IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Fast recovery of releasable vesicles and formation of non-releasable endosomes follow intense exocytosis in chromaffin cells
Autor/es:
ANDRÉS PÉREZ BAY; FERNANDO D. MARENGO
Lugar:
Sestri Levante, Italia
Reunión:
Congreso; 14th Internacional Symposium of Chromaffin Cell Biology; 2007
Institución organizadora:
Internacional Symposium of Chromaffin Cell Biology
Resumen:
After exocytosis, neurons and neuroendocrine cells have to retrieve the excess of plasmamembrane and refill the depleted pools of vesicles. To perform this task various endocytotic/recycling mechanisms can potentially operate in these cells. It is expected that the selection of a particular endocytosis/recycling pathway will impact in the recycling time of releasable vesicles. In this work we tried to discriminate the endocytosis-recycling responses produced by a variety of stimuli respect to their ability to rapidly generate releasable vesicles. Using FM type styryl dyes in chromaffin cells, we evaluated in the same experiment the Ca2+-triggered exocytosis, the resulting endocytosis and the internalized membrane fraction cycled to releasable vesicles. Exocytosis induced by cholinergic agonists was followed by total recovery of releasable vesicles. If a stronger stimulus (50mM K+, 2mM Ca2+) provoking more intense exocytosis was applied, endocytosis still retrieved all the fused membrane, but only a fraction was releasable by a second stimulus. Using the chelator ADVASEP-7 or the quencher bromophenol-blue after exocytosis to quickly eliminate fluorescence coming from non-internalized FM1-43, we determined that this fraction became releasable in < 2 min, indicating that vesicular cycling is completed in this short timeframe. This was demonstrated for cholinergic and high potassium stimulation. As the remaining non-releasable fraction was mainly distributed as big fluorescent spots (~0.7 mm), it was selectively labeled by 40kD and 70 kD dextrans, there is a 80% co-localization between FM and dextran labeled fractions, and its formation was inhibited by a PI 3-kinase inhibitor, we believe that it might be the result of a bulk retrieval process. Thus, chromaffin cells can rapidly recover important fractions of their vesicle population, and this pathway prevails when cholinergic agonists were used as secretagogues. Masive exocytosis also triggers an additional mechanism, which is unable to quickly produce secretory vesicles but seems to be crucial for membrane surface homeostasis.