Ca2+ oscillations and Ca2+ influx in Xenopus oocytes expressing a novel 5-hydroxytryptamine receptor.
1. We expressed a novel 5-hydroxytryptamine receptor (SRL) in Xenopus oocytes and monitored cytosolic Ca2+ through the endogenous Ca(2+)-dependent Cl- channel activity using the double electrode voltage-clamp technique. 2. 5-Hydroxytryptamine (5-HT; 200 nM) led to an initial rapid oscillatory current followed by a pronounced secondary one, which lasted long after 5-HT wash-out (20-40 min) and was not affected by the receptor antagonist yohimbine. 3. Both phases of the current were abolished by heparin demonstrating a key role for IP3-induced Ca2+ release. 4. Caffeine (10 mM) alone did not evoke a current but reduced both phases of the current evoked by 5-HT. Ryanodine had no effect. No evidence for Ca(2+)-induced Ca2+ release was found. 5. The secondary current activated by 5-HT was sensitive to changes in extracellular Ca2+, suggesting it was evoked by Ca2+ influx. Reducing external Na+ did not affect this current, demonstrating that it was rather specific for Ca2+. 6. The Ca2+ influx pathway was much more sensitive to Cd2+ than other divalent ions (Co2+, Mn2+, Sr2+, Ba2+). It was insensitive to verapamil. 7. Injection of D-myo-inositol 1,4,5-trisphosphate, 3-deoxy-3-fluoro (IP3-F; an analogue not metabolized to D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4)), evoked either an oscillatory current or a rapid current followed by a sustained secondary one. The latter was sensitive to external Ca2+ and was blocked by Cd2+. Heparin dramatically reduced the IP3-F-evoked current. 8. Perfusion in Ca(2+)-free solution, once a secondary current had been generated, significantly decreased the amount of intracellular Ca2+ mobilized by 5-HT, indicating that the Ca2+ influx pathway plays an important role in pool refilling. 9. Block of Ca2+ influx by Cd2+ in cells that were oscillating transiently increased the amplitude and then either abolished the oscillations or made them irregular. This effect was also elicited by increasing external Ca2+. 10. These results demonstrate that 5-HT, acting via IP3, both releases Ca2+ from internal stores and evokes a pronounced Ca2+ influx. This last step is activated by pool depletion and is important for both refilling of the agonist-sensitive stores and modifying the oscillatory pattern.
Parekh
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Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Foguet
M
M
Lübbert
H
H
Stühmer
W
W
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D007293:Inosine Triphosphate / Q000378:metabolism; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D018075:RNA, Complementary / Q000378:metabolism; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D014981:Xenopus; D015016:Yohimbine / Q000494:pharmacology
D018118:Chloride Channels; D018075:RNA, Complementary; D011985:Receptors, Serotonin; D002104:Cadmium; D007293:Inosine Triphosphate; D015016:Yohimbine; D012701:Serotonin; D002110:Caffeine; D006493:Heparin; D019204:GTP-Binding Proteins; D002118:Calcium