Mechanism of release of Ca2+ from intracellular stores in response to ionomycin in oocytes of the frog Xenopus laevis.

1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole‐cell currents using the two‐electrode voltage‐clamp method. 2. The extracellular application of ionomycin, a selective Ca2+ ionophore, evoked an inward current consisting of a spike‐like fast component followed by a long‐lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose‐dependent manner and was dependent on Cl‐. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0.1 microM). In contrast, a larger ionomycin response (1 microM), consisting of fast and slow components, was followed by refractory period. Washing for 50‐90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis‐(O‐aminophenoxy)‐ethane‐N,N,N',N'‐tetraacetic acid (BAPTA AM, 1‐10 microM), a membrane‐permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX, 0.3‐2.0 microgram/ml), a blocker of guanine nucleotide‐binding regulatory proteins (G proteins). In contrast, the response to acetylcholine (ACh), which is known to occur via a G protein, was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mM EGTA), whereas the slow component was suppressed. 7. Injection of inositol 1,4,5‐trisphosphate (IP3) following a response to extra‐cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast, ionomycin evoked a small inward current when it was applied after an inward‐current response evoked by IP3 injection, whereas a second injection of IP3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins, resulting in activation of Ca(2+)‐activated Cl‐ channels, (b) ionomycin mainly acts on the same intracellular Ca2+ stores as IP3, and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response, whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores.