Ca2+ influx modulation of temporal and spatial patterns of inositol trisphosphate-mediated Ca2+ liberation in Xenopus oocytes.
Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger by liberating intracellular Ca2+ and by promoting influx of extracellular Ca2+. We examined the effects of Ca2+ influx on the temporal and spatial patterns of intracellular Ca2+ liberation in Xenopus oocytes by fluorescence imaging of cytosolic free Ca2+ together with voltage clamp recording of Ca(2+)-activated Cl- currents. Oocytes were injected with a poorly metabolized InsP3 analogue (3-F-InsP3; see Introduction) to induce sustained activation of InsP3 signalling, and Ca2+ influx was controlled by applying voltage steps to change the driving force for Ca2+ entry. Positive- and negative-going potential steps (corresponding, respectively, to decreases and increases in Ca2+ influx) evoked damped oscillatory Cl- currents, accompanied by cyclical changes in cytosolic free Ca2+. The source of this Ca2+ was intracellular, since oscillations persisted when Ca2+ entry was suppressed by removing extracellular Ca2+ or by polarization close to the Ca2+ equilibrium potential. Fluorescence recordings from localized (ca 5 microns) spots on the oocyte showed repetitive Ca2+ spikes. Their frequency increased at more negative potentials, but they became smaller and superimposed on a sustained 'pedestal' of Ca2+. Spike periods ranged from about 50 s at +20 mV to 4s at potentials between -60 and -120 mV. Ca2+ spike frequency decreased after removing extracellular Ca2+, but the spike amplitude was not reduced and low frequency spikes continued for at least 30 min in the absence of extracellular Ca2+. Membrane current oscillations decayed in amplitude following voltage steps, while locally recorded Ca2+ spikes did not. This probably arose because Ca2+ release was initially synchronous across the cell, leading to large Ca(2+)-activated Cl- currents, but the currents then diminished as different areas of the cell began to release Ca2+ asynchronously. Fluorescence imaging revealed that Ca2+ liberation in 3-F-InsP3-loaded oocytes occurred as transient localized puffs and as propagating waves. Polarization to more negative potentials increased the frequency of puffs and the number of sites at which they were seen, and enhanced their ability to initiate waves. The frequency and velocity of Ca2+ waves increased at more negative potentials. When the potential was returned to more positive levels, repetitive Ca2+ spikes at first occurred synchronously across the recording area, but this synchronization was gradually lost and Ca2+ waves began at several foci. We conclude that influx of extracellular Ca2+ regulates the temporal and spatial patterns of Ca2+ liberation from InsP3-sensitive intracellular stores, probably as a result of dual excitatory and inhibitory actions of cytosolic Ca2+ on the InsP3 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Yao
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Y
Department of Psychobiology, University of California Irvine 92717.
Parker
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I
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000378:metabolism*; D009930:Organic Chemicals; D014982:Xenopus laevis
D015220:Calcium Channels; D018118:Chloride Channels; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C071336:calcium green; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium