Abstract
Calcium dependent exocytosis from pituitary lactotrophs is known to be regulated by G protein coupled receptors (GPCRs) that regulate intracellular [cAMP]. We have used fura-2 measurements of intracellular calcium concentration along with both population biochemical cAMP measurements and single cell FRET-based cAMP measurements to investigate the relationship between [Ca2+]i and cAMP in clonal rat MMQ lactotrophs. Both VIP and PACAP activation of GPCR coupled to Gs lead to a Ca2+ influx as evident by increase in Ca2+-oscillations. Bypass of GPCRs with the adenylyl cylclase activator forskolin also resulted in strong in increase in Ca2+ oscillations consistent with measured forskolin-induced increase in [cAMP]. Pharmacological activation of PKA using 6-Bn-cAMPor activation of EPAC using 8-cpt-cAMP each resulted in increase in Ca2+ oscillations indicating a role for both cAMP binding proteins in control of calcium dynamics although with differing lag times likely reflecting distinct signaling pathways. The broad-spectrum cAMP phosphodiesterase (PDE) inhibitor IBMX caused an increase in Ca2+ influx without delay. The PDE3 inhibitor milrinone caused a strong increase in Ca2+ after a delay whereas the PDE4 inhibitor rolipram caused an immediate increase in Ca2+. FRET-based measurements of [cAMP] are being used to further analyze these differences. These results suggest a possible different relationship between the various PDE sensitive pools of cAMP and the L-type Ca2+-channels responsible for Ca2+ influx as well as distinct roles for PKA and EPAC in control of Ca2+ dynamics and prolaction secretion.
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