Ethanol triggers neural crest apoptosis through the selective activation of a pertussis toxin-sensitive G protein and a phospholipase Cbeta-dependent Ca2+ transient.
BACKGROUND
Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient.


METHODS
Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes.


RESULTS
Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells.


CONCLUSIONS
We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.
Garic-Stankovic
Ana
A
Department of Nutritional Sciences, University of Wisconsin-Madison, Madison Wisconsin 53706, USA.
Hernandez
Marcos R
MR
Chiang
Po Jen
PJ
Debelak-Kragtorp
Katherine A
KA
Flentke
George R
GR
Armant
D Randall
DR
Smith
Susan M
SM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002642:Chick Embryo; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000187:drug effects; D004963:Estrenes / Q000494:pharmacology; D000431:Ethanol / Q000633:toxicity*; D005260:Female; D063647:Fetal Alcohol Spectrum Disorders / Q000503:physiopathology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D009432:Neural Crest / Q000187:drug effects* / Q000473:pathology; D037342:Pertussis Toxin / Q000494:pharmacology*; D054799:Phospholipase C beta; D010742:Phospholipid Ethers / Q000494:pharmacology; D011247:Pregnancy; D011760:Pyrrolidinones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology*
D004963:Estrenes; D007527:Isoenzymes; D010742:Phospholipid Ethers; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C026659:edelfosine; D000431:Ethanol; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium