Intracellular Cl- modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins

Abstract

We used the whole-cell patch-clamp technique to monitor changes in membrane capacitance (Cm) 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 3.2microM at 4mM [Cl-]i to 0.7microM at 154mM [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], 100microM) the maximal rate of Cm increase (dCm/dt) was enhanced at 4 and 14mM [Cl-]i, but it was not affected at 154mM [Cl-]i. In contrast, the secretory response, measured as a percentage of resting Cm 10min after the start of recordings, was reduced at 154mM [Cl-]i, but not affected at 4mM [Cl-]i. Only with 154mM [Cl-]i did intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) (GDP[beta-S], 500microM) inhibit dCm/dt as well as relative secretory responses. The presence of AlF4(-) (30microM) or a 7-h pretreatment of cells with pertussis toxin (250ng/ml) significantly reduced both the maximal dCm/dt and relative secretory responses, but only in the presence of 154mM [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 GTP-binding proteins in the secretory activity of melanotrophs.