title: Reduced membrane cholesterol limits pulmonary endothelial Ca2+entry after chronic hypoxia.
abstract: Chronic hypoxia (CH)-induced pulmonary hypertension is associated with diminished production of endothelium-derived Ca2+-dependent vasodilators such as nitric oxide. Interestingly, ATP-induced endothelial Ca2+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 Ca2+entry (SOCE) and depolarization-induced Ca2+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 Ca2+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 Ca2+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 Ca2+influx were observed in response to a depolarizing stimulus of KCl. Furthermore, KCl-induced Ca2+entry was inhibited by the T-type Ca2+channel antagonist mibefradil but not the L-type Ca2+channel inhibitor diltiazem. We conclude that PAEC membrane Chol is required for ATP-induced Ca2+entry and its two components, SOCE and depolarization-induced Ca2+entry, and that reduced Ca2+entry after CH may be due to loss of this key regulator.NEW & NOTEWORTHYThis research is the first to examine the direct role of membrane cholesterol in regulating pulmonary endothelial agonist-induced Ca2+entry and its components. The results provide a potential mechanism by which chronic hypoxia impairs pulmonary endothelial Ca2+influx, which may contribute to pulmonary hypertension.
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
authors.forename: Bojun
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
authors.forename: Jay S
authors.identifier: http://orcid.org/0000-0002-5725-4664
authors.initials: JS
authors.lastname: Naik
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
authors.forename: Nikki L
authors.initials: NL
authors.lastname: Jernigan
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
authors.forename: Benjimen R
authors.initials: BR
authors.lastname: Walker
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico TResta@salud.unm.edu.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Resta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology; D002908:Chronic Disease; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D000255:Adenosine Triphosphate; D002784:Cholesterol; D002118:Calcium
keywords: ATP; T-type calcium channels; depolarization-induced Ca2+ entry; pulmonary hypertension; store-operated Ca2+ entry