Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA

Abstract

A new method is described for measuring intracellular free calcium concentrations, [(Ca²⁺_i)], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca²⁺)_i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca²⁺)_i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca²⁺)_i] and show three times as large a response to cAMP stimulation as prespore cells.