Caffeine-induced Ca2+ transients and exocytosis in Paramecium cells. A correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis.
Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within </=3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9-28 degrees C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent >/=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.
Klauke
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N
Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany.
Plattner
H
H
D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects*; D005614:Freeze Fracturing; D007700:Kinetics; D008854:Microscopy, Electron; D008954:Models, Biological; D016805:Paramecium tetraurelia / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure
D002110:Caffeine; D002118:Calcium