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

Abstract

Aims: Ca²⁺ 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 Ca²⁺ currents and proliferation in pituitary tumor GH3 cells.

Main methods: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca²⁺ 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 48h) induced a significant increase in total Ca²⁺ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca²⁺ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. 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 Ca²⁺ 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 Ca²⁺ current density and this phenomenon impacts proliferation rate in GH3 cells.

Keywords: Ca(2+) channels; Cell proliferation; FRET-imaging; Forskolin; Patch-clamp; cAMP.