Increase in Ca2 + current by sustained cAMP levels enhances proliferation rate in GH3 cells

Abstract Aims Ca 2 + and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca 2 + currents and proliferation in pituitary tumor GH3 cells. Main methods Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca 2 + current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. Key findings Sustained forskolin treatment (24 and 48 h) induced a significant increase in total Ca 2 + current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca 2 + current density. However, the maximum effect of dbcAMP occurred only after 72 h incubation, whereas forskolin showed maximal effect at 48 h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca 2 + channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. Significance We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca 2 + current density and this phenomenon impacts proliferation rate in GH3 cells.