Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.
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-3T3t 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-trisphosphate (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 (greater than 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, approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Cheek
T R
TR
Agricultural and Food Research Council Unit of Insect Neurophysiology and Pharmacology, Department of Zoology, Cambridge, England.
Jackson
T R
TR
O'Sullivan
A J
AJ
Moreton
R B
RB
Berridge
M J
MJ
Burgoyne
R D
RD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002450:Cell Communication; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism* / Q000648:ultrastructure; D002838:Chromaffin System / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis; D005456:Fluorescent Dyes; D016257:Fura-2; D051379:Mice; D009538:Nicotine / Q000494:pharmacology
D001572:Benzofurans; D005456:Fluorescent Dyes; D000804:Angiotensin II; D009538:Nicotine; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2