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

Abstract

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.

CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells

“…Calcium-dependent secretion was confirmed in LNCaP cells using FM1-43 and TMA-DPH imaging assays. These lipophilic dyes have been widely used to measure exocytotic release in neurons, endocrine, or exocrine cells (21,22,34). As noticed from our experiments, FM1-43 fluorescence increased after ionomycin or thapsigargin stimulation in the absence or presence of extracellular calcium, although more efficiently in the latter condition.…”

Involvement of SNAP-25 in TRH-induced exocytosis in pituitary GH4C1 cells

“…The fluorescent excitation beam was targeted on the cells, and then the emis¬ sion fluorescence was recorded by digital imaging and ana¬ lyzed with a computer. Measurement of exocytosis and prolactin release To evaluate exocytosis, the impermeable fluorescent mem¬ brane probe TMA-DPH was used (Masumoto et al 1993, Shorte et al 1993, Masumoto et al 1995. GH4C1 cells were loaded with a final concentration of 3 µ TMA-DPH, and the 430 nm fluorescence of single cells at 360 nm excitation was measured by a fluorescence microscope system with computer analysis (Mu-1000, Scholar Tec, Japan).…”

Calcium‐induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals

“…The excitation and observation wavelengths were 360 f 10 run and 510 f 20 run, respectively. When TMA-DPH interacts with intact nerve terminals in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium, ie the amount of the probe incorporated is proportional to the available membrane surface (Shorte et al, 1993;Haugland, 1996).…”