Endocannabinoids And The Control Of Vascular Tone

适量饮酒与降低心血管疾病发病率有关，而大量饮酒则有相反的效果。我们研究了乙醇（EtOH）对培养的人脐静脉内皮细胞（HUVEC）中促进细胞存活的PI 3激酶/Akt通路的影响。将细胞暴露于低浓度（2-20 mM）的EtOH导致Akt磷酸化的快速诱导，这可以被百日咳毒素（PTX）或PI 3激酶抑制剂阻止。PI 3激酶/akt的几种下游效应物，包括p70 S6激酶、糖原合成酶激酶3 α/β和IkB-α，也被EtOH磷酸化，后者导致NF κ B活化3倍。EtOH还以PI 3激酶依赖性方式激活p42/44 MAP激酶。EtOH（2-20 mM）增加内皮NO合酶活性，这可以通过转染显性阴性Akt的HUVEC来阻断。腺苷A1受体拮抗剂DPCX阻止2-20 mM EtOH或腺苷引起的Akt磷酸化。相反，HUVEC与高浓度（50-100 mM）的乙醇孵育导致线粒体通透性转换和半胱天冬酶-3激活，随后凋亡，如DNA片段化和TUNEL测定所证实的。EtOH诱导的细胞凋亡不受DCPX的影响，并通过PI 3激酶抑制剂增强。因此，低浓度的EtOH通过腺苷依赖性机制激活内皮细胞中促进细胞存活的PI 3 K/akt途径，而高浓度的EtOH通过腺苷非依赖性机制激活促细胞凋亡的半胱天冬酶途径可以掩盖或抵消此类作用。这些影响可能与乙醇饮用对心血管发病率的双峰效应有关。 我们先前的研究已经表明在血管内皮中存在新的大麻素（CB）受体（Jarai等人，PNAS 96：14136，1999）。我们最近发现CB类似物？异常大麻二酚（abn-cbd）不与两种已知的CB受体CB 1和CB 2结合，但在松弛大鼠离体肠系膜动脉段中充当完全激动剂。由abn-cbd引起的血管舒张是内皮依赖性的，PTX敏感的，并且被BKCa通道抑制剂Charybdotoxin抑制，但不被NO合酶抑制剂L-NAME或香草酸VR 1拮抗剂辣椒平抑制。大麻二酚类似物O-1918不与CB 1或CB 2受体结合，在高达30微摩尔的浓度下不引起血管舒张，但引起浓度依赖性（1-30微摩尔）抑制abn-cbd和大麻素的血管舒张作用。在麻醉小鼠中，O-1918剂量依赖性地抑制abn-cbd的促炎作用，但不抑制CB 1激动剂HU-210的促炎作用。在HUVEC中，abn-cbd诱导蛋白激酶B/akt和p42/44 MAP激酶的磷酸化，其被O-1918、PTX和PI 3激酶抑制剂抑制。这些研究结果表明，abn-cbd是一种选择性激动剂和O-1918一种新的，沉默的拮抗剂的内皮？花生四烯酸受体？它与CB 1和CB 2受体不同，通过Gi/Go与PI 3激酶/Akt信号通路偶联。

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.