Background: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging.

Results: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions: Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.


Biological Sciences

Keywords and Phrases

Calcium; Carbachol; Guanine Nucleotide; Inositol Trisphosphate; Ligand; Methylscopolamine; Muscarinic M3 Receptor; Tert Butyl Hydroperoxide; Thapsigargin; Allosterism; Animal Cell; Binding Affinity; Calcium Depletion; Calcium Signaling; Calcium Transport; Cell Viability; Controlled Study; Cytosol; Cytotoxicity; Endoplasmic Reticulum; Extracellular Calcium; Fluorescence Imaging; Ligand Binding; Nonhuman; Oxidative Stress; Receptor Binding; Sensitivity Analysis; Cell Survival; CHO Cell Line; Cricetulus; Cytoplasm; Drug Effects; Genetics; Hamster; Ion Transport; Metabolism; Signal Transduction; Animals; Carrier Proteins; CHO Cells; Cricetinae; Cricetulus; Cytoplasm; Endoplasmic Reticulum; Tert-Butylhydroperoxide; Inositol Trisphosphate (IP3); Muscarinic Acetylcholine Receptor; Phospholipase C[beta]; Store-Operated Calcium Entry (SOCE)

International Standard Serial Number (ISSN)

1021-7770; 1423-0127

Document Type

Article - Journal

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Final Version

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This work is licensed under a Creative Commons Attribution 2.0 License.

Publication Date

01 Jul 2013

PubMed ID


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