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

• Published in: Alcoholism, clinical and experimental research Vol. 29; no. 7; pp. 1237 - 1246
• Main Authors: Garic-Stankovic, Ana, Hernandez, Marcos R., Chiang, Po Jen, Debelak-Kragtorp, Katherine A., Flentke, George R., Armant, D Randall, Smith, Susan M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.07.2005; Lippincott Williams & Wilkins
• Subjects: Alcoholism and acute alcohol poisoning; Biological and medical sciences; Medical sciences; Toxicology; Fetal alcohol syndrome; Gβγ Protein; Embryo; Calcium; Ethanol; Enzyme; Phospholipase Cp; Islet activating protein; Esterases; Activation; Inorganic element; Neural crest; Chick Embryo; Transitory; Pertussis toxin; Alcoholic beverage; Phospholipase; Newborn diseases; Ca2+ Transients; Cell death; Hydrolases; Gai/o Protein; Apoptosis; G protein
• ISSN: 0145-6008; 1530-0277
• DOI: 10.1097/01.ALC.0000172460.05756.D9

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.