Ethanol Triggers Neural Crest Apoptosis through the Selective Activation of a Pertussis Toxin–Sensitive G Protein and a Phospholipase Cβ–Dependent Ca2+ Transient

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Abstract
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‐Gα protein antagonist GDPβS, 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. Gαi2 was identified by immunostaining in the neural crest cells.Conclusion: We propose a role for Gαi/o protein activation and subsequent interaction of Gβγ with PLCβ in mediating the proapoptotic effects of ethanol upon the developing neural crest.

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