Li L, Bressler B, Prameya R, Dorovini-Zis K, Van Breemen C
The Vancouver Vascular Biology Research Center, Vancouver General Hospital, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Microvasc Res. 1999 May;57(3):211-26. doi: 10.1006/mvre.1998.2131.
Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital imaging microscopy. Agonists ATP (100 micro), thrombin (10 units/ml), and histamine (25 microM) induced a transient [Ca2+]i increase. Histamine (100 microM) induced a biphasic [Ca2+]i increase with an initial [Ca2+]i peak followed by a [Ca2+]i plateau. The [Ca2+]i plateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to the [Ca2+]i plateau. However, this [Ca2+]i plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization with 80K+ or application of the VGC agonist BAY K 8644 did not alter the resting [Ca2+]i; but 80K+ reduced the histamine (100 microM) induced [Ca2+]i plateau. These results show that HCMEC are devoid of functional VGC. Thus the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i. Blockade of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+]i. This effect was dependent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide synthase (eNOS), which stimulates cyclic GMP production. The final result that the eNOS inhibitor L-NAME enhanced the histamine (100 microM) induced [Ca2+]i plateau suggests a negative feedback loop (via cGMP) of endothelial NO on its own synthesis in the regulation of endothelial [Ca2+]i signal.
人脑微血管内皮细胞（HCMEC）的原代培养细胞用fura-2进行负载。通过数字成像显微镜测量细胞内游离钙离子浓度（[Ca2+]i）。激动剂ATP（100微摩尔）、凝血酶（10单位/毫升）和组胺（25微摩尔）可诱导[Ca2+]i短暂升高。组胺（100微摩尔）可诱导[Ca2+]i双相升高，先是出现一个初始的[Ca2+]i峰值，随后是一个[Ca2+]i平台期。该[Ca2+]i平台期被受体操纵性钙离子通道（ROC）阻滞剂SK&F 96365和NCDC阻断，表明通过ROC的钙离子内流对[Ca2+]i平台期有贡献。然而，该[Ca2+]i平台期未被电压门控钙离子通道（VGC）阻滞剂地尔硫䓬（DTZ）阻断。用80K钾进行K+进行去极化或应用VGC激动剂BAY K 8644并未改变静息[Ca2+]i；但80K+降低了组胺（100微摩尔）诱导的[Ca2+]i平台期。这些结果表明HCMEC缺乏功能性VGC。因此，膜电位（Em）主要通过增强电化学钙离子梯度来调节钙离子内流，使得超极化时[Ca2+]i升高，而去极化时[Ca2+]i降低。环磷腺苷（CPA）对肌浆网/内质网钙离子-ATP酶（SERCA）的阻断增加了[Ca2+]i。这种效应依赖于细胞外钙离子，并被iberiotoxin（IBTX）对钙离子激活的钾通道（Kca）的阻断所减弱，提示Kca在调节钙离子内流中起作用。钙离子是内皮型一氧化氮合酶（eNOS）的主要激活剂，eNOS可刺激环磷酸鸟苷（cGMP）的产生。eNOS抑制剂L-NAME增强组胺（100微摩尔）诱导的[Ca2+]i平台期这一最终结果表明，在内皮[Ca2+]i信号的调节中，内皮一氧化氮（NO）通过cGMP对其自身合成存在负反馈回路。
