Ca2+-dependent exocytotic pathways in Chinese hamster ovary fibroblasts revealed by a caged-Ca2+ compound

Abstract

Ca2+-dependent exocytosis and endocytosis of Chinese hamster ovary (CHO) fibroblasts were investigated using capacitance measurement and rapid photolysis of a caged-Ca2+ compound, dimethoxynitrophenamine tetrasodium salt. CHO cells exhibited large and fast increases in membrane capacitance (1.9 ± 1 picofarads, or 13 ± 7% of total membrane area, mean ± S.D., n = 37) upon Ca2+ jumps to [Ca2+](i) larger than 20 μM. The fast exocytosis occurred with a delay (20-80 ms), and exhibited a rate constant that was strongly dependent on [Ca2+](i). The maximal rate constant of exocytosis was 2.8/s, and a half-maximal rate was achieved at 30 μM. The fast exocytosis was followed by rapid endocytosis in 28% of the cells. The endocytosis often began after a delay of 0.5-2 s. Ca2+ jumps also induced stepwise increases in membrane capacitance of 10-134 femtofarads in 40% of the cells, indicating fusion of large vesicles with diameters of 0.4-1.5 μm. The exocytosis of the large vesicles could selectively be induced with smaller Ca2+ jumps (6-20 μM), and occurred slowly with a rate constant of 0.3/s. These data indicate that CHO fibroblasts possess Ca2+-dependent exocytotic mechanisms. Moreover, two parallel exocytotic pathways may exist reminiscent of those of neurons and endocrine cells. A kinetic model was constructed to account for the fast exocytosis of CHO cells.