Endocannabinoids And The Control Of Cardiovascular Function

我们早期的研究表明，anandamide和一些非典型大麻素配体（如异常大麻二酚（abn-cbd））的内皮依赖性血管扩张作用是由百日咳毒素敏感的G蛋白偶联受体介导的，与CB1或CB2不同。在与Mechoulam小组的合作中，我们早些时候发现了一种新的内源性大麻素样脑脂质，花生四烯醇酰l -丝氨酸（ARA-S）。与anandamide相反，ARA-S对CB1， CB2或香草酸TRPV1受体没有亲和力，并且在大鼠离体肠系膜动脉和主动脉制剂中产生部分内皮依赖性血管舒张。在最近一项刚刚提交发表的研究中，我们分析了ARA-S对人胚胎肾细胞中Ca2+激活K+电流的影响，该细胞稳定转染了人α亚基，大电导Ca2+激活K+ (BKCa)通道（HEK293hSlo细胞）。ARA-S引起大鼠离体、完整和脱落的肠系膜小动脉松弛（pEC50分别为5.49和5.14）。两种制剂均被100 nM的iberiotoxin抑制。在HEK293hSlo细胞中，ARA-S及其对映体n -花生四烯酰基- d -丝氨酸以相似的效力增强全细胞外向K+电流（pEC50分别为5.63和5.32）。增强不是由ARA-S代谢物、已知大麻素受体、G蛋白、蛋白激酶或Ca2+依赖过程的刺激介导的，并且在斑块切除或膜胆固醇消耗后丢失，但在胆固醇重建后恢复。anandamide （AEA, pEC50: 5.27）也能增强BKCa电流，但被另一种内源性大麻素virodhamine （pIC50: 6.35）或合成大麻素O-1918 （pIC50: 6.59）抑制，后者能阻断ara - s诱导的血管舒张（pIC50: 6.59）。这些发现表明(i)内源性大麻素直接调节BKCa通道或通道相关成分的活性。（ii）这种相互作用不涉及胞质因子，而是依赖于膜胆固醇的存在（iii）直接BKCa通道激活可能有助于ara - s诱导的肠系膜血管松弛的内皮独立成分。（iv）根据头部群的结构，对BKCa电流的影响是刺激的还是抑制的。(v) O-1918是一种有效的BKCa通道抑制剂。

Our earlier studies have indicated that the endothelium-dependent vasodilator effect of the anandamide and of some atypical cannabinoid ligands, such as abnormal cannabidiol (abn-cbd) is mediated by a pertussis toxin-sensitive, G protein-coupled receptor distinct from CB1 or CB2. In collaboration with Mechoulam's group we have earlier identified a novel, endocannabinoid-like brain lipid, arachidonoyl L-serine (ARA-S). Contrary to anandamide, ARA-S has no affinity for CB1, CB2 or vanilloid TRPV1 receptors, and it produces partially endothelium-dependent vasodilation in rat isolated mesenteric artery and aorta preparations. In a more recent study just submitted for publication, we have analyzed the effect of ARA-S on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca2+-activated K+ (BKCa) channel (HEK293hSlo cells). ARA-S caused relaxation of isolated, intact and denuded, small mesenteric arteries (pEC50: 5.49 and 5.14, respectively) of the rat. In both preparations the response was inhibited by 100 nM iberiotoxin. In HEK293hSlo cells, ARA-S and its enantiomer N-arachidonoyl-D-serine enhanced the whole cell outward K+ current with similar potency (pEC50: 5.63 and 5.32, respectively). The potentiation was not mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases or Ca2+-dependent processes, and it was lost after patch excision or following membrane cholesterol depletion, but was restored after cholesterol reconstitution. BKCa currents were also enhanced by anandamide (AEA, pEC50: 5.27) but inhibited by another endocannabinoid, virodhamine (pIC50: 6.35), or by the synthetic cannabinoid O-1918, which blocks ARA-S-induced vasodilation (pIC50: 6.59). These findings indicate that (i) endocannabinoids directly modulate the activity of BKCa channels or a channel-associated component. (ii) This interaction does not involve cytosolic factors but is dependent on the presence of membrane cholesterol (iii) Direct BKCa channel activation likely contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. (iv) Depending on the structure of the head group, the effect on BKCa currents is either stimulatory or inhibitory. (v) O-1918 is a potent BKCa channel inhibitor. We have earlier reported that CB1 cannabinoid receptor antagnists, which do not affect blood pressure in healthy, normotensive rats, increase blood pressure as well as cardiac contractile function in 3 different models of experimental hypertension in rats. In contrast, a commercially available inhibitor of fatty acid amidohydrolase (FAAH), the enzyme responsible for the in vivo degradation of the endocannabinoid anandamide normalizes both the elevated blood pressure and the inappropriately high cardiac contractility in hypertensive rats. We have now extended this study by developing our own proprietary FAAH antagonist, which is more potent in producing similar effects both in anesthetized as well as in conscious, chronically instrumented animals. We further verified that these effects are mediated by CB1 receptors, as they can be prevented or acutely reversed by a CB1 receptor antagonist. The hypotensive and negative inotropic effects of the FAAH antagonist are substantially reduced following ganglionic blockade and restoration of blood pressure by vasopressin infusion, suggesting the a sympatholytic mechanism of action. Ongoing studies in spontaneously hypertensive rats are aimed to test whether chronic treatment with the FAAH antagonist can delay/prevent the development of cardiac hypertrophy.