Confocal microfluorimetry of Ca2+ signals evoked in Xenopus oocytes by photoreleased inositol trisphosphate.
1. The subcellular characteristics of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ liberation were studied in Xenopus oocytes by the use of confocal microfluorimetry to monitor Ca2+ signals from minutely localized region of the cell in response to photorelease of InsP3 from a caged precursor. 2. Photorelease of increasing amounts of InsP3 by progressively longer light flashes evoked transient Ca2+ responses that appeared abruptly at a certain threshold duration, and then grew steeply over a narrow range of flash durations to reach a maximum. Further lengthening of flash duration gave no increase in size of the Ca2+ signals, but their rate of rise continued to increase and their duration became longer. Simultaneous measurements of Ca(2+)-activated Cl- currents showed a slightly higher threshold than the Ca2+ signal, and a more graded dependence upon flash duration. 3. The threshold flash durations required to evoke Ca2+ and membrane current signals grew by more than 100-fold as the area of the oocyte exposed to photolysis light was reduced from a square of 140 microns to 5 microns. 4. Ca2+ signals evoked by photoreleased InsP3 began following a dose-dependent latency that was as long as several seconds with low intensity light, but shortened to about 50 ms at maximum intensity. The extrapolated minimum latency with infinite photorelease of InsP3 was about 30 ms. 5. InsP3-evoked membrane currents began 30 ms or longer after the corresponding Ca2+ signals, whereas currents evoked by photorelease of Ca2+ from a caged precursor began within 5 ms of the onset of the light flash. 6. No differences in duration of InsP3-evoked Ca2+ signals were apparent when the confocal measuring spot was positioned close to the plasma membrane or about 10 microns more deeply into the oocyte. At both locations the Ca2+ signals were more prolonged than the associated membrane current signals. 7. Ca2+ signals to a test light flash were suppressed for about 2 s following a conditioning suprathreshold flash, but recovered almost completely after 6 s. The associated membrane current signals were facilitated at short intervals, suppressed at intervals between 0.5 and 3 s, and subsequently recovered more slowly than the Ca2+ signals. 8. Photorelease of InsP3 during 30 s exposures of low intensity evoked trains of repetitive Ca2+ spikes. The overall amplitudes of these responses changed little with increasing in frequency, and became smaller and superimposed on a more sustained elevation of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Parker
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Department of Psychobiology, University of California Irvine 92717.
Ivorra
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003592:Cytophotometry / Q000379:methods; D004305:Dose-Response Relationship, Drug; D005260:Female; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000378:metabolism*; D010782:Photolysis; D011930:Reaction Time / Q000502:physiology; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; C068483:rhod-2; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium