title: Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia.
abstract: Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 μM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-β-cyclodextrin (mβCD; 10 mM). Cholesterol depletion with mβCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mβCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 μM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Paffett
authors.forename: Jay S
authors.initials: JS
authors.lastname: Naik
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Riddle
authors.forename: Steven D
authors.initials: SD
authors.lastname: Menicucci
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Gonzales
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Resta
authors.forename: Benjimen R
authors.initials: BR
authors.lastname: Walker
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020747:Calcium Channels, T-Type / Q000378:metabolism; D002469:Cell Separation; D002784:Cholesterol / Q000378:metabolism / Q000502:physiology; D002908:Chronic Disease; D042783:Endothelial Cells / Q000187:drug effects; D004727:Endothelium / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D016257:Fura-2; D000860:Hypoxia / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008297:Male; D008345:Manganese / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism / Q000502:physiology*; D008854:Microscopy, Electron; D018408:Patch-Clamp Techniques; D011651:Pulmonary Artery / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D015220:Calcium Channels; D020747:Calcium Channels, T-Type; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D008345:Manganese; D000255:Adenosine Triphosphate; D002784:Cholesterol; D002118:Calcium; D016257:Fura-2