Garic-Stankovic Ana, Hernandez Marcos R, Chiang Po Jen, Debelak-Kragtorp Katherine A, Flentke George R, Armant D Randall, Smith Susan M
Department of Nutritional Sciences, University of Wisconsin-Madison, Madison Wisconsin 53706, USA.
Alcohol Clin Exp Res. 2005 Jul;29(7):1237-46. doi: 10.1097/01.alc.0000172460.05756.d9.
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.
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.
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.
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.
酒精是一种强效神经毒素，可引发发育中胎儿神经元群体的选择性凋亡。对于神经嵴细胞，临床相关的乙醇水平（0.3%）会迅速引发磷脂酶C（PLC）依赖性的细胞内Ca2+瞬变，这足以激活细胞凋亡。我们研究了这种Ca2+瞬变的生化起源。
在乙醇刺激前，用PLC信号通路的激动剂和拮抗剂预处理三对体节的鸡胚（第8阶段-）。使用Fluo-3对产生的细胞内Ca2+释放进行定量；使用活性染料评估细胞凋亡。
用PLC拮抗剂U73122或ET-18-OCH3预处理胚胎证实，磷酸肌醇特异性PLC对于乙醇依赖性Ca2+瞬变和随后的细胞死亡都是必需的。乙醇迅速升高了胚胎头部含有神经嵴祖细胞部分的细胞内肌醇-1,4,5-三磷酸[Ins(1,4,5)P3]水平。Ins(1,4,5)P3受体拮抗剂西司他汀C阻断了乙醇依赖性Ca2+瞬变的出现。用泛Gα蛋白拮抗剂GDPβS预处理，但不用酪氨酸激酶拮抗剂染料木黄酮预处理，可抑制乙醇引发Ca2+瞬变的能力，这表明PLC活性和Ins(1,4,5)P3浓度的升高源于异源三聚体G蛋白的刺激。为了探究这种G蛋白的身份，用G蛋白拮抗剂处理胚胎。百日咳毒素和NF023抑制了乙醇诱导的Ca2+瞬变和随后的神经嵴细胞凋亡，而苏拉明的抑制作用较弱。C3外切酶在很宽的浓度范围内具有胚胎致死性，这与Rho家族GTP酶参与神经嵴发育的观点一致。通过免疫染色在神经嵴细胞中鉴定出Gαi2。
我们提出Gαi/o蛋白激活以及随后Gβγ与PLCβ的相互作用在介导乙醇对发育中的神经嵴的促凋亡作用中起作用。
