HUMAN CEREBROMICROVASCULAR ENDOTHELIAL RECEPTORS

鉴于观察到的ET-1刺激离子转运系统， 欧洲独联体区域局，必须确定这些系统是否也存在， 人脑微血管内皮细胞（HBMEC）。摄取 ~（86）Rb ~+（0.2 γ C/孔）作为K ~+吸收的示踪剂， 用抑制剂或拮抗剂预孵育（30 min）的汇合HBMEC， 在室温下单独或与ET-1或ET-3在无血清培养基中孵育（5分钟） 温度同样的程序用于缺氧暴露的HBMEC (95 ET-1呈剂量依赖性升高，而ET-3无剂量依赖性升高 ET/A受体拮抗剂BQ 123可抑制K+摄取， ET/B受体拮抗剂IRL 1038则无此作用。 哇巴因（Na+K+-ATP酶 抑制剂）在更大程度上降低了ET-1刺激的K+摄取 (94%）比布美他尼[Na+K+Cl-共转运抑制剂] 30%。 N-乙基-N- N+/H+交换的抑制剂异丙基氨基苯丙氨酸（EIPA）降低了 仅在布美他尼存在下ET-1刺激的K+摄取。维拉帕米， Ca ~（2+）通道抑制剂可抑制ET-1刺激的K ~+吸收 而不是布美他尼。 与此相反， staurosporine [蛋白激酶c（PKC）抑制剂]降低ET-1 在布美他尼而不是哇巴因的存在下刺激K+摄取。 HMBEC在氮气氛中的过夜暴露呈剂量依赖性 增强ET-1对K+摄取的刺激，影响哇巴因- 敏感的K+吸收。 结果表明：（1）Na+K+-AT_（10）活性和Na+K+Cl- ET-1通过激活ET/A受体刺激共转运 ET-1对Na+-K+-AT β活性的刺激作用， 介导的Ca 2+离子和连接到Na+/H+交换，而 Na+K+Cl-共转运与PKC有关; 3）缺氧使ET增加 刺激Na+K+-AT β活性。这项研究代表了第一个 HBMEC中离子传输系统的演示。 观察到的ET-1 Na+K+-ATP酶活性和Na+K+Cl-共转运的调节表明， ET-1可能在调节电解质跨膜转运中发挥作用， 血脑屏障（BBB）。 缺氧增强ET-1刺激 Na+K+-ATP酶活性强烈提示ET-1（从 血管、血液和/或脑细胞）可能参与这些紊乱 水电解质稳态的病理条件下，如 缺血

In view of the observed ET-1 stimulation of ion transport systems in RBEC, it has been important to establish whether these systems also exist in microvascular endothelium derived from human brain (HBMEC). Uptake of 86Rb+ (0.2 gammaC/well) as a tracer of K+ uptake was determined in confluent HBMEC preincubated (30 min) with inhibitor or antagonist and incubated (5 min) alone or with ET-1 or ET-3 in serum-free medium at room temperature. The same procedures were used for HBMEC exposed to hypoxia (95% N2, 5% CO2) for 24 hr. ET-1 but not ET-3 dose-dependently increased K+ uptake which was inhibited with BQ123 (ET/A receptor antagonist) but not with IRL1038 (ET/B receptor antagonist). Ouabain (Na+K+-ATPase inhibitor) reduced the ET-1-stimulated K+ uptake to a greater degree (94%) than bumetanide [Na+K+Cl- cotransport inhibitor) 30%. N-ethyl-n- isopropyl amelinide (EIPA) the inhibitor of N+/H+ exchange reduced the ET-1-stimulated K+ uptake in the presence of bumetanide only. Verapamil, the inhibitor of Ca2+ channels decreased the ET-1 stimulated K+ uptake in the presence of ouabain but not with bumetanide. In contrast, staurosporine [inhibitor of protein kinase c (PKC)] reduced the ET-1 stimulated K+ uptake in the presence of bumetanide but not ouabain. Overnight exposure of HMBEC in nitrogen atmosphere dose-dependently augmented the ET-1 stimulation of K+ uptake affecting the ouabain- sensitive K+ uptake only. The data indicated that: 1) Na+K+-AT Pase activity and Na+K+Cl- cotransport are stimulated by ET-1 through activation of ET/A receptors in HBMEC; 2) the ET-1 stimulation of the Na+K+-AT Pase activity is mediated by Ca2+ ions and is linked to Na+/H+ exchange, whereas the Na+K+Cl- cotransport is linked to PKC; and 3) hypoxia amplifies the ET stimulation of Na+K+-AT Pase activity. This study represents the first demonstration of ionic transport systems in HBMEC. The observed ET-1 modulation of Na+K+-ATPase activity and Na+K+Cl- cotransport indicate that ET-1 may play a role in regulating electrolytes transport across the blood-brain barrier (BBB). The hypoxic augmentation of ET-1 stimulated Na+K+-ATPase activity strongly suggests that ET-1 (released from vascular, blood and/or brain cells) may participate in the disturbances of water electrolytes homeostasis under pathological conditions such as ischemia.