In general, calcium has been believed to control a variety of cellular processes as a signal transducer, with a high degree of spatial and temporal precision. For the determination of intracellular free-calcium concentrations [( Ca2+]i), the highly selective Ca2+ indicators, quin2/AM and fura2/AM, have been widely used in many mammalian and plant cells. However, intact cells of the cellular slime mold Dictyostelium discoideum Ax-2 are generally impermeable to externally added drugs, thus resulting in a failure to determine [Ca2+]i. Introduction of quin2/AM and fura2/AM by electroporation allowed us to measure [Ca2+]i in D. discoideum cells. The fluorescence images of fura2-loaded single cells showed that resting [Ca2+]i in vegetative and aggregation-competent cells is around 50 nM. Caffeine (10 mM) gave a transient increase in [Ca2+]i, which illustrated a normal responsive ability of electroporated cells to the externally added stimulus. Application of the chemoattractant, cAMP (20 nM), to aggregation-competent cells induced a rapid increase in [Ca2+]i within 1-2 s, and the [Ca2+]i level increased to about four-fold higher than the resting [Ca2+]i within 30 s of chemotactic stimulation. This was followed by a gradual decrease of [Ca2+]i to the basal level. These results strongly suggest that [Ca2+]i is a primary messenger in signal transduction, particularly during the chemotactic response of Dictyostelium cells.
Caffeine Calcium