Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
Dev Neurosci. 2010 Mar;32(1):1-18. doi: 10.1159/000235758. Epub 2009 Nov 12.
Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.
研究了海胆（Lytechinus variegatus、Strongylocentrotus droebachiensis、Strongylocentrotus purpuratus、Dendraster excentricus）和海星（Pisaster ochraceus）的胚胎和幼虫中功能性内源性大麻素系统的存在。通过液相色谱/质谱法测量了早期 L. variegatus 胚胎中的花生四烯酰乙醇胺（arachidonoyl ethanolamide，AEA）。AEA 表现出强烈的发育动态，在 8-16 细胞和中囊胚 2 期之间增加了 5 倍以上。在不同的海胆物种和海星中进行的“干扰和挽救”实验表明，AEA 阻止胚胎从囊胚过渡到原肠胚阶段，但即使在高剂量下，对卵裂分裂也没有影响。非选择性大麻素受体激动剂 CP55940 也具有相似的作用，但与 AEA 不同，它还阻断了卵裂分裂。CB1 拮抗剂、AEA 转运抑制剂和阳离子通道瞬时膜电位受体 V1（TrpV1）激动剂 arachidonoyl vanillic 酸（arvanil）以及 arachidonoyl 血清素和多巴胺（AA-5-HT、AA-DA）作为挽救物质，部分或完全阻止了 AEA 或 CP55940 引起的异常胚胎表型。用 [3H]CP55940 对来自胚胎/幼虫的膜制剂进行放射性配体结合未能显示出显著的结合，这与海胆基因组中缺乏 CB 受体同源物一致。然而，当在全细胞裂解物上进行结合时，在 pluteus 阶段观察到少量 [3H]CP55940 结合，该结合可被 CB2 拮抗剂 SR144528 置换。由于已知 AEA 与 TrpV1 和某些 G 蛋白偶联受体（GPCR）具有高亲和力结合，arvanil、AA-5-HT 和 AA-DA 能够挽救胚胎免受 AEA 致畸作用，这表明在海胆中，AEA 和其他内源性大麻素可能同时利用 Trp 和 GPCR 同源物。使用生物信息学和系统发育工具来鉴定 S. purpuratus 海胆基因组中的候选同源物，探讨了这种可能性。鉴定出候选 TrpA1 和 TrpV1 同源物。TrpA1 同源物位于单系群内，包括脊椎动物和无脊椎动物的同源物，而 TrpV1 同源物位于两个不同的 TrpV 样无脊椎动物进化枝中。使用轮廓隐藏 Markov 模型（HMM）搜索确定，海胆 TrpV 同源物中的一个与脊椎动物上皮钙通道（TrpV5-6 家族）的关系比与脊椎动物 TrpV1-4 家族的关系更密切。在海胆基因组中鉴定出候选多巴胺和肾上腺素能 GPCR 同源物，但没有发现大麻素 GPCR，这与早期研究一致。使用 HMM 搜索鉴定出候选多巴胺 D1、D2 或 alpha1-肾上腺素能受体同源物作为脊椎动物大麻素受体的潜在前体，这取决于 CB 受体序列的多重序列比对是否仅由尾索动物和头索动物序列组成，或者还包括脊椎动物序列。
