CYCLIC-AMP STIMULATES LUTEINIZING-HORMONE (LUTROPIN) EXOCYTOSIS IN PERMEABILIZED SHEEP ANTERIOR-PITUITARY-CELLS - SYNERGISM WITH PROTEIN-KINASE-C AND CALCIUM

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus α-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+](free)) is clamped by Ca2+ buffers. At resting [Ca2+](free) (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 μM. cAMP-stimulated exocytosis required millimolar concentrations of MgAtp, as has been found with Ca2+- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2+-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7 fold and decreasing the aC50 for cAMP to 3 μM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+](free). Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [C12+](free), our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.