Endocannabinoids And The Control Of Vascular Tone

我们先前的研究表明，血管内皮细胞中存在一种新的大麻素受体。我们最近报道，CB类似物“异常大麻二醇”(ABN-CBD)不能与两种已知的CB受体CB1和CB2结合，但在松弛大鼠离体肠系膜动脉节段时起到完全的激动剂作用。ABN-CBD的血管扩张作用是内皮依赖性的，对甲氨蝶呤敏感，可被BKCa通道阻滞剂查氏毒素抑制，但不能被一氧化氮合酶抑制剂L-NAME或香草素VR1拮抗剂卡萨西平所抑制。大麻二醇类似物O-1918不与CB1或CB2受体结合，在浓度达到30微摩尔时不引起血管松弛，但对ABN-CBD和ANANDAME的血管松弛作用有浓度依赖性(1-30微摩尔)抑制作用。在麻醉小鼠中，O-1918呈剂量依赖性地抑制ABN-CBD的降压作用，但不能抑制CB1激动剂HU-210的降压作用。在HUVEC中，ABN-CBD可诱导蛋白激酶B/AKT和p42/44 MAP激酶的磷酸化，该作用可被O-1918、PTX和PI3激酶抑制剂所抑制。这些结果表明，ABN-CBD是一种选择性激动剂，O-1918是一种新的内皮“anandamide受体”的无声拮抗剂，该受体不同于CB1和CB2受体，并通过Gi/Go偶联到PI3激酶/AKT信号通路。 在最近发表的另一项研究中，我们检测了ABN-CBD对人脐静脉内皮细胞(HUVEC)的电生理效应。电压阶跃产生非失活的外向电流，可被硫杆菌毒素或细胞内BAPTA消除。该作用可被PTX或O-1918抑制，但不受CB1或CB2拮抗剂的影响。CGMP也增强了这一电流，ABN-CBD增加了细胞内cGMP的水平。ABN-CBD对外向电流的增强作用可被PKG或可溶性鸟苷环化酶抑制剂阻断。我们的结论是，在HUVEC中激活对ABN-CBD敏感的GPCR通过通过PKG发出信号来增强BKCa电流，从而增加通道的可用性或对钙的敏感性。 我们以前的研究表明，巨噬细胞衍生的内源性大麻素与内毒素(LPS)诱导的低血压有关。我们现在提供的证据表明，内毒素通过CD14/MAP激酶/PI-3激酶/核因子-kappaB途径诱导巨噬细胞合成花生胺，而不依赖于血小板激活因子(PAF)。相反，2-花生四烯基甘油的合成不受内毒素的影响，但被PAF促进。来自脂肪酸氨基水解酶(FAAH)缺陷小鼠的巨噬细胞对LPS的反应是，与来自野生型小鼠的类似处理的细胞相比，正常受体大鼠的ANANDAME增加更多，并引起更大的低血压。结果表明，内毒素引起的降压与β-氨基酚的合成不同，而α-氨基酚参与了内毒素诱导的降压。 在与N·斯特拉博士的一项合作研究中，ABN-CBD受体在小胶质细胞中的存在及其参与细胞迁移的控制已被记录在案。这一结果为这些受体拮抗剂在神经炎症性疾病中的应用提供了可能。

Our previous study has indicated the existence of a novel cannabinoid (CB) receptor in vascular endotheliu. We recently reported that the CB analog "abnormal cannabidiol" (abn-cbd) does not bind to the two known CB receptors, CB1 and CB2, yet acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasodilation by abn-cbd is endothelium-dependent, PTX-sensitive, and is inhibited by the BKCa channel inhibitor charybdotoxin, but not by the NO synthase inhibitor L-NAME or the vanilloid VR1 antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB1 or CB2 receptors and does not cause vasorelaxation at concentrations up to 30 micromolar, but causes concentration-dependent (1-30 micromolar) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd, but not of the CB1 agonist HU-210. In HUVEC, abn-cbd induces phosphorylation of protein kinase B/akt and p42/44 MAP kinase, which is inhibited by O-1918, by PTX and by PI3 kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and O-1918 a novel, silent antagonist of an endothelial "anandamide receptor" which is distinct from CB1 and CB2 receptors and is coupled thropugh Gi/Go to the PI3 kinase/akt signaling pathway. In another recently published study we examined the electrophysiological effects of abn-cbd in human umbilical vein endothelial cells (HUVEC). Voltage steps produced non-inactivating outward currents abolished by iberiotoxin or intracellular BAPTA. ABN-cbd potentiated this current, and this effect was inhibited by PTX or O-1918, but was unaffected by CB1 or CB2 antagonists. cGMP also potentiated this current, and abn-cbd increased the cellular levels of cGMP. Potentiation of the outward current by abn-cbd was blocked by inhibitors of PKG or soluble guanylate cyclase. We conclude that activation of an abn-cbd sensitive GPCR in HUVEC potentiates BKCa current by signalling through PKG to increase channel availability or sensitivity to calcium. Our previous studies implicated macrophage-derived endocannabinoids in endotoxin (LPS)-induced hypotension. We now provide evidence that LPS induces anandamide synthesis in macrophages via a CD14/MAP kinase/PI-3 kinase/NF-kappaB pathway, independently of platelet activating factor (PAF). In contrast, the synthesis of 2-arachidonoyl glycerol is unaffected by LPS but increased by PAF. Macrophages from mice deficient in fattyacid amido hydrolase (FAAH) respond to LPS with a greater increase in anandamide and elicit greater hypotension in normal recipient rats than similarly treated cells from wild-type mice. We conclude that anandamide and 2-AG synthesis are differentially regulaterd and anandamide rather than @-AG is involved in LPS-induced hypotension. In a collaborative study with Dr. N. Stella, the presence of abn-cbd receptors in microglia and their involvement in the control of cell migration has been documented. The results raise the possible therapeutic use of antagonist of these receptors in neuroinflammatory diseases.