Refilling of cortical XXXX stores in Paramecium cells: in situ analysis in correlation with store-operated @entity460 influx.

@entity141 @entity460 @entity18650
This is the first thorough study of refilling of a cortical @entity460 store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a @entity141 -influx" superimposed to @entity141 -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [ @entity141 ](o) in the medium, from approximately 20-25% at [ @entity141 ](o) < or = 0.25 microM to 100% at [ @entity141 ](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [ @entity141 ] during membrane fusion. Next, we analyzed @entity141 -specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on @entity141 -signaling. In double trigger experiments, with @entity18650 in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical @entity141 -signal (due solely to @entity141 released from cortical stores) recovered with t(1/2) approximately 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+ @entity141 (o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45 @entity141 -uptake by isolated ASs. In sum we find, (i) exogenous @entity141 increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) @entity141 -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical @entity460 stores goes on over hours and thus is much slower than expected.
