3 years ago

Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Orai1-Mediated Pulmonary Endothelial Ca(2+) Entry.

Thomas C Resta, Bojun Zhang, Jay S Naik, Benjimen R Walker, Nikki L Jernigan
Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca(2+) entry (SOCE) and diminished Ca(2+)-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAEC) is tightly regulated by membrane cholesterol, and that decreased membrane cholesterol is responsible for impaired SOCE following CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAEC through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 Torr) as a physiological stimulus to decrease PAEC cholesterol. Effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats following cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4 and Orai1 siRNA each inhibited SOCE compared to their respective controls. Epichol had no additional effect following knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interaction assessed by proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol following CH limits Orai1-mediated SOCE.

Publisher URL: http://doi.org/10.1152/ajpheart.00540.2017

DOI: 10.1152/ajpheart.00540.2017

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