Protein kinase C activates the respiratory burst of fertilization, but not cortical granule exocytosis, in ionophore-stimulated sea urchin eggs.
In a "respiratory burst", fertilized sea urchin eggs consume oxygen to produce H2O2 as an extracellular oxidant to crosslink their protective surface envelopes. The egg generates H2O2 via a NADPH-specific oxidase that requires protein kinase C for activation. To further study the physiological regulation of the respiratory burst and cortical granule exocytosis, we have measured azide-insensitive oxygen uptake and fertilization envelope assembly in ionophore-stimulated eggs. Procaine, trifluoperazine, staurosporine, and H-7, which inhibit protein kinase C by different mechanisms, suppressed egg oxygen consumption without affecting fertilization envelope assembly. In contrast, both exocytosis and oxygen uptake were blocked in N-ethylmaleimide-treated eggs. When the eggs were stimulated with ionophore in Na-free artificial seawater, which prevents the increase in pHi, oxidase activity was inhibited. This effect was reversed by elevation of cytoplasmic pH with the membrane-permeant base NH4Cl. We conclude that protein kinase C was not involved in the events downstream from the ionophore-dependent elevation of Ca2+ which induced cortical granule exocytosis. However, the respiratory burst was inhibited despite the increase in Ca2+ that triggered exocytosis. The likely target for inhibition of the burst was protein kinase C. Cytoplasmic alkalinization was necessary for optimal rates of H2O2 synthesis, further implicating pHi as a regulator of the egg oxidase.
Heinecke
J W
JW
Department of Medicine, University of Washington, Seattle 98195.
Shapiro
B M
BM
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D005089:Exocytosis; D005306:Fertilization / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010063:Ovum / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D010101:Oxygen Consumption / Q000187:drug effects*; D010640:Phenothiazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D012617:Sea Urchins; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D010640:Phenothiazines; D013438:Sulfhydryl Compounds; D000001:Calcimycin; D004247:DNA; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D002118:Calcium