FAST RECOVERY OF EXOCYTOSIS AFTER ACTION POTENTIAL LIKE STIMULUS IN MOUSE CHROMAFFIN CELLS

JOSÉ MOYA DÍAZ; YANINA D. ALVAREZ; FERNANDO D. MARENGO

Rouen

Congreso; 17th International Symposium of Chromaffin Cell Biology; 2013

International Symposium of Chromaffin Cell Biology

The immediately releasable pool (IRP) is a group of vesicles highly coupled to voltage dependent Ca2+ channels. Because IRP is selectively released by short length stimuli, it is expected that the application of an action potential like stimulus (APs) evokes specifically the exocytosis of vesicles from this pool. In fact, when IRP was assessed immediately after a single APs application, IRP exocytosis was decrease in a magnitude similar to APs associated exocytosis (APAE), which was in average 11±2 fF (~8 vesicles). Therefore, IRP might be responsible to provide vesicles during basal low frequency physiological stimulation. However, the process of replenishment after total depletion of IRP is too slow (τ=7±1s) to allow sustainable exocytosis, even during lowest physiological frequencies. In this work we analyzed the process of exocytosis recovery after APAE. We applied a pair of APs separated by a variable time period, and expressed the recovery of APAE as the ratio between the exocytosis evoked by the second stimulus (Cm2) and the exocytosis evoked by the first stimulus (Cm1). The analysis of the time dependence of Cm2/Cm1 showed that APAE recovered much faster than total IRP (τ=1±0.1s). This exponential time constant suggests that under our experimental conditions (room temperature, 5 mM external Ca2+) that APAE can sustain secretion at moderate frequencies of APs. In fact, the application of APs at 0.2 Hz provoked no reduction of the synchronous exocytosis measured at each stimuli along the train, APs at 0.5 Hz produced just a 30% stable reduction, , and APs at frequencies > 1 Hz provoked an important decreased of this parameter. We wonder about the mechanism of APAE recovery: it may be vesicle mobilization from the rest of IRP (or some other upstream pool), or recovery ?in situ? by a kiss and run like mechanism. In reference to the second possibility, it is important to mention that we often observed fast endocytosis (τ=711±21 ms) after APAE. To evaluate the first hypothesis we completely depleted IRP by a 50 ms depolarization, and after a variable time we applied a APs to test APAE. In these conditions APAE recovered slowly, with similar kinetics as IRP recovery. On the other hand, when we inhibited completely fast endocytosis with 80 μM dynasore or 10 μM nitrendipine, APAE recovery was not modified respect to the control condition. Therefore, or results suggest that APAE is associated to a small group of vesicles rapidly recovered from IRP.