Sustained stimulation of exocytosis triggers continuous membrane retrievalin rat pituitary somatotrophs

1. We studied the relationship between exocytosis and endocytosis in rat pi tuitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence ima ging and amperometry. 2. Stimulation of exocytosis through voltage-dependent Ca2+ channels by dep olarizations (1-5 s) increased the capacitance by 4.3 +/- 0.9% and the fluo rescence by 6.6 +/- 1.1% (10 cells). The correlation between the capacitanc e and fluorescence changes indicated that the cell membrane and granule mem brane 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 Ca2+ (1.5-100 muM) ev oked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluoresce nce (56 +/- 10%; 21 cells). Interestingly during Ca2+ dialysis the capacita nce did not significantly change (2 +/- 1%; 31 cells), indicating that endo cytosis efficiently retrieved increased cell membrane. 4. Sustained endocytosis was not blocked when the intracellular GTP (300 mu M) was replaced with GTP gammaB. Replacing intracellular Ca2+ (100 muM) wit h Ba2+ (300 muM) or Sr2+ (200 muM), 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 unde rgo con exocytosis and membrane retrieval that persist in whole-cell record ings.