Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx.
This is the first thorough study of refilling of a cortical calcium 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 Ca(2+) -influx" superimposed to Ca(2+) -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca(2+)](o) in the medium, from approximately 20-25% at [Ca(2+)](o) < or = 0.25 microM to 100% at [Ca(2+)](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [Ca(2+)] during membrane fusion. Next, we analyzed Ca(2+)-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca(2+) -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca(2+) -signal (due solely to Ca(2+) 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 (+Ca(2+)(o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45Ca(2+) -uptake by isolated ASs. In sum we find, (i) exogenous Ca(2+) increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) Ca(2+) -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.
Mohamed
I
I
Universität Konstanz, Fachbereich Biologie, P.O. Box 5560, 78457 Konstanz, Germany.
Husser
M
M
Sehring
I
I
Hentschel
J
J
Hentschel
C
C
Plattner
H
H
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*; D010247:Paramecium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D002118:Calcium