of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest. Exocytosis in response to nicotine occurred over the entire surface of the cell, whereas exocytosis due to angiotensin II was polarized, as was confirmed by immunofluorescent localization of dopamine-B-hydroxylase, a chromaflin granule protein that becomes incorporated into the plasma membrane during fusion.These results directly demonstrate, for the first time, that intact chromaffin cells can undergo a large, agonist-induced transient rise in [Ca2+]i without this stimulating secretion and, furthermore, show that the location of exocytosis around the cell can vary depending on the nature of the stimulus. XOCYTOSlS, the process by which intracellular vesicles fuse with the inner surface of the plasma membrane and release their contents into the surrounding medium, is the mechanism underlying the secretion of many physiologically important mediators such as hormones, enzymes, and neurotransmitters. The process is often regulated by an external signal which stimulates release by altering the level of an intracellular second messenger.The pivotal role that Ca 2÷ plays in triggering exocytosis was first noted nearly 30 yr ago with the demonstration that depolarized chromaffin cells would not secrete catecholamines in the absence of extraceUular Ca 2÷ (13). The involvement of Ca 2÷ in exocytosis was advanced by subsequent information obtained from studies with 45Ca 2÷ (12) All reprint requests should be...

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

“…The first examples for TRH and bradykinin stimulation of pituitary cells show a multiphasic progression of exocytosis. This is consistent with previous results obtained with both stimuli (32) (38). All these results are consistent with the postulate Cytosolic free calcium and exocytosis (37).…”

The caffeine‐sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

“…Imaging of intracellular Ca 2+ in single cells was carried out as described in detail elsewhere [3,4]. After differential plating, cells were resuspended in fresh DMEM and seeded onto 22 mm diameter glass coverslips at a density of 1 x 105 cells/ml.…”

“…Both of these stimuli lead to Ca 2+ activation of the subplasmalemmal exocytotic sites [2,3]. In contrast, the InsPrmobilizing agonist angiotensin II results in either no secretion or a polarized secretory response as the InsP3-sensitive store [3,5] and the intracellular Ca 2÷ signal [4] are spatially restricted. We have previously shown that, in addition to the InsP3-sensitive store, these cells also possess a caffeinesensitive Ca 2÷ store that is diffusely distributed throughout the cell [5].…”

ATP release from non-excitable cells

“…After stimulation with nicotine, the chromaffin cells released ATP, which was sensed via a P2 receptor-dependent [Ca 2+ ] i increase by the neighbouring fibroblasts [39]. Extracellular ATP and other nucleotides commonly produce elevations of cytosolic Ca 2+ via activation of either P2Y or P2X receptors [40].…”