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.
SUMMARY1. We have investigated the use of TMA-DPH (1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene) as an indicator of exocytosis in individual bovine anterior pituitary cells using microfluorimetric imaging.2. TMA-DPH was photolabile in artificial and cell membranes. In cells incubated in TMA-DPH the distribution of fluorescence depended both on the incubation time and the illumination schedule. If the dye was added while the cells were subjected to repeated cycles of 0-36 s light intermittent with 1-15 s dark, the fluorescence of the peripheral annulus and the central region of individual cells rose in parallel and reached a steady state within 200 s; the annulus was always brighter than the central region. However, using long intervening dark periods (200 s), the central region continued to incorporate dye after the annulus had reached a plateau.3. When the cells were loaded with TMA-DPH using intermittent light with short dark periods, the dye washed out of the central region and the annulus in parallel when external dye was removed. However, if the cells had been loaded using long dark periods, the dye was washed out of the central region more slowly than from the annulus.4. When cells were incubated in TMA-DPH in the dark for 1 min and then exposed to constant illumination in the presence of external dye, the fluorescence of the central region and the annulus both decayed in parallel to a new steady state. If the cells were incubated in TMA-DPH in the dark for 240 min the fluorescence from each region fell to a steady state but the falls were larger and were not in parallel.5. We suggest that TMA-DPH fluorescence was derived from plasma membraneassociated and internalized dye and that the amount of fluorescence from the latter varied because TMA-DPH was photobleached. Thus, when illumination was interrupted by short dark intervals, annular fluorescence was high compared to central fluorescence because bleached dye in the plasma membrane was rapidly replaced by unbleached dye from the medium. However, long dark intervals permitted the dye to be internalized before it was bleached and fluorescence was therefore also present in central regions.* To whom reprint requests should be addressed.
Despite extensive study [I], the relationship between second messenger generation and the triggering of exocytosis in lactotroph
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