Simultaneous Measurements of Cytosolic Calcium and Secretion in Single Bovine Adrenal Chromaffin Cells by Fluorescent Imaging of Fura-2 in Cocultured Cells

• Published in: The Journal of cell biology 1989-09, Vol.109 (3), p.1219-1227
• Main Authors: Cheek, Timothy R., Jackson, Trevor R., O'Sullivan, Antony J., Moreton, Roger B., Berridge, Michael J., Burgoyne, Robert D.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Adrenal Medulla - cytology; Adrenal Medulla - drug effects; Adrenal Medulla - metabolism; Angiotensin II - pharmacology; Animals; Benzofurans; Biological and medical sciences; Calcium; Calcium - metabolism; Cell Communication; Cell physiology; Cells; Cells, Cultured; Cheek; Chromaffin cells; Chromaffin Granules - metabolism; Chromaffin Granules - ultrastructure; Chromaffin System - metabolism; Cultured cells; Cytosol - metabolism; Exocytosis; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fura-2; Mice; Molecular and cellular biology; Nicotine - pharmacology; NIH 3T3 cells; Perfusion; Secretion; Secretion. Exocytosis; Ungulates
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.109.3.1219

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. Chromaffin cell [ Ca2+] i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3 T3 t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ 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-triphosphate ( InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin 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 chromaffin 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.