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.
BackgroundHonokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.ResultsHonokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding.ConclusionsIt is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.
Cell‐permeable biphenols, such as honokiol, present in herbal teas, inhibit autonomic responses. The effects of biphenols on calcium signaling activated by human M3 muscarinic receptors was studied in Chinese hamster ovary (CHO) cells. Receptor binding was determined by radiolabelled ligand binding; intracellular calcium concentrations were determined using a fura‐2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. Activation of M3 muscarinic receptors with carbachol induced a biphasic increase in [Ca2+]i: an initial, IP3‐ mediated release of Ca2+ from endoplasmic reticulum stores followed by a sustained phase of steady Ca2+ entry (i.e., store operated calcium entry, SOCE). Biphneols had potent (EC50 ≈ 5 μM) inhibitory effects on SOCE that was induced by the activation of the M3 receptor. SOCE was also examined by acute exposure to thapsigarin (2 μM) in a calcium‐free medium, followed by reintroduction of calcium. Again, SOCE was severely depressed by biphenols. The effect of honokiol on SOCE was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor‐mediated calcium release, or depletion of ER calcium stores. Moreover, biphenols did not disrupt the ligand binding properties of M3 receptors. An inhibition of SOCE probably contributes to biphenol disruption of parasympathetic functions.
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