Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells

• Published in: American Journal of Physiology: Cell Physiology Vol. 293; no. 5; pp. C1509 - C1522
• Main Authors: Perez Bay, Andres E, Ibanez, Lorena I, Marengo, Fernando D
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.11.2007
• Subjects: Acetylcholine - pharmacology; Adrenal Glands - cytology; Adrenal Glands - drug effects; Adrenal Glands - metabolism; Animals; Bromphenol Blue - chemistry; Calcium - metabolism; Cells; Cells, Cultured; Cholinergic Agonists - pharmacology; Chromaffin Cells - drug effects; Chromaffin Cells - enzymology; Chromaffin Cells - metabolism; Cyclodextrins - chemistry; Endocytosis; Endocytosis - drug effects; Endosomes - drug effects; Endosomes - metabolism; Exocytosis - drug effects; Fluorescent Dyes - chemistry; Homeostasis; Membrane Fusion; Membranes; Mice; Neurons; Nicotine - pharmacology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphatidylinositol 3-Kinases - metabolism; Potassium - metabolism; Protein Kinase Inhibitors - pharmacology; Pyridinium Compounds - chemistry; Quaternary Ammonium Compounds - chemistry; Rodents; Signal transduction; Staining and Labeling - methods; Time Factors; Transport Vesicles - drug effects; Transport Vesicles - metabolism
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00632.2006

Laboratorio de Fisiología y Biología Molecular, Instituto de Fisiología, Biología Molecular y Neurociencias (Consejo Nacional de Investigaciones Científicas y Técnicas), Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina Submitted 26 December 2006 ; accepted in final form 2 August 2007 Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of 20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K + and 2 mM Ca 2+ ) provoking intense exocytosis (51 ± 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 ± 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots ( 0.7 µm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions. endocytosis; mouse chromaffin cells; calcium signal; FM1-43; ADVASEP-7; bromophenol blue Address for reprint requests and other correspondence: F. D. Marengo, Laboratorio de Fisiología y Biología Molecular, Instituto de Fisiología, Biología Molecular y Neurociencias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria-Pabellón II-2° piso, Buenos Aires, Argentina (CP 1428) (e-mail: fernando{at}fbmc.fcen.uba.ar )