Cytosolic Cl- ions in the regulation of secretory and endocytotic activity in pituitary tissue slices

Cl- ions are known regulators of Ca2+-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 Ca2--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-gamma-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 Ca2+ channels.