Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs

Abstract

1. We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry.2. Stimulation of exocytosis through voltage-dependent Ca 2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 ± 0.9 % and the fluorescence by 6.6 ± 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner.3. Intracellular dialysis (0.5-4.5 min) with elevated Ca 2+ (1.5-100 µM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 ± 10 %; 21 cells). Interestingly during Ca 2+ dialysis the capacitance did not significantly change (2 ± 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane.4. Sustained endocytosis was not blocked when the intracellular GTP (300 µM) was replaced with GTPyS. Replacing intracellular Ca 2+ (100 µM) with Ba 2+ (300 µM) or Sr 2+ (200 µM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. 5. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

Calcium-Dependent Exocytosis of Atrial Natriuretic Peptide from Astrocytes

“…To test this hypothesis further, we monitored cumulative exocytosis by using the FM 4-64 styryl fluorescent probe, which stains membranes (Betz et al, 1992;Smith and Betz, 1996;Kilic et al, 2001). If the ionomycin-induced decline in green fluorescence intensity is attributable to regulated exocytosis, this should be associated with the incorporation of the new vesicle membrane into the plasma membrane (Smith and Betz, 1996); therefore, the intensity of FM 4-64 fluorescence should increase.…”

“…Using FM 4-64 to monitor cumulative exocytosis (Betz et al, 1992;Smith and Betz, 1996;Kilic et al, 2001), we found a Ca 2ϩ -dependent increase in total FM 4-64 fluorescence intensity.…”

“…Astrocyte-loaded coverslips were transferred into the recording chamber on a confocal microscope (model LSM 510; Zeiss, Jena, Germany) and supplied with 400 l of extracellular solution containing FM 4-64 (4 M; Molecular Probes, Leiden, The Netherlands). Cumulative exocytosis was monitored by measuring the intensity of FM 4-64 fluorescence (Betz et al, 1992;Smith and Betz, 1996;Kilic et al, 2001). To achieve this objective, cells were maintained in 4 M FM 4-64, which initially stains total plasma membrane.…”

Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells

“…Smith and Neher, 1997), pancreatic β-cells (Eliasson et. al., 1996), melanotrophs (Thomas et al, 1994), corticotrophs (Lee and Tse, 2001) and somatotrophs (Kilic et. al., 2001).…”

Heterotrimeric G-proteins Activate Cl− Channels through Stimulation of a Cyclooxygenase-dependent Pathway in a Model Liver Cell Line

“…To stimulate heterotrimeric G-proteins more specifically, cells were dialyzed with an aluminum fluoride solution that contained 120 mM KCl, 10 mM NaCl, 10 mM NaF, 0.03 mM AlCl 3 , 1 mM EGTA, 0.5 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES/NaOH. Under these conditions, the concentration of AlF 4 Ϫ was 30 M (20 ] i was determined as described previously (21). The pH of all solutions was 7.25.…”