Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB₁ (CB₁XS) or CB₂ (CB₂XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/β-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB₁ or CB₂ expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/β hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB₁ receptor expression increased lengths of short (<30 μm) and long (>30 μm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gβγ inhibitor gallein, and β-arrestin inhibitor barbadin did not significantly alter long projection length in CB₁XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB₁ receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB₁ receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB₂ receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB₁ receptor signaling through Gi/o, Gβγ, and β-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

开发了稳定表达外源性 CB ₁ (CB ₁ XS) 或 CB ₂ (CB ₂ XS) 受体的人类 SH-SY5Y 神经母细胞瘤细胞，以研究神经元投射延伸中的内源性大麻素信号传导。与亲本 SH-SY5Y 相比，大麻素受体的表达不会改变增殖率、活力或细胞凋亡。CB ₁或 CB ₂未改变内源性大麻素系统酶（二酰基甘油脂肪酶、单酰基甘油脂肪酶、含有蛋白质 6 和 12 的α/β-水解酶结构域、N-酰基磷脂酰乙醇胺-磷脂酶 D 和脂肪酸酰胺水解酶）的转录本表达。在 SH-SY5Y 细胞中对内源性大麻素配体 2-花生四烯酸甘油 (2-AG) 和 anandamide 进行了定量，二酰基甘油脂肪酶抑制剂四氢脂抑素将 2-AG 丰度降低了 90%，但并未改变 anandamide 丰度。M3 毒蕈碱激动剂 oxotremorine M 和单酰基甘油脂肪酶抑制剂和含有 α/β 水解酶结构域的蛋白质 6 和 12 增加了 2-AG 丰度。CB ₁受体表达增加了短 (<30 μm) 和长 (>30 μm) 投射的长度，四氢脂抑素显着降低了这种效果，表明受到内源性 2-AG 的刺激。百日咳毒素、Gβγ 抑制剂gallein 和β-arrestin 抑制剂barbadin 没有显着改变CB _{1 中的}长投射长度XS，但显着减少了短投影，其中鸡肝素具有最大的抑制作用。rho 激酶抑制剂 Y27632 增加了 CB ₁受体介导的长投射延伸，表明肌动蛋白细胞骨架受累。CB ₁受体表达增加 GAP43 和 ST8SIA2 mRNA 并减少 ITGA1 mRNA，而 CB ₂受体表达增加 NCAM 和 SYT mRNA。我们建议 2-AG 的基础内源性产生通过 Gi/o、Gβγ 和 β-抑制蛋白机制提供 CB ₁受体信号传导的自分泌刺激，以促进神经发生，并且 rho 激酶影响过程扩展。