Dynasore inhibits rapid endocytosis in bovine chromaffin cells

• Published in: American Journal of Physiology: Cell Physiology Vol. 297; no. 2; pp. C397 - C406
• Main Authors: Tsai, Chia-Chang, Lin, Chih-Lung, Wang, Tzu-Lun, Chou, Ai-Chuan, Chou, Min-Yi, Lee, Chia-Hsueh, Peng, I-Wei, Liao, Jia-Hong, Chen, Yit-Tsong, Pan, Chien-Yuan
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.08.2009
• Subjects: Animals; Calcium - metabolism; Calcium Channels - metabolism; Cattle; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cells; Chromaffin Cells - drug effects; Chromaffin Cells - physiology; Clathrin - metabolism; Effects; Electric Capacitance; Endocytosis; Endocytosis - drug effects; Exocytosis - physiology; Fluorescent Dyes - metabolism; Hydrazones - pharmacology; Membranes; Microscopy, Atomic Force; Patch-Clamp Techniques; Plasma; Potassium - metabolism; Pyridinium Compounds - metabolism; Quaternary Ammonium Compounds - metabolism; Transport Vesicles - metabolism
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00562.2008

1 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; 2 Institute of Zoology, 3 Department of Chemistry, and 4 Department of Life Science, National Taiwan University, Taipei, Taiwan, Republic of China Submitted 4 November 2008 ; accepted in final form 29 May 2009 Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all 110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis. actin; atomic force microscope; calcium current; clathrin-mediated endocytosis; dynamin; FM4-64 Address for reprint requests and other correspondence: C.-Y. Pan, Institute of Zoology, National Taiwan Univ., Rm. 730, Life Science Bldg., 1 Roosevelt Rd., Sec. 4, Taipei 106, Taiwan (e-mail: cypan{at}ntu.edu.tw )