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Oxidative stress disruption of receptor-mediated calcium signaling mechanisms.

Tang TH, Chang CT, Wang HJ, Erickson JD, Reichard RA, Martin AG, Shannon EK, Martin AL, Huang YW, Aronstam RS - J. Biomed. Sci. (2013)

Bottom Line: 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 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.

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ABSTRACT

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.

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Influence of tBHP on the concentration of calcium in the lumen of the ER. The relative level of [Ca2+]L was determined in cells in which cytosolic calcium had been depleted by permeabilization with 0.005% saponin. a Mag-Fura2 380/340 fluorescence ratio in a typical experiment. Saponin (0.005%) was added at the time indicated by the first arrow; carbamylcholine was added at the time indicated by the second arrow. Prior to permeabilization, the fluorescent signal associated with ER calcium is obscured by the dominant signal from cytosolic calcium. Data are the average from 20 cells in a typical experiment. [Ca2+]L was inferred from the average ratio measured between 100 and 110 sec. b The ratio of fluorescence intensity of Mag-Fura2 following excitation of at 380 and 340 nm was determined in control cells and in cells exposed to 1, 10 or 20 mM tBHP for 90 min prior to imaging, as indicated. This ratio is an indication of [Ca2+]L. The column and bars represent the mean and standard deviation from 3 experiments that involved separate determinations in 20 cells. The asterisks indicate a statistical difference from the control level as determined by ANOVA with a Tukey post-test.
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Figure 7: Influence of tBHP on the concentration of calcium in the lumen of the ER. The relative level of [Ca2+]L was determined in cells in which cytosolic calcium had been depleted by permeabilization with 0.005% saponin. a Mag-Fura2 380/340 fluorescence ratio in a typical experiment. Saponin (0.005%) was added at the time indicated by the first arrow; carbamylcholine was added at the time indicated by the second arrow. Prior to permeabilization, the fluorescent signal associated with ER calcium is obscured by the dominant signal from cytosolic calcium. Data are the average from 20 cells in a typical experiment. [Ca2+]L was inferred from the average ratio measured between 100 and 110 sec. b The ratio of fluorescence intensity of Mag-Fura2 following excitation of at 380 and 340 nm was determined in control cells and in cells exposed to 1, 10 or 20 mM tBHP for 90 min prior to imaging, as indicated. This ratio is an indication of [Ca2+]L. The column and bars represent the mean and standard deviation from 3 experiments that involved separate determinations in 20 cells. The asterisks indicate a statistical difference from the control level as determined by ANOVA with a Tukey post-test.

Mentions: The influences of tBHP on [Ca2+]i during the initial response to IP3 receptor- or thapsigargin-mediated release from the ER could reflect changes in the luminal concentration of calcium ([Ca2+]L). Accordingly, [Ca2+]L was measured using an alternate calcium-sensitive dye (Mag-Fura2) and depletion of cytosolic [Ca2+]I by permeabilization of the plasma membrane (Figure 7). The increase in ratio of fluorescent emission at 512 nm following excitation at 340 and 380 nm (R340/380) reflected ER Ca2+ levels insofar as: 1) R340/380 was immediately diminished following exposure to 10 μM carbamylcholine, and 2) pretreatment with the ER Ca2+-ATPase inhibitor, thapsigargin, prevented the increase in R340/380 observed upon permeabilization. Pretreatment with tBHP (1–20 mM) for 90 min caused a decrease in [Ca2+]L to ≈ 55% of control levels.


Oxidative stress disruption of receptor-mediated calcium signaling mechanisms.

Tang TH, Chang CT, Wang HJ, Erickson JD, Reichard RA, Martin AG, Shannon EK, Martin AL, Huang YW, Aronstam RS - J. Biomed. Sci. (2013)

Influence of tBHP on the concentration of calcium in the lumen of the ER. The relative level of [Ca2+]L was determined in cells in which cytosolic calcium had been depleted by permeabilization with 0.005% saponin. a Mag-Fura2 380/340 fluorescence ratio in a typical experiment. Saponin (0.005%) was added at the time indicated by the first arrow; carbamylcholine was added at the time indicated by the second arrow. Prior to permeabilization, the fluorescent signal associated with ER calcium is obscured by the dominant signal from cytosolic calcium. Data are the average from 20 cells in a typical experiment. [Ca2+]L was inferred from the average ratio measured between 100 and 110 sec. b The ratio of fluorescence intensity of Mag-Fura2 following excitation of at 380 and 340 nm was determined in control cells and in cells exposed to 1, 10 or 20 mM tBHP for 90 min prior to imaging, as indicated. This ratio is an indication of [Ca2+]L. The column and bars represent the mean and standard deviation from 3 experiments that involved separate determinations in 20 cells. The asterisks indicate a statistical difference from the control level as determined by ANOVA with a Tukey post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716919&req=5

Figure 7: Influence of tBHP on the concentration of calcium in the lumen of the ER. The relative level of [Ca2+]L was determined in cells in which cytosolic calcium had been depleted by permeabilization with 0.005% saponin. a Mag-Fura2 380/340 fluorescence ratio in a typical experiment. Saponin (0.005%) was added at the time indicated by the first arrow; carbamylcholine was added at the time indicated by the second arrow. Prior to permeabilization, the fluorescent signal associated with ER calcium is obscured by the dominant signal from cytosolic calcium. Data are the average from 20 cells in a typical experiment. [Ca2+]L was inferred from the average ratio measured between 100 and 110 sec. b The ratio of fluorescence intensity of Mag-Fura2 following excitation of at 380 and 340 nm was determined in control cells and in cells exposed to 1, 10 or 20 mM tBHP for 90 min prior to imaging, as indicated. This ratio is an indication of [Ca2+]L. The column and bars represent the mean and standard deviation from 3 experiments that involved separate determinations in 20 cells. The asterisks indicate a statistical difference from the control level as determined by ANOVA with a Tukey post-test.
Mentions: The influences of tBHP on [Ca2+]i during the initial response to IP3 receptor- or thapsigargin-mediated release from the ER could reflect changes in the luminal concentration of calcium ([Ca2+]L). Accordingly, [Ca2+]L was measured using an alternate calcium-sensitive dye (Mag-Fura2) and depletion of cytosolic [Ca2+]I by permeabilization of the plasma membrane (Figure 7). The increase in ratio of fluorescent emission at 512 nm following excitation at 340 and 380 nm (R340/380) reflected ER Ca2+ levels insofar as: 1) R340/380 was immediately diminished following exposure to 10 μM carbamylcholine, and 2) pretreatment with the ER Ca2+-ATPase inhibitor, thapsigargin, prevented the increase in R340/380 observed upon permeabilization. Pretreatment with tBHP (1–20 mM) for 90 min caused a decrease in [Ca2+]L to ≈ 55% of control levels.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus