Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.
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 microM) 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 microM) was replaced with GTP[gamma]S. Replacing intracellular Ca2+ (100 microM) with Ba2+ (300 microM) or Sr2+ (200 microM), 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.
Kilic
G
G
Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, CO 80262, USA. gordan.kilic@uchsc.edu
Angleson
J K
JK
Cochilla
A J
AJ
Nussinovitch
I
I
Betz
W J
WJ
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology*; D004298:Dopamine / Q000378:metabolism; D004553:Electric Conductivity; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D017551:Microdialysis; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000166:cytology*; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000502:physiology; D013324:Strontium / Q000494:pharmacology
C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D001464:Barium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013007:Growth Hormone-Releasing Hormone; D002118:Calcium; D004298:Dopamine; D013324:Strontium