Monitoring receptor mediated regulation of cytosolic calcium in single pituitary cells by dual excitation microfluorimetry.
Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.
Schlegel
W
W
Department of Medicine, University of Geneva, Switzerland.
Winiger
B P
BP
Wuarin
F
F
Zahnd
G R
GR
Wollheim
C B
CB
D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005470:Fluorometry / Q000295:instrumentation; D016257:Fura-2; D008297:Male; D008832:Microchemistry / Q000295:instrumentation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D051381:Rats
D001572:Benzofurans; D002118:Calcium; D016257:Fura-2