Endocannabinoids And The Control Of Vascular Tone

适度饮酒与降低心血管发病率有关，而大量饮酒则有相反的效果。研究乙醇(Etoh)对体外培养的人脐静脉内皮细胞(HUVEC)PI3K/AKT通路激活后细胞存活的影响。细胞暴露于低浓度(2-20 mM)的乙醇可迅速诱导Akt的磷酸化，这可被百日咳毒素(PTX)或PI3激酶抑制剂所阻止。PI3激酶/AKT的几个下游效应分子，包括p70S6激酶、糖原合成酶激酶3α/β和IKB-α，也被乙醇磷酸化，后者导致NFkappaB激活3倍。Etoh还以PI3依赖的方式激活p42/44 MAP激酶。乙醇(2-20 mM)可增加内皮细胞一氧化氮合酶活性，该作用可被Akt显性阴性的人脐静脉内皮细胞所阻断。腺苷A1受体拮抗剂DPCX可通过2-20 mM乙醇或腺苷阻止Akt的磷酸化。相反，高浓度(50-100 mM)的乙醇与HUVEC孵育后，DNA片段化和TUNEL检测证实，HUVEC发生线粒体通透性转变和caspase-3激活，继而发生细胞凋亡。乙醇诱导的细胞凋亡不受DCPX的影响，但可被PI3K抑制剂增强。因此，低浓度的乙醇通过腺苷依赖的机制激活内皮细胞的PI3K/AKT通路，而高浓度的乙醇通过腺苷不依赖的机制激活促凋亡的caspase通路可以掩盖或抵消这种作用。这些效应可能与饮用乙醇对心血管发病率的双峰效应有关。 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(CB)受体(Jarai等人，PNAS96：14136,1999年)。我们最近发现了CB类似物？异常大麻二酚？(ABN-CBD)不与两个已知的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激动剂的作用。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。这些发现表明，ABN-CBD是一种选择性激动剂，而O-1918是一种新型的内皮细胞ANANDAME受体的无声拮抗剂。它不同于CB1和CB2受体，通过GI/GO偶联到PI3激酶/AKT信号通路。

Moderate drinking has been associated with reduced cardiovascular morbidity, whereas heavy drinking has an opposite effect. We investigated the effect of ethanol (EtOH) on the cell survival promoting PI3 kinase/akt pathway in cultured human umbilical vein endothelial cells (HUVEC). Exposure of cells to low concentrations (2-20 mM) of EtOH resulted in rapid induction of Akt phosphorylation that could be prevented by pertussis toxin (PTX) or PI3 kinase inhibitors. Several downstream effectors of PI3 kinase/akt, including p70S6 kinase, glycogen synthase kinase 3 alpha/beta, and IkB-alpha, were also phosphorylated by EtOH, the latter resulting in a 3-fold activation of NFkappaB. EtOH also activated p42/44 MAP kinase in a PI3 kinase-dependent manner. EtOH (2-20 mM) increased endothelial NO synthase activity, which could be blocked by transfection of HUVEC with dominant negative Akt. The adenosine A1 receptor antagonist DPCX prevented Akt phosphorylation by 2-20 mM EtOH or adenosine. In contrast, incubation of HUVEC with high concentrations (50-100 mM) of EtOH resulted in mitochondrial permeability transition and caspase-3 activation, followed by apoptosis, as verified by DNA fragmentation and TUNEL assays. EtOH-induced apoptosis was unaffected by DCPX and was potentiated by PI3kinase inhibitors. Thus, low concentrations of EtOH activate the cell survival promoting PI3K/akt pathway in endothelial cells by an adenosine-dependent mechanism, and activation of the proapoptotic caspase pathway by high concentrations of EtOH via an adenosine-independent mechanism can mask or counteract such effects. These effects may be relevant to the bimodal effects of EtOH drinking on cardiovascular morbidity. Our previous study has indicated the existence of a novel cannabinoid (CB) receptor in vascular endothelium (Jarai et al., PNAS 96:14136, 1999). We recently found 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.