THYROLIBERIN-INDUCED CHANGES IN THE FLUORESCENCE OF A MEMBRANE PROBE IN INDIVIDUAL BOVINE ANTERIOR-PITUITARY-CELLS

1. We have investigated the use of TMA-DPH rimethylammonio)phenyl]-6-p henylhexa-1,3,5-triene) as an indicator of exocytosis in individual bo vine anterior pituitary cells using microfluorimetric imaging. 2. TMA- DPH was photolabile in artificial and cell membranes. In cells incubat ed in TMA-DPH the distribution of fluorescence depended both on the in cubation time and the illumination schedule. If the dye was added whil e the cells were subjected to repeated cycles of 0.36 s light intermit tent 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 ce ntral region. However, using long intervening dark periods (200 s), th e central region continued to incorporate dye after the annulus had re ached a plateau. 3. When the cells were loaded with TMA-DPH using inte rmittent light with short dark periods, the dye washed out of the cent ral 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 annulu s. 4. When cells were incubated in TMA-DPH in the dark for 1 min and t hen 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-DP H 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 membrane-as sociated and internalized dye and that the amount of fluorescence from the latter varied because TMA-DPH was photobleached. Thus, when illum ination 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 be fore it was bleached and fluorescence was therefore also present in ce ntral regions. 6. The total cell fluorescence, observed using 15 s dar k intervals, was increased 5-40% (in single cells) in a dose-dependent fashion by addition of TRH (tripeptide thyrotrophin-releasing hormone ; 1-200 nm). The increase could be reproduced several times in the sam e cell. The numbers of cells responding to TRH (20 nm) was inhibited i n a dose-dependent fashion by acute addiction of quinpirole (1-100 mum ), a dopamine D, receptor agonist which inhibits prolactin secretion. The sensitivity of cells to inhibition was enhanced considerably when cells were grown in the presence of quinpirole (1-10 mum; 12-48 h). 7. Changes in TMA-DPH fluorescence and then [Ca2+]i were measured succes sively in sixty-five cells; all fifty-one cells subsequently shown to contain prolactin showed rises in [Ca2+]i, but of these only thirty-se ven showed rises in TMA-DPH fluorescence, on addition of TRH. We concl ude that TRH increases [Ca2+]i only in prolactin-containing cells and that in some of these cells the increase was associated with an increa sed incorporation of TMA-DPH which we suggest reflects an increase in exocytosis.