SIMULTANEOUS MONITORING OF CYTOSOLIC FREE CALCIUM AND EXOCYTOSIS AT THE SINGLE CELL LEVEL

Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca2+]i. With an appropriate selection of the excitation wavelengths, in dual excitation microfluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca2+]i versus quinacrine exocytosis. Transient elevations of [Ca2+]i were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca2+]i, coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca2+]i, Concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca2+]i, suggesting that in both cellular systems, an increase in [Ca2+]i is absolutely necessary, but not sufficient to induce secretion.