Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine

Abstract

A transient rise in intracellular Ca 2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca 2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP 3-sensitive and -insensitive Ca 2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca 2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca 2+ release that was insensitive to heparin or ruthenium red. Ca 2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ ATPase. Variable Ca 2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca 2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca 2+ release during fertilization was measured. Unlike the caffeineand ryanodine-sensitive Ca 2+-induced Ca 2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca 2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca 2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca 2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca 2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca 2+-induced Ca 2+ release of somatic cells.