Membrane cycling after the excess retrieval mode of rapid endocytosis in mouse chromaffin cells

Aim: After exocytosis, neuroendocrine cells and neurones keep constant the plasma membrane and the releasable vesicle pools by performing endocytosis and vesicular cycling. Patch-clamp capacitance measurements on chromaffin cells showed that strong Ca+2 entry activates excess retrieval: a rapid endocytosis process that retrieves more membrane than the one fused by preceding exocytosis. The main purpose of the present experiments was to study the recycling pathway that follows excess retrieval, which is unknown. Methods: Membrane recycling after exocytosis-endocytosis can be studied by fluorescence imaging assays with FM1-43 (Perez Bay et al. Am J Physiol Cell Physiol 2007; 293, C1509). In this work, we used this assay in combination with fluorescent dextrans and specific organelle-targeted antibodies to study the membrane recycling after excess retrieval in mouse chromaffin cells. Results: Excess retrieval was observed after the application of high-K+ or cholinergic agonists during 15 or 30 s in the presence of FM1-43. We found that the excess retrieval membrane pool (defined as endocytosis-exocytosis) was associated with the generation of a non-releasable fraction of membrane (up to 30% of plasma membrane surface) colocalizing with the lysosomal compartment. The excess retrieval membrane pool followed a saturable cytosolic Ca2+ dependency, and it was suppressed by inhibitors of L-type Ca2+ channels, endoplasmic reticulum Ca2+ release and PKC. Conclusion: Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non-releasable endosomes. This process is activated by a concerted contribution of Ca2+ entry through L-channels and Ca2+ release from endoplasmic reticulum.