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

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Abstract
We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ 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. Intracellular dialysis (0.5-4.5 min) with elevated Ca2+ (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 Ca2+ dialysis the capacitance did not significantly change (2 ± 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. Sustained endocytosis was not blocked when the intracellular GTP (300 μm) was replaced with GTPγS. Replacing intracellular Ca2+ (100 μm) with Ba2+ (300 μm) or Sr2+ (200 μm), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.