CA2-OOCYTES EXPRESSING A NOVEL 5-HYDROXYTRYPTAMINE RECEPTOR( OSCILLATIONS AND CA2+ INFLUX IN XENOPUS)

1. We expressed a novel 5-hydroxytryptamine receptor (SRL) in Xenopus oocytes and monitored cytosolic Ca2+ through the endogenous Ca2+-depen dent Cl- channel activity using the double electrode voltage-clamp tec hnique. 2. 5-Hydroxytryptamine (5-HT; 200 nM) led to an initial rapid oscillatory current followed by a pronounced secondary one, which last ed long after 5-HT wash-out (20-40 min) and was not affected by the re ceptor antagonist yohimbine. 3. Both phases of the current were abolis hed by heparin demonstrating a key role for IP3-induced Ca2+ release. 4. Caffeine (10 mM) alone did not evoke a current but reduced both pha ses of the current evoked by 5-HT. Ryanodine had no effect. No evidenc e for Ca2+-induced Ca'' release was found. 5. The secondary current ac tivated by 5-HT was sensitive to changes in extracellular Ca2+, sugges ting it was evoked by Ca2+ influx. Reducing external Na+ did not affec t this current, demonstrating that it was rather specific for Ca2+.6. The Ca2+ influx pathway was much more sensitive to Cd2+ than other div alent ions (Co2+, Mn2+, Sr2+, Ba2+). It was insensitive to verapamil. 7. Injection of D-Myo-inositol 1,4,5-trisphosphate,3-deoxy-3-fluoro (I P3-F; an analogue not metabolized to D-Myo-inositol 1,3,4,5-tetrakisph osphate (IP4)), evoked either an oscillatory current or a rapid curren t followed by a sustained secondary one. The latter was sensitive to e xternal Ca2+ and was blocked by Cd2+. Heparin dramatically reduced the IP3-F-evoked current. 8. Perfusion in Ca2+-free solution, once a seco ndary current had been generated, significantly decreased the amount o f intracellular Ca2+ mobilized by 5-HT, indicating that the Ca2+ influ x pathway plays an important role in pool refilling. 9. Block of Ca2influx by Cd2+ in cells that were oscillating transiently increased th e amplitude and then either abolished the oscillations or made them ir regular. This effect was also elicited by increasing external Ca2+. 10 . These results demonstrate that 5-HT, acting via IP3, both releases C a2+ from internal stores and evokes a pronounced Ca2+ influx. This las t step is activated by pool depletion and is important for both refill ing of the agonist-sensitive stores and modifying the oscillatory patt ern.