Monitoring Receptor Mediated Regulation of Cytosolic Calcium in Single Pituitary Cells by Dual Excitation Microfluorimetry

Abstract

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca²⁺]_i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluori-metry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, λ₁ and λ₂, at which an increase in [Ca²⁺]_i causes either a rise or a fall in fluorescence; the ratio of fluorescence at λ₁ and λ₂ (R =Fλ₁/Fλ₂) 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.