Ca2+ oscillations and Ca2+ influx in Xenopus oocytes expressing a novel 5‐hydroxytryptamine receptor.

Abstract

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. © 1993 The Physiological Society