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Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Depolarization‐induced Ca 2+ Entry In Pulmonary Arterial Endothelial Cells
Author(s) -
Zhang Bojun,
Naik Jay,
Jernigan Nikki,
Resta Thomas,
Walker Benjimen
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.774.20
Subject(s) - depolarization , chemistry , nitric oxide , membrane potential , endocrinology , medicine , biophysics , endothelium , biochemistry , biology
Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with diminished production of endothelium‐derived Ca 2+ ‐dependent vasodilators such as nitric oxide. Interestingly, ATP‐induced endothelial Ca 2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from rats exposed to CH (4 wk, PB = 380 torr) compared to normoxic control rats. Furthermore, Chol supplementation restores ATP‐induced Ca 2+ entry to the level of controls, suggesting that reduced membrane Chol following CH limits the activity of ion channels required for agonist‐mediated Ca 2+ entry. Depolarization‐induced Ca 2+ entry is a major component of the response to ATP and is also decreased after CH. However, the effect of Chol on this component of Ca 2+ entry is unknown. We hypothesized that depolarization‐induced entry also requires membrane Chol, and that CH attenuates this response by decreasing membrane Chol. To test this hypothesis, we administered Chol (2 mM) or epicholesterol (EpiCh; epimeric form of cholesterol, 2 mM) to acutely isolated pulmonary arterial endothelial cells (PAEC) from control and CH rats to either supplement or replace native Chol. The efficacy of membrane Chol manipulation was confirmed by filipin staining in isolated PAECs. Ca 2+ influx in response to a depolarizing stimulus of KCl (60 mM) was assessed by Mn 2+ ‐quenching of fura‐2 fluorescence. Whereas KCl‐induced Ca 2+ entry was observed in cells from control rats, exposure to CH nearly abolished this response. EpiCh treatment greatly reduced KCl‐induced Ca 2+ influx in PAECs from control rats (p<0.05 vs. vehicle) without further effect in those from CH animals. Chol treatment, however, restored depolarization‐induced Ca 2+ entry in PAECs from CH rats (p<0.05 vs. vehicle). Ca 2+ influx in response to KCl was inhibited (p<0.05 vs. vehicle) by the T‐type Ca 2+ channel antagonist, mibefradil (10 μM), but not by the L‐type Ca 2+ channel inhibitor, diltiazem (50 μM). We conclude that membrane Chol is required for depolarization‐induced Ca 2+ entry through T‐type channels in PAECs and that reduced Ca 2+ entry after CH may be due to loss of this key regulator. Support or Funding Information AHA Southwest Affiliate Award #15GRNT21080001