Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg.
The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.
Department of Zoology and Genetics, Iowa State University, Ames 50011-3223, USA. ssshen@iastate.edu
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005306:Fertilization; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism; D008935:Mitoguazone / Q000494:pharmacology; D010063:Ovum / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012617:Sea Urchins / Q000166:cytology*; D010738:Type C Phospholipases / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015544:Inositol 1,4,5-Trisphosphate; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D008935:Mitoguazone; D002118:Calcium