Endocannabinoids And The Control Of Cardiovascular Funct

我们早期的研究表明，内源性大麻素Anandamide和一些非典型大麻素配体如异常大麻二醇(ABN-CBD)的内皮依赖性血管扩张作用是由不同于CB1或CB2的百日咳毒素敏感的G蛋白偶联受体介导的。在最近与Raphael Mechoulam的研究小组的合作中，一种新的内源性大麻样脑成分花生四烯酰L丝氨酸(Ara-S)被鉴定和表征。与Anandamide相反，Ara-S对CB1、CB2或香草样TRPV1受体的亲和力很低或没有亲和力。然而，ARA-S在大鼠离体肠系膜动脉和主动脉标本上产生内皮依赖性的血管扩张，并刺激人脐静脉内皮细胞的p44/42 MAPK和蛋白激酶B/AKT的磷酸化。阿糖胞苷-S还抑制脂多糖诱导的小鼠巨噬细胞系和野生型小鼠以及CB1或CB2受体缺陷小鼠的肿瘤坏死因子α的形成。这些影响中的许多与ABN-CBD的报道相似。今年发表的这些发现表明，Ara-S可能是可能的ABN-CBD敏感受体的内源性配体。 为了识别这种受体，我们与一家欧洲生物技术公司建立了合作关系，该公司为我们提供了稳定转染不同孤儿GPCRs的细胞。通过检测ARA-S和ABN-CBD诱导p44/42蛋白激酶和AKT磷酸化以及以剂量依赖、百日咳毒素敏感的方式刺激GTP-Gamma结合的能力来筛选这些细胞。一旦鉴定出阳性克隆，在Stephen Ikeda博士的实验室中，通过产生给定GPCR的cDNA和绿色荧光蛋白的融合构建体，将该构建体导入原代培养的大鼠交感神经节神经元中，GI/GO偶联受体被证明抑制由N型钙通道携带的钙电流，从而验证了结果。一旦证实了受体的正确表达和膜定位，然后通过膜片钳记录Ara-S和ABN-CBD抑制这一钙电流的能力来测试这种细胞。 我们早些时候已经证实，晚期肝硬变的血管扩张状态是由作用于血管CB1受体的内源性大麻素介导的(NAT Med 7：827,2001)。众所周知，肝硬变还与心功能改变有关，其特征是收缩能力和对儿茶酚胺的收缩反应降低。肝硬化性心肌病最近被认为也是由于内源性大麻素刺激CB1受体所致，这是基于一项来自对照组和肝硬变大鼠的分离乳头肌的研究。我们正在通过在CCl4诱导的大鼠肝硬变的活体模型中测试CB1拮抗剂的心血管效应来验证这一假设，在该模型中，通过使用Millar压力/容量系统直接分析心脏血流动力学，并通过死后量化肝硬变大鼠和其非肝硬变对照组的心脏内源性大麻素和CB1受体含量来验证这一假说。 在第四项研究中，我们一直在研究内源性大麻系统在心血管调节中的作用，并将其作为高血压的潜在治疗靶点。我们早些时候曾报道(Batkai等人，《循环》，2004年)，内源性大麻素/CB1受体系统在3种不同的高血压大鼠模型中作为一种代偿机制变得活跃。在使用麻醉的仪器化动物的这些模型中，阻断CB1受体被发现由于心肌收缩能力和外周血管收缩都增加而导致血压进一步升高。相比之下，用一种商用的脂肪酸氨基水解酶(FAAH)抑制剂(FAAH)治疗动物，这种酶负责在体内降解ANANDAME，使血压正常化，心脏收缩能力不适当地增加。这些结果提示，抑制FAAH可能是一种治疗高血压的新方法，也可能预防/逆转相关的心肌肥厚。我们现在已经(与Alex Makriyannis博士合作)开发出一种新型的、体内有效的FAAH抑制剂，它比我们以前使用的抑制剂URB597更有效，而且有更长的持久作用。这种抑制剂目前在慢性模型中进行测试，使用未麻醉的高血压大鼠，植入遥测记录探针，用于非侵入性监测血压和心率。此外，通过超声监测心脏壁厚度，以测试新型FAAH抑制剂慢性治疗对心肌肥厚发展的影响。

Our earlier studies, confirmed by others, have indicated that the endothelium-dependent vasodilator effect of the endocannabinoid 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 a recent collaboration with Raphael Mechoulam's group a novel, endocannabinoid-like brain constituent, arachidonoyl L-serine (ARA-S) has been identified and characterized. Contrary to anandamide, ARA-S has very low or no affinity for CB1, CB2 or vanilloid TRPV1 receptors. However, ARA-s produces endothelium-dependent vasodilation in rat isolated mesenteric artery and aorta preparations and stimulates p44/42 MAP kinase and protein kinase B/Akt phosphorylation in human umbilical vein endothelial cells (HUVEC). ARA-S also suppresses LPS-induced formation of TNFalpha in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel those reported for abn-cbd. These findings, published this year, suggest that ARA-S may be an endogenous ligand for the putative abn-cbd sensitive receptor. In an effort to identify this receptor, we set up a collaboration with a European biotech company that provides us with cells stably transfected with different orphan GPCR. These cells are screened by testing the ability of ARA-S and abn-cbd to induce p44/42 MAP kinase and Akt phosphorylation and stimulate GTPgammaS binding in a dose-dependent, pertussis toxin sensitive manner. Once a positive clone is identified, the results are verified in Dr. Stephen Ikeda's laboratory by generating a fusion construct of the cDNA of the given GPCR and of green fluorescent protein, transfecting this construct into primary cultured rat sympathetic ganglion neurons, in which Gi/Go-coupled receptors have been shown to inhibit calcium currents carried by an N type calcium channel. Once proper expression and membrane localization of the receptor is verified, such cells are then tested by patch clamp recording of the ability of ARA-S and abn-cbd to inhibit this calcium current. We have earlier established that the vasodilated state in advanced liver cirrhosis is mediated by endocannabinoids acting at vascular CB1 receptors (Nat Med 7:827, 2001). Cirrhosis is also known to be associated with altered cardiac function characterized by reduced contractility and contractile response to catecholamines. The cirrhotic 'cardiomyopathy' was recently proposed to be also due to stimulation of CB1 receptors by an endocannabinoids, based on a study using isolated papillary muscles from control and cirrhotic rats. We are testing this hypothesis by testing the cardiovascular effects of CB1 antagonists in an in vivo model of CCl4-induced cirrhosis in rats, in which cardiac hemodynamics are analyzed directly by using the Millar pressure/volume system, and by post-mortem quantification of cardiac endocannabinoid and CB1 receptor content in cirrhotic rats and their non-cirrhotic controls. In a fourth study, we have been investigating the role of the endocannabinoid system in cardiovascular regulation and as a potential therapeuic target in hypertension. We have earlier reported (Batkai et al., Circulation, 2004) that the endocannabinoid/CB1 receptor system becomes tonically active as a compensatory mechanism in 3 different rat models of hypertension. In these models using anesthetized, instrumented animals, blocking CB1 receptors was found to cause a further increase in blood pressure due to both increased cardiac contractility and peripheral vasoconstriction. In contrast, treatment of the animals with a commercially available inhibitor of fatty acid amidohydrolase (FAAH), the enzyme responsible for the in vivo degradation of anandamide, normalized blood pressure and the inappropriately increased cardiac contractility. These findings suggested that inhibition of FAAH may be a novel approach to treat hypertension and also to possibly prevent/reverse the associated cardiac hypertrophy. We have now developed (in collaboration with Dr. Alex Makriyannis) a novel, in vivo effective FAAH inhibitor which is both more potent and has a much longer lasting effect than URB597, the inhibitor we used before. This inhibitor is now tested in a chronic model using unanesthetized hypertensive rats implanted with telemetric recording probes for the non-invasive monitoring of blood pressure and heart rate. In addition, cardiac wall thickness is monitored by ultrasound to test the effect of chronic treatment with the novel FAAH inhibitor on the development of cardiac hypertrophy.