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

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 μ m ) 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 μ m ) was replaced with GTPγS. Replacing intracellular Ca 2+ (100 μ m ) with Ba 2+ (300 μ m ) or Sr 2+ (200 μ m ), 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.