Zhang Bojun, Naik Jay S, Jernigan Nikki L, Walker Benjimen R, Resta Thomas C
Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
Am J Physiol Heart Circ Physiol. 2017 Jun 1;312(6):H1176-H1184. doi: 10.1152/ajpheart.00097.2017. Epub 2017 Mar 31.
Chronic hypoxia (CH)-induced pulmonary hypertension is associated with diminished production of endothelium-derived Ca-dependent vasodilators such as nitric oxide. Interestingly, ATP-induced endothelial Ca entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from CH rats (4 wk, barometric pressure = 380 Torr) compared with normoxic controls. Store-operated Ca entry (SOCE) and depolarization-induced Ca entry are major components of the response to ATP and are similarly decreased after CH. We hypothesized that membrane Chol facilitates both SOCE and depolarization-induced pulmonary endothelial Ca entry and that CH attenuates these responses by decreasing membrane Chol. To test these hypotheses, we administered Chol or epicholesterol (Epichol) to acutely isolated pulmonary arterial endothelial cells (PAECs) from control and CH rats to either supplement or replace native Chol, respectively. The efficacy of membrane Chol manipulation was confirmed by filipin staining. Epichol greatly reduced ATP-induced Ca influx in PAECs from control rats. Whereas Epichol similarly blunted endothelial SOCE in PAECs from both groups, Chol supplementation restored diminished SOCE in PAECs from CH rats while having no effect in controls. Similar effects of Chol manipulation on PAEC Ca influx were observed in response to a depolarizing stimulus of KCl. Furthermore, KCl-induced Ca entry was inhibited by the T-type Ca channel antagonist mibefradil but not the L-type Ca channel inhibitor diltiazem. We conclude that PAEC membrane Chol is required for ATP-induced Ca entry and its two components, SOCE and depolarization-induced Ca entry, and that reduced Ca entry after CH may be due to loss of this key regulator. This research is the first to examine the direct role of membrane cholesterol in regulating pulmonary endothelial agonist-induced Ca entry and its components. The results provide a potential mechanism by which chronic hypoxia impairs pulmonary endothelial Ca influx, which may contribute to pulmonary hypertension.
慢性缺氧（CH）诱导的肺动脉高压与内皮源性钙依赖性血管舒张剂（如一氧化氮）生成减少有关。有趣的是，与常氧对照组相比，CH大鼠（4周，气压 = 380托）肺动脉中ATP诱导的内皮细胞钙内流以及膜胆固醇（Chol）均减少。储存性钙内流（SOCE）和去极化诱导的钙内流是对ATP反应的主要组成部分，CH后同样减少。我们假设膜Chol促进SOCE和去极化诱导的肺内皮细胞钙内流，且CH通过降低膜Chol减弱这些反应。为验证这些假设，我们分别向来自对照和CH大鼠的急性分离肺动脉内皮细胞（PAECs）给予Chol或表胆固醇（Epichol），以分别补充或替代天然Chol。通过制霉菌素染色证实了膜Chol操作的有效性。Epichol极大地减少了对照大鼠PAECs中ATP诱导的钙内流。虽然Epichol同样减弱了两组PAECs中的内皮SOCE，但补充Chol恢复了CH大鼠PAECs中减少的SOCE，而对对照组无影响。在对氯化钾的去极化刺激反应中，观察到Chol操作对PAECs钙内流有类似影响。此外，氯化钾诱导的钙内流被T型钙通道拮抗剂米贝拉地尔抑制，但未被L型钙通道抑制剂地尔硫卓抑制。我们得出结论，PAEC膜Chol是ATP诱导的钙内流及其两个组成部分（SOCE和去极化诱导的钙内流）所必需的，CH后钙内流减少可能是由于这种关键调节因子的丧失。本研究首次探讨了膜胆固醇在调节肺内皮激动剂诱导的钙内流及其组成部分中的直接作用。结果提供了一种潜在机制，通过该机制慢性缺氧损害肺内皮细胞钙内流，这可能导致肺动脉高压。
