title: Oxidative stress disruption of receptor-mediated calcium signaling mechanisms.
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.
authors.forename: Tso-Hao
authors.initials: TH
authors.lastname: Tang
authors.forename: Chiung-Tan
authors.initials: CT
authors.lastname: Chang
authors.forename: Hsiu-Jen
authors.initials: HJ
authors.lastname: Wang
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Erickson
authors.forename: Rhett A
authors.initials: RA
authors.lastname: Reichard
authors.forename: Alexis G
authors.initials: AG
authors.lastname: Martin
authors.forename: Erica K
authors.initials: EK
authors.lastname: Shannon
authors.forename: Adam L
authors.initials: AL
authors.lastname: Martin
authors.forename: Yue-Wern
authors.initials: YW
authors.lastname: Huang
authors.forename: Robert S
authors.initials: RS
authors.lastname: Aronstam
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D017136:Ion Transport / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects*; D015398:Signal Transduction / Q000235:genetics; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D002352:Carrier Proteins; D020122:tert-Butylhydroperoxide; D002118:Calcium