Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells

Abstract

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 microM), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K+. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

“…FM1-43 and other styryl dyes have been used to monitor exocytosis, endocytosis, and endosomal traffic in a variety of cell types, including motor nerve terminals in amphibia (1)(2)(3), mammals (4), and Drosophila larvae (5); cultured hippocampal neurons (6)(7)(8)(9); pituitary cells (10); Dictyostelium (11); and yeast (12). The amphipathic structure of the dyes, with ionically charged heads and hydrophobic tails, has led to the following proposed mechanism of action: From the extracellular fluid dye molecules reversibly partition into the outer leaflet of all surface membranes, but they cannot penetrate to the cytoplasm, owing to their charged headgroups.…”

Membrane trafficking in pituitary cells: studies using a fluorescent dye

“…On stimuIation of secretion a large increase of ftuorcscence is sea which may spread a c r w the cell [3]. We have suggested that this is due to a combination of increased dye uptake into membranes of secretory vesicles exposed on exocytosis, and the subsequent increase in endocytosis of labelled membrane [3]. Fig.2 shows that PMA increased fluorescence whereas ionomycin had little effect on its own, but potentiated the PMA response.…”

“…On incubation with unstimulated cells, FM1-43 is normally restricted to the outer leaflet of the plasma membrane because of its charge. On stimuIation of secretion a large increase of ftuorcscence is sea which may spread a c r w the cell [3]. We have suggested that this is due to a combination of increased dye uptake into membranes of secretory vesicles exposed on exocytosis, and the subsequent increase in endocytosis of labelled membrane [3].…”

The Anti-Botulism Triterpenoid Toosendanin Elicits Calcium Increase and Exocytosis in Rat Sensory Neurons

“…FM1-43 has been used to monitor exocytosis in both neurons and neuroendocrine cells (Ryan and Smith 1995;Stafford et al 1993). In this study, attached nodose ganglion neurons were first loaded with Fura-2 AM (5 lM), then perfused in standard buffer containing FM1-43 (4 lM).…”