INTRACELLULAR CL- MODULATES CA2+-INDUCED EXOCYTOSIS FROM RAT MELANOTROPHS THROUGH GTP-BINDING PROTEINS

We used the whole-cell patch-clamp technique to monitor changes in membrane capacitance (C-m) to study the influence of cytosolic concentration ([Cl-](i)) on the secretory activity of rat melanotrophs. The sensitivity of the secretory machinery to Ca2+ was enhanced in the presence of a high [Cl-](i). The free concentration of Ca2+ required for half-maximal secretory activity was reduced from 3.2 mu M at 4 mM [Cl-](i) to 0.7 mu M at 154 mM [Cl-](i). To study whether the modulation of secretory activity by Cl- involves guanosine 5'-triphosphate-(GTP-) binding proteins, cells were dialysed with non-hydrolysable GTP and GDP analogues, fluoroaluminate (AlF4-), or were pretreated with pertussis toxin, With guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S], 100 mu M) the maximal rate of C-m increase (dC(m)/dt) was enhanced at 4 and 14 mM [Cl-](i), but it was not affected at 154 mM [Cl-](i). In contrast, the secretory response, measured as a percentage of resting C-m 10 min after the start of recordings, was reduced at 154 mM [Cl-](i), but not affected at 4 mM [Cl-](i). Only with 154 mM [Cl-](i) did intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) (GDP[beta-S], 500 mu M) inhibit dC(m)/dt as well as relative secretory responses. The presence of AlF4- (30 mu M) or a 7-h pretreatment of cells with pertussis toxin (250 ng/ml) significantly reduced both the maximal dC(m)/dt and relative secretory responses, but only in the presence of 154 mM [Cl-](i). Since the effects of GDP[beta-S], AlF4-, and pertussis toxin pretreatment were only detected with a high [Cl-](i), we conclude that modulation by Cl- of secretory activity of rat melanotrophs is mediated through GTP-binding proteins. Furthermore, the effects of AlF4- and pertussis toxin indicate a role of heterotrimeric CTP-binding proteins in the secretory activity of melanotrophs.