Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilizationfront cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than tota l Ca concentrations, [Ca], govern cellular responses, analysis of total cal cium fluxes would be important to fully understand the very complex Ca2+ dy namics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mob ilization from cortical stores during synchronous (less than or equal to 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitu tion (modified for Ca retention) and X-ray microanalysis which registers to tal calcium concentrations, [Ca], When the extracellular free calcium conce ntration, [Ca2+](e), is adjusted to similar to 30 nM, i.e. slightly below t he normal free intracellular calcium concentration, [Ca2+](i) = 65 nM, exoc ytosis stimulation causes release of 52% of calcium from stores within 80 m s. At higher extracellular calcium concentration, [Ca2+](c) = 500 mu M, Ca2 + release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal value s within 1 s, This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization fro m stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stim ulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, partic ularly with increasing extracellular free calcium, [Ca2+](e). Cortically en riched mitochondria rapidly gain Ca signals during stimulation. Balance cal culations indicate that total Ca2+ flux largely exceeds values of intracell ular free calcium concentrations locally required for exocytosis (as determ ined previously). Our approach and some of our findings appear relevant als o for some other secretory systems.