Abstract
BackgroundOxidative 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.ResultsActivation 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.ConclusionsOxidative 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.
Highlights
Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins
Chinese hamster ovary (CHO) cells not transfected with the M3 receptor gene did not respond to carbamylcholine
The response of CHO-M3 cells to carbamylcholine was completely blocked by preincubation for 10 min with 10 μM atropine, a receptor antagonist
Summary
Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. 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. Exposure to nanoparticles causes an increase in resting intracellular calcium levels in human bronchial epithelial cells [13]. These findings led us to examine the effects of ZnO nanoparticles on muscarinic signaling through the M3 receptor – phospholipase Cβ pathway [16] that involves IP3-stimulated calcium release from the ER and subsequent depletion-induced calcium entry (i.e., store-operated calcium entry; SOCE) [17,18,19]. The extent to which these actions reflected oxidative stress as opposed to specific chemical effects was not assessed, providing the impetus for the present study
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