A Method for Studying cAMP-relay in Dictyostelium discoideum: the Effect of Temperature on cAMP-relay

摘要

A simple assay has been developed to quantify the cAMP-relay in Dictyostelium discoideum. The assay is based on the stimulation of cells, in the presence of a phosphodiesterase inhibitor, with 2'-deoxyadenosine 3',5'-monophosphate (dcAMP) at a concentration which saturates cell surface cAMP receptors. The accumulation of cAMP is measured by an isotope dilution assay using a cAMP-binding protein with such a low affinity for dcAMP that purification of cAMP can be omitted. The accumulation of extracellular and total cAMP was measured at 20 °C and at 0 °C. At 20 °C the rate of cAMP synthesis increased immediately after stimulation, reaching a maximum after 1 min, with a return to undetectable levels after about 4 min. The secretion of cAMP at this temperature was proportional to the intracellular cAMP concentration and reached a maximum at about 2 min. At 20 °C the cAMP-relay was essentially completed within 3-4 min, yielding about 35 pmol cAMP per 10^7 cells. At 0 °C the same amount of cAMP was relayed, but the rate of cAMP synthesis was reduced about 2.5 fold. The secretion rate depended on the intracellular cAMP concentration with a delay period of 1.25 ± 0.5 min, and was reduced about 5.5-fold compared to secretion at 20 °C. Due to the relatively slow secretion and fast synthesis of cAMP, an approximately twofold higher intracellular cAMP concentration was reached at 0 °C compared to 20 °C, and this persisted for about 15 min. Taking into account that the secretion rate is proportional to intracellular cAMP concentration, these results indicate that the mechanism underlying cAMP secretion is slowed 13-fold when the temperature is reduced from 20 °C to 0 °C.