Journal
LIFE SCIENCES
Volume 192, Issue -, Pages 144-150Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.lfs.2017.11.040
Keywords
Patch-clamp; FRET-imaging; cAMP; Forskolin; Ca2+ channels; Cell proliferation
Funding
- Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [308007/2013-1]
- Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) [10274/16, 00056/15]
- Fundacao Mineira de Educacao e Cultura (Universidade FUMEC)
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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 48 h) 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 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 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.
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