Cytosolic Cl − ions in the regulation of secretory and endocytotic activity in melanotrophs from mouse pituitary tissue slices

Cl − ions are known regulators of Ca 2 + ‐dependent secretory activity in many endocrine cells. The suggested mechanisms of Cl − action involve the modulation of GTP‐binding proteins, voltage‐activated calcium channels or maturation of secretory vesicles. We examined the role of cytosolic Cl − ([Cl − ] i ) and Cl − currents in the regulation of secretory activity in mouse melanotrophs from fresh pituitary tissue slices by using the whole‐cell patch‐clamp. We confirmed that elevated [Cl − ] i augments Ca 2− ‐dependent exocytosis and showed that Cl − acts on secretory vesicle maturation. The latter process was abolished by a V‐type H − ‐ATPase blocker (bafilomycin), intracellular 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS), a Cl − channel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Cl − current by DIDS reversibly enhanced endocytosis. The direct activation of G‐proteins by GTP‐γ‐S, a non‐hydrolysable GTP analogue, did not restore the impaired secretion observed in low [Cl − ] i conditions. The amplitude of voltage‐activated calcium currents was unaffected by the [Cl − ] i . Furthermore, two Cl − ‐permeable channels, calcium‐activated Cl − channels and GABA A receptors, appeared as major regulators of intracellular Cl − homeostasis. In conclusion, the predominant underlying mechanism of Cl − action is mediated by intracellular Cl − fluxes during vesicle maturation, rather than activation of G‐proteins or modulation of voltage‐activated Ca 2 + channels.