CONICET | Buscador de Institutos y Recursos Humanos

Neurons and neuroendocrine cells have to retrieve excess of plasma membrane 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, during 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 ¡Ü20% of plasma membrane (provoked by nicotine/acetylcloline) was followed by total recovery of releasable vesicles. If a stronger stimulus (50mM K+, 2mM Ca2+) provoking intense exocytosis (51¡À7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19¡À2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol-blue to quickly eliminate fluorescence from non-internalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining non-releasable fraction was distributed mainly as fluorescent spots (~0.7mm) selectively labeled by 40-70 kD dextrans and was supressed by a PI3-kinase inhibitor, suggesting it had been formed by 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 timeframe but seemed crucial to maintain membrane surface homeostasis under extreme conditions.