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 alpha-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 microM. 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 EC50 for cAMP to 3 microM. 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 [Ca2+]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.
Macrae
M B
MB
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
Davidson
J S
JS
Millar
R P
RP
van der Merwe
P A
PA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics; D002463:Cell Membrane Permeability / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis / Q000494:pharmacology* / Q000502:physiology; D004357:Drug Synergism; D005089:Exocytosis / Q000187:drug effects*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D007424:Intracellular Fluid / Q000502:physiology; D007986:Luteinizing Hormone / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000502:physiology*; D011493:Protein Kinase C / Q000493:pharmacokinetics; D012756:Sheep; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007987:Gonadotropin-Releasing Hormone; D000255:Adenosine Triphosphate; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine