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

Authors


Corresponding author
G. Kilic: Department of Medicine, University of Colorado Health Sciences Center, Campus Box B-158, 4200 East 9th Avenue, Denver, CO 80262, USA.

Abstract

  • 1We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry.
  • 2Stimulation 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.
  • 3Intracellular 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.
  • 4Sustained 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.
  • 5Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

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