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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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The influence of tBHP on allosteric regulation of M3 muscarinic receptors expressed in CHO cells is illustrated. a The dissociation of [3H]MS was measured by incubating CHO-M3 cell membranes with 1 nM [3H]MS for 1 hour before adding an excess of atropine (10 μM) to block the forward binding reaction. The off rate was measured in the absence (circles) and presence (triangles) of 10 μM gallamine in control (open symbols) or tBHP-exposed (2 mM 90 min) cells (closed symbols). Dissociation rate constants are listed in Table 1. b Similar responses in CHO cells expressing human M2 muscarinic receptors are shown. M2 receptors were examined because they typically display a more pronounced allosteric effect.
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Figure 9: The influence of tBHP on allosteric regulation of M3 muscarinic receptors expressed in CHO cells is illustrated. a The dissociation of [3H]MS was measured by incubating CHO-M3 cell membranes with 1 nM [3H]MS for 1 hour before adding an excess of atropine (10 μM) to block the forward binding reaction. The off rate was measured in the absence (circles) and presence (triangles) of 10 μM gallamine in control (open symbols) or tBHP-exposed (2 mM 90 min) cells (closed symbols). Dissociation rate constants are listed in Table 1. b Similar responses in CHO cells expressing human M2 muscarinic receptors are shown. M2 receptors were examined because they typically display a more pronounced allosteric effect.

Mentions: Muscarinic receptors can also be regulated by ligands that bind to an allosteric site. This allosteric effect is clearly evident in a decrease in the rate of [3H]MS dissociation in the presence of the allosteric ligand (e.g., [22]). Accordingly, we measured ligand dissociation from muscarinic receptors after exposure to 2 mM tBHP for 90 min (Figure 9). Exposure to tBHP had no effect on atropine-induced dissociation of an M3 receptor-[3H]MS complex in CHO-M3 cells, or on the ability of the allosteric ligand gallamine to slow the rate of dissociation (Figure 9a; Table 1). M2 muscarinic receptors display more pronounced allosteric effects than M3 receptors. Therefore, we also examined the influence of tBHP on allosteric muscarinic responses in CHO cells stably transfected with the human M2 muscarinic receptor (Figure 9b). Again, allosteric receptor actions were not affected by exposure to 2 mM tBHP for 90 min.


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)

The influence of tBHP on allosteric regulation of M3 muscarinic receptors expressed in CHO cells is illustrated. a The dissociation of [3H]MS was measured by incubating CHO-M3 cell membranes with 1 nM [3H]MS for 1 hour before adding an excess of atropine (10 μM) to block the forward binding reaction. The off rate was measured in the absence (circles) and presence (triangles) of 10 μM gallamine in control (open symbols) or tBHP-exposed (2 mM 90 min) cells (closed symbols). Dissociation rate constants are listed in Table 1. b Similar responses in CHO cells expressing human M2 muscarinic receptors are shown. M2 receptors were examined because they typically display a more pronounced allosteric effect.
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Figure 9: The influence of tBHP on allosteric regulation of M3 muscarinic receptors expressed in CHO cells is illustrated. a The dissociation of [3H]MS was measured by incubating CHO-M3 cell membranes with 1 nM [3H]MS for 1 hour before adding an excess of atropine (10 μM) to block the forward binding reaction. The off rate was measured in the absence (circles) and presence (triangles) of 10 μM gallamine in control (open symbols) or tBHP-exposed (2 mM 90 min) cells (closed symbols). Dissociation rate constants are listed in Table 1. b Similar responses in CHO cells expressing human M2 muscarinic receptors are shown. M2 receptors were examined because they typically display a more pronounced allosteric effect.
Mentions: Muscarinic receptors can also be regulated by ligands that bind to an allosteric site. This allosteric effect is clearly evident in a decrease in the rate of [3H]MS dissociation in the presence of the allosteric ligand (e.g., [22]). Accordingly, we measured ligand dissociation from muscarinic receptors after exposure to 2 mM tBHP for 90 min (Figure 9). Exposure to tBHP had no effect on atropine-induced dissociation of an M3 receptor-[3H]MS complex in CHO-M3 cells, or on the ability of the allosteric ligand gallamine to slow the rate of dissociation (Figure 9a; Table 1). M2 muscarinic receptors display more pronounced allosteric effects than M3 receptors. Therefore, we also examined the influence of tBHP on allosteric muscarinic responses in CHO cells stably transfected with the human M2 muscarinic receptor (Figure 9b). Again, allosteric receptor actions were not affected by exposure to 2 mM tBHP for 90 min.

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.

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Related in: MedlinePlus