Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Abstract

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,

Spontaneous Intracellular Calcium Oscillations and Gsα Subunit Expression are Inversely Correlated with Secretory Granule Content in Pituitary Cells

“…As has been shown previously, quinacrine fluorescence permits quantification of granule content, as well as granule release upon stimulation (17). In this study we used quinacrine to quantify the granule content of GH4C, cells, having verified that the distribution of quinacrine fluorescence colocalizes with that of PRL immunostaining in the same cells, exactly as shown previously with pituitary cells in primary culture (1 7).…”

Real‐time studies of zymogen granule exocytosis in intact rat pancreatic acinar cells

“…To confirm that the spots observed in the time‐differential analysis reflect exocytotic activity, we carried out experiments in which we labelled zymogen granules with the fluorescent marker quinacrine. Quinacrine is a weak base that accumulates in acidic compartments such as secretory vesicles (Chiavaroli et al 1991; Titievsky et al 1996), and has previously been used to validate the measurement of exocytosis using either complex impedance analysis (Breckenridge & Almers, 1987; Zimmerberg et al 1987) or voltammetry (Tatham et al 1991). In acinar cells incubated with quinacrine the apical pole of the cells became brightly fluorescent (Fig.…”

Thyroliberin‐induced changes in the fluorescence of a membrane probe in individual bovine anterior pituitary cells.

“…This suggests that a rise in [Ca2+]i may be necessary but not sufficient to lead to increased incorporation of TMA-DPH. Such a conclusion was recently used to explain why only 25 % of rat anterior pituitary cells gave secretory responses to either 100 nm TRH or electrical depolarization as assessed by the quinacrine method (Chiavaroli et al 1991); however, this discrepancy may be due to the lower sensitivity of the method. Interestingly, in these studies, none of the prolactin-negative cells showed TRH-induced rises in [Ca2+]i or TMA-DPH fluorescence, indeed some cells in this group even showed transient falls in [Ca2+]i on exposure to TRH suggesting the presence of TRH receptors coupled to falls in [Ca2+]i on some prolactin-negative cells.…”

“…Some use the ability of either basic (Breckenridge & Almers, 1987; Chiavaroli et al 1991) or acidic (Kawasaki, Saitoh, Okabe, Kumakura & Ohara-Imaizumi, 1991) fluorescent molecules to stain secretory vesicles before or after secretion respectively. However, labelling of anterior pituitary cells with basic dyes requires long incubation (Chiavaroli et al 1991) and the dye is not necessarily limited to secretory granules; whereas, acidic probes require the focal plane resolution of a confocal microscope to resolve cell fluorescence against a large 192 background of out-of-focus fluorescence (Kawasaki et al 1991). The latter workers also used a fluorescence membrane probe TMA-DPH (1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene) to measure exocytosis.…”