Tsukahara H, Ende H, Magazine H I, Bahou W F, Goligorsky M S
Division of Nephrology and Hypertension, State University of New York, Stony Brook 11794.
J Biol Chem. 1994 Aug 26;269(34):21778-85.
There is accumulating evidence that endothelial cells express a non-isopeptide-selective endothelin (ET) receptor, ETB, which may be responsible for ET-1-induced transient vasorelaxation. The purpose of the present study was to seek direct evidence for ETB receptor expression in human umbilical vein endothelial cells (HUVEC) and to characterize its functional role in HUVEC and in Chinese hamster ovary cells stably transfected with ETB receptor cDNA (CHO-ETB). Reverse polymerase chain reaction using HUVEC total RNA and ETB receptor-specific oligonucleotide primers firmly demonstrated the presence of an endogenous transcript of the appropriate molecular size. Next, a biotinylated ligand specifically recognizing the ETB receptor, IRL-1620, was synthesized, and immunocytochemical mapping of binding sites was performed in CHO-ETB cells. Specific binding of biotinylated IRL-1620 was evident in CHO-ETB cells, confirming appropriate cell surface receptor expression. Continuous nitric oxide (NO) monitoring with NO-selective electrode revealed a dose-dependent ET-1 stimulation of NO production by HUVEC. Stable transfection of CHO-ETB cells with endothelial nitric oxide synthase (NOS), but not mock-transfection, imparted responsiveness to ET-1 similar to that for HUVEC and was characterized by the immediate release of NO. Protein tyrosine kinase-dependent and calcium-calmodulin-dependent pathways were involved in ET-1-induced activation of the constitutive NOS in CHO-ETB/NOS cells, but coupling of the receptor to the enzyme in HUVEC appeared to be predominantly protein tyrosine kinase-dependent. Although sufficient, calcium/calmodulin system was not an obligatory prerequisite for the ET-1-induced activation of NOS in HUVEC. In conclusion, using two cell systems, we demonstrated that the ETB receptor is functionally coupled to NOS and coordinates the generation of NO via a tyrosine kinase-dependent and a calcium/calmodulin-dependent pathway.
越来越多的证据表明，内皮细胞表达一种非异肽选择性内皮素（ET）受体ETB，它可能是ET-1诱导的短暂血管舒张的原因。本研究的目的是寻找人脐静脉内皮细胞（HUVEC）中ETB受体表达的直接证据，并表征其在HUVEC和稳定转染ETB受体cDNA的中国仓鼠卵巢细胞（CHO-ETB）中的功能作用。使用HUVEC总RNA和ETB受体特异性寡核苷酸引物进行的逆转录聚合酶链反应有力地证明了存在适当分子大小的内源性转录本。接下来，合成了一种特异性识别ETB受体的生物素化配体IRL-1620，并在CHO-ETB细胞中进行了结合位点的免疫细胞化学定位。生物素化的IRL-1620在CHO-ETB细胞中具有明显的特异性结合，证实了适当的细胞表面受体表达。用NO选择性电极连续监测NO显示，ET-1对HUVEC产生NO有剂量依赖性刺激。用内皮型一氧化氮合酶（NOS）稳定转染CHO-ETB细胞，而不是空转染，赋予其对ET-1的反应性，类似于HUVEC，其特征是立即释放NO。蛋白酪氨酸激酶依赖性和钙调蛋白依赖性途径参与了ET-1诱导的CHO-ETB/NOS细胞中组成型NOS的激活，但在HUVEC中受体与酶的偶联似乎主要是蛋白酪氨酸激酶依赖性的。虽然钙/钙调蛋白系统足够，但它不是ET-1诱导HUVEC中NOS激活的必要先决条件。总之，使用两种细胞系统，我们证明了ETB受体在功能上与NOS偶联，并通过酪氨酸激酶依赖性和钙/钙调蛋白依赖性途径协调NO的生成。
