Stimulation of Ca(2+)-independent exocytosis in rat pituitary gonadotrophs by G-protein.

We employed the whole-cell recording technique in conjunction with fluorometry to measure cytosolic Ca(2+) concentration ([Ca(2+)](i)) and exocytosis (capacitance measurement) in single, identified rat gonadotrophs. Direct activation of G-protein (via intracellular dialysis of non-hydrolysable analogues of GTP, but not of GDP) triggered a slow rise in capacitance even in the presence of a fast intracellular Ca(2+) chelator. The broad-spectrum kinase inhibitors H7 and staurosporine did not prevent this Ca(2+)-independent exocytosis, ruling out the involvement of the cAMP and PKC pathways. AlF(4)(-), a potent stimulator of heterotrimeric G-proteins, failed to stimulate any exocytosis when the intracellular Ca(2+) store was depleted, implicating the involvement of AlF(4)(-)-insensitive G-protein(s). Maximal stimulation of Ca(2+)-independent exocytosis by GTP analogues did not reduce the number of readily releasable granules that were available subsequently for Ca(2+)-dependent release. The last finding raises the possibility that the G-protein-stimulated Ca(2+)-independent exocytosis may regulate a pool of granules that is distinct from the Ca(2+)-dependent pool.