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Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels

Research output: Contribution to journalArticle

Original languageEnglish
Pages (from-to)2531-2538
JournalTHE FASEB JOURNAL : OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY
Volume32
Early online date26 Apr 2018
DOIs
Publication statusE-pub ahead of print - 26 Apr 2018

King's Authors

Abstract

Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

It is well established that routine cell culture under standard conditions (18% O2 at sea level) is far removed from the physiologic milieu with respect to O2 (1, 2). In comparison with the culture of cells under physiologic (3–5%) O2, a significant impact of hyperoxic culture has been demonstrated in macrophages (3), endothelial cells (ECs) (4–6), stem cells (7), and leukocytes (8). We have recently described the consequences of long-term culture of ECs under physiologic (5%) O2 levels on nuclear factor E2–related factor 2 (4) and NO signaling (6).

The influence of O2 on Ca2+ signaling has been widely studied in specialized O2-sensing cells such as those in the carotid body and pulmonary arteries (2). These cells possess O2-sensitive channels (Kv1.2/5) through which a rapid and sustained inward current is detectable upon reduction in cytosolic O2 (9). In the absence of Kv1.2/5 channel expression, the influence of O2 on Ca2+ signaling in nonexcitable cells is more complex. During acute hypoxia (1–3% O2), increased influx through the Na+:Ca2+ exchanger (NCX) has been demonstrated in HUVECs (10) and chondrocytes (11), whereas a verapamil-sensitive influx has been observed in mesangial cells (12). Reactive oxygen species–mediated alterations in the sensitivity of ryanodine receptors also contribute to changes in Ca2+ homeostasis in saphenous vein ECs (13) and C2C12 skeletal muscle microtubules (14) under hypoxic conditions. We have previously reported reduced plateau [Ca2+]i in response to agonist stimulation in HUVECs cultured at 5% O2, conditions under which no change in basal redox phenotype is detectable (4). In this study, we characterize the mechanisms underlying this reduced plateau [Ca2+]i and provide evidence that physiologic normoxia enhances cytoplasmic Ca2+ clearance via the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), protecting ECs against Ca2+-induced cell death.

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