CONICET | Buscador de Institutos y Recursos Humanos

Action potential like stimulus (APls) applied to chromaffin cells induces synchronous exocytosis (EXO) of few vesicles followed by fast, dynamin-dependent, endocytosis. We studied the effect produced by the variation of the stimulus in a range that release IRP vesicles (5ms APls and 5-50ms squared-depolarizing-pulses (SQPs)) on fast endocitosis (ENDO). ENDO compensated completely EXO for APls, since ENDO/EXO was 1.1±0.1. However, this parameter decreased monotonically with the length of depolarization, reaching a value of 0.43±0.09 for 50ms SQP. On the other hand, the ENDO time constant (τ) showed a biphasic behavior, increasing from 0.8±0.1 for APls to 1.1±0.1s for 10 and 25ms SQPs, and decreasing to 0.7±0.1s for 50ms SQP. Similarly, the application of double and triple 25ms SQPs (20 Hz) decreased significantly τ (i.e. accelerated endocytosis) respect to individual 25ms SQP. This change is correlated with the monotonic increase in total Ca2+ entry, without significant changes in EXO, opening the possibility of a modulation of endocytosis by Ca2+. In other set of experiments we tested the participation of dynamin in endocytosis by application of an anti-dynamin monoclonal antibody (7nM) or a peptide competing with endogenous dynamin (GST-Dyn, 30 µM). Both treatments decreased ENDO/EXO associated to APls to 40% and 20% of control values respectively, but they did not modify the same parameter for 50ms SQPs. We also tested a possible kinase-dependent regulation of fast endocytosis by application of 100nM staurosporine, which did not affect APls-induced endocytosis but reduced ENDO/EXO associated to 50ms SQPs to 30% of control values. These results confirm that fast endocytosis associated to APls is dependent on dynamin, and suggest that the endocytosis related to longer depolarizations that release completely IRP is dynamin independent but can be regulated by an unknown protein kinase.