Biomedical Sciences ETDs

Publication Date

Winter 12-13-2017

Abstract

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) is associated with diminished ATP-induced endothelial Ca2+ entry as well as membrane cholesterol in pulmonary arteries. Store-operated Ca2+ entry (SOCE) and depolarization-induced Ca2+ entry are major components of the response to ATP and are similarly decreased after CH. Because endothelium-dependent vasodilation is closely associated with pulmonary endothelial [Ca2+]i, the blunted agonist-induced Ca2+ influx in pulmonary artery endothelial cells (PAEC) may contribute to the development of CH-induced PH. Interestingly, impaired agonist-induced Ca2+ influx in PAEC following CH can be restored by membrane cholesterol supplementation. In the current studies, we hypothesized that impaired Ca2+ entry in PAEC following CH is due to decreased membrane cholesterol. We demonstrated that substitution of cholesterol with its functionally inactive epimer epicholesterol, greatly attenuated ATP-induced Ca2+ influx in PAEC from control rats. Whereas epicholesterol similarly blunted endothelial SOCE in PAEC from both groups, cholesterol supplementation restored diminished SOCE in PAEC from CH rats while having no effect in controls. Similar effects of cholesterol manipulation on T-type Ca2+ vii channel-mediated Ca2+ influx were observed in PAEC. Additionally, the role of cholesterol in SOCE mediated by Orai1, a Ca2+ selective ion channel, was examined in PAEC. Whereas cholesterol restored endothelial SOCE in CH rats, both epicholesterol and the Orai1 inhibitor, AnCoA4, attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with epicholesterol. In cultured pulmonary endothelial cells, using pharmacological inhibition and siRNA knockdown of Orai1, we found that epicholesterol, AnCoA4 and Orai1 siRNA each inhibited SOCE compared to their respective controls. Epicholesterol had no additional effect following knockdown of Orai1. Finally, we found that endothelial stromal interaction molecule 1 (STIM1)-Orai1 interaction, which is essential for SOCE, requires membrane cholesterol. Our studies support a novel regulatory role for membrane cholesterol in agonist-induced Ca2+ entry and its components. Our observation also demonstrated that altered membrane cholesterol homeostasis may contribute to impaired endothelial Ca2+ influx pathways following CH.

Keywords

pulmonary hypertension, T-type calcium channels, ATP, store-operated Ca2+ entry, depolarization-induced Ca2+ entry, STIM1

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Thomas C. Resta

Second Committee Member

Benjimen R. Walker

Third Committee Member

Nikki L. Jernigan

Fourth Committee Member

William S. Garver

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