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Am J Physiol Endocrinol Metab 273: E850-E858, 1997;
0193-1849/97 $5.00
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Vol. 273, Issue 5, E850-E858, November 1997

Stimulation of Ca2+ influx in alpha T3-1 gonadotrophs via the cAMP/PKA signaling system

Marjan Hezareh, Werner Schlegel, and Stephen R. Rawlings

Fondation pour Recherches Médicales, University of Geneva, CH-1211 Geneva 4, Switzerland

To investigate the regulation of free cytosolic calcium concentration ([Ca2+]i) by the adenosine 3',5'-cyclic monophosphate (cAMP) signaling system in clonal gonadotrophs, microfluorimetric recordings were made in single indo 1-loaded alpha T3-1 cells. Forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or a low concentration (100 pM) of the hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated Ca2+ step responses or repetitive Ca2+ transients, which were blocked by the removal of extracellular Ca2+ by the dihydropyridine (DHP) (+)PN 200-110 or by preincubation with the protein kinase A (PKA) antagonist H-89 (10 µM). Thus activation of the cAMP/PKA system in alpha T3-1 gonadotrophs stimulates Ca2+ influx through DHP-sensitive (L-type) Ca2+ channels. In contrast, high PACAP concentrations (100 nM) stimulated biphasic Ca2+ spike-plateau responses. The Ca2+ spike was independent of extracellular Ca2+, and similar responses were observed by microperfusion of individual cells with D-myo-inositol 1,4,5-trisphosphate, suggesting the involvement of the phospholipase C (PLC) signaling pathway. The Ca2+ plateau depended on Ca2+ influx, was blocked by (+)PN 200-110, but was only partially blocked by H-89 pretreatment. In conclusion, PACAP stimulates [Ca2+]i increases in alpha T3-1 gonadotrophs through both the PLC and adenylate cyclase signaling pathways. Furthermore, this is the first clear demonstration that the cAMP/PKA system can mediate changes in [Ca2+]i in gonadotroph-like cells.

pituitary adenylate cyclase-activating polypeptide; adenosine 3',5'-cyclic monophosphate; protein kinase A; vasoactive intestinal polypeptide; H-89; anterior pituitary cells; L-type calcium channels


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