Spatz M, Kawai N, Merkel N, Bembry J, McCarron R M
Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4128, USA.
Am J Physiol. 1997 Jan;272(1 Pt 1):C231-9. doi: 10.1152/ajpcell.1997.272.1.C231.
This report describes the fractional separation of microvessels from human brain for establishment of segmentally derived endothelial cell (EC) cultures. The investigation comprised evaluation of media constituents and purity of the cell culture and focused on functional biochemical characterization of endothelium derived from large microvessels (EC) Cells contained endothelial marker factor VIII (von Willebrand antigen), secreted endothelin-1 (ET-1) and prostaglandins, and took up 86Rb+ as a measure of K+. Exogenous ET-1 stimulated phosphatidylinositol hydrolysis and K+ uptake; BQ-123 (selective ETA receptor antagonist) but not IRL-1038 or BQ-788 (selective ETB receptor antagonists) inhibited both. Ouabain (inhibitor of Na(+)-K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (74-80 and 20-40%, respectively) the ET-1-stimulated K+ uptake. Staurosporine [protein kinase C (PKC) inhibitor] selectively reduced Na(+)-K(+)-Cl- cotransport, whereas verapamil but not nifedipine (L-type voltage-dependent Ca2+ channel blockers) decreased Na(+)-K(+)-ATPase activity induced by ET-1. Phorbol 12-myristate 13-acetate (PMA; activator of PKC) stimulated K+ uptake, which was only decreased with bumetanide. N-ethylisopropylamiloride (inhibitor of Na+/H+ exchange) reduced the ET-1-stimulated but not the PMA-induced K+ uptake. Results indicate that phosphatidylinositol hydrolysis and ion transport systems in large microvascular EC are stimulated by ET-1 through activation of ETA receptors. The findings also suggest that the ET-1-stimulated Na(+)-K(+)-ATPase activity, in contrast to Na(+)-K(+)-Cl- cotransport, is not mediated by PKC. In addition, the data suggest a linkage between Na(+)-K(+)-ATPase activity and Na+/H+ exchange.
本报告描述了从人脑中分离微血管以建立分段来源的内皮细胞（EC）培养物的分级分离方法。该研究包括对培养基成分和细胞培养纯度的评估，并着重于源自大脑微血管的内皮细胞（EC）的功能生化特性。细胞含有内皮标记因子VIII（血管性血友病因子抗原），分泌内皮素-1（ET-1）和前列腺素，并摄取86Rb+作为钾离子的测量指标。外源性ET-1刺激磷脂酰肌醇水解和钾离子摄取；BQ-123（选择性ETA受体拮抗剂）而非IRL-1038或BQ-788（选择性ETB受体拮抗剂）可抑制两者。哇巴因（钠钾ATP酶抑制剂）和布美他尼（钠钾氯共转运抑制剂）分别使ET-1刺激的钾离子摄取减少74-80%和20-40%。星形孢菌素[蛋白激酶C（PKC）抑制剂]选择性降低钠钾氯共转运，而维拉帕米而非硝苯地平（L型电压依赖性钙通道阻滞剂）降低ET-1诱导的钠钾ATP酶活性。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA；PKC激活剂）刺激钾离子摄取，仅布美他尼可使其降低。N-乙基异丙基阿米洛利（钠氢交换抑制剂）降低ET-1刺激的钾离子摄取，但不降低PMA诱导的钾离子摄取。结果表明，大微血管EC中的磷脂酰肌醇水解和离子转运系统通过ETA受体的激活受到ET-1的刺激。研究结果还表明，与钠钾氯共转运相反，ET-1刺激的钠钾ATP酶活性不受PKC介导。此外，数据表明钠钾ATP酶活性与钠氢交换之间存在联系。
