Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Orai1 ‐Mediated Pulmonary Endothelial Ca2+ Entry

Abstract

Diminished Ca2+‐dependent production of endothelium‐derived vasodilators like nitric oxide is observed in chronic hypoxia (CH)‐induced pulmonary hypertension. Our previous studies show that endothelial store‐operated calcium entry (SOCE) as well as membrane cholesterol (Chol) are reduced in pulmonary arteries from rats exposed to CH (4 wk, 380 Torr) compared to their normoxic controls. Interestingly, Chol supplementation restores both membrane Chol and endothelial SOCE in cells from CH rats. Substitution of membrane Chol with epicholesterol (EpiChol, epimeric form of Chol) in freshly isolated pulmonary artery endothelial cell (PAEC) sheets mimics the effect of CH to blunt endothelial SOCE. These data suggest that reduced membrane Chol following CH limits the activity of ion channels required for endothelial SOCE. However, the ion channels involved in Chol‐sensitive SOCE have not been identified. We hypothesized that membrane Chol facilitates Orai1‐mediated endothelial Ca2+ entry, and that reduced Ca2+ entry following CH is due to loss of membrane Chol. To test this hypothesis, we pretreated PAEC sheets isolated from control and CH rats with Chol (2 mM) or EpiChol (2 mM), and administered the Orai1 inhibitor AnCoA4 (20 μM), or vehicle. Ca2+ influx in response store depletion with cyclopiazonic acid (CPA, 10 μM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas Chol supplementation restored diminished SOCE in PAEC sheets from CH rats, both EpiChol and AnCoA4 significantly reduced endothelial SOCE in normoxic controls, but not in cells from CH rats. However, the Orai1 inhibitor had no further effect in cells pretreated with EpiChol. Additional experiments examined SOCE following Orai1 knockdown using siRNA in cultured rat pulmonary microvascular endothelial cells (PMVEC). We found that both EpiChol and Orai1 siRNA significantly inhibited SOCE compared to vehicle or non‐targeting siRNA, respectively. EpiChol treatment had no additional effect in cells following knockdown of Orai1. Furthermore, using a proximity ligation assay, we observed that EpiChol treatment significantly reduced the STIM1‐Orai1 interaction required for SOCE when compared to either vehicle or Chol treated groups. We conclude that membrane Chol is required for STIM1‐Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced Ca2+ entry after CH is due to loss of membrane Chol thereby limiting Orai1 activity.Support or Funding InformationNIH R01 HL132883, NIH R01 HL111084, AHA 16GRNT27700010, AHA 15GRNT21080001,