Cyclic AMP- and Ins(1,4,5)P3-induced Ca2+ fluxes in permeabilised cells of Dictyostelium discoideum: cGMP regulates Ca2+ entry across the plasma membrane

Abstract

Transduction of chemotactic signals in Dictyostelium discoideum apparently involves a precise regulation of the cytosolic Ca2+ concentration. Cyclic AMP stimulation causes Ca2+ influx followed by Ca2+ extrusion, the magnitude of extrusion depending on the state of differentiation. Here, we show that the cAMP receptor controls Ca2+ influx both at the level of entry across the plasma membrane and at the level of transport into Ca2+-sequestering organelles. The use of permeabilised cells allowed us to discriminate between both fluxes. Permeabilised cells still showed the cAMP-induced Ca2+ uptake. The flux across the plasma membrane was more sensitive to Ba2+ and Mn2+, respectively, than Ca2+ sequestration. We have shown previously, using stmF mutants, that cGMP regulates Ca2+ influx. We confirmed this result with the membrane-permeant cGMP-analogue, Sp-8-Br-cGMPS, which enhanced the cAMP-induced Ca2+ influx in intact cells but not the uptake in permeabilised cells, indicating that cGMP regulates Ca2+ influx across the plasma membrane. Occasionally, a fast transient Ca2+ efflux, preceding the influx, occurred in intact cells. A small cAMP-induced Ca2+ release was also found in permeabilised cells. A similarly sized Ca2+ release was elicited by Ins(1,4,5)P3 and could be substituted for by GTP or GTPgammaS. This result suggests that rapid Ca2+ release can be mediated by Ins(1,4,5)P3.