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

Abstract

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.