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ABSTRACT: Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.
Journal of Biological Chemistry 04/2007; 282(12):8667-77. · 4.77 Impact Factor
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ABSTRACT: Glutathione plays many critical roles within the cell, including offering protection from reactive chemicals. The bioactivated toxicant naphthalene forms chemically reactive intermediates that can deplete glutathione and covalently bind to cellular proteins. Naphthalene selectively injures the nonciliated epithelial cells of the intrapulmonary airways (i.e., Clara cells). This study attempted to define what role glutathione loss plays in naphthalene cytotoxicity by comparing Swiss-Webster mice treated with naphthalene with those treated with the glutathione depletor diethylmaleate. High-resolution imaging techniques were used to evaluate acute changes in Clara cell ultrastructure, membrane permeability, and cytoskeleton structure. A single dose of either diethylmaleate (1000 mg/kg) or naphthalene (200 mg/kg) caused similar glutathione losses in intrapulmonary airways (< 20% of control). Diethylmaleate did not increase membrane permeability, disrupt mitochondria, or lead to cell death--hallmark features of naphthalene cytotoxicity. However, diethylmaleate treatment did cause Clara cell swelling, plasma membrane blebs, and actin cytoskeleton disruptions similar to naphthalene treatment. Structural changes in mitochondria and Golgi bodies also were noted. Changes in ATP levels were measured as an indication of overall cell function, in isolated airway explants incubated with diethylmaleate, naphthalene, or naphthalene metabolites in vitro. Only the reactive metabolites of naphthalene caused significant ATP losses. Unlike the lethal injury caused by naphthalene, the disruptive cellular changes associated with glutathione loss from diethylmaleate seemed to be reversible after recovery of glutathione levels. This suggests that glutathione depletion may be responsible for some aspects of naphthalene cytotoxicity, but it is not sufficient to cause cell death without further stresses.
Journal of Pharmacology and Experimental Therapeutics 09/2005; 314(2):506-13. · 3.83 Impact Factor
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ABSTRACT: Using a highly sensitive membrane permeability assay, a viral infection was discovered in the lungs of virus antibody free (VAF) Swiss-Webster mice purchased for respiratory toxicology studies. The assay is based on the uptake of a charged fluorescent compound by cells lacking an intact plasma membrane. Lungs from 74% of the untreated animals from a single vendor tested positive for injury in this assay. High-resolution histopathologic analysis of 1-microm epoxy resin sections from affected animals identified increased peribronchiolar lymphocytic infiltration and markers of epithelial cell injury. Viral particles were directly observed to be budding from the membranes of infiltrating lymphocytic cells by transmission electron microscopy. Standard histological analysis of paraffin-embedded tissues from lungs of the same mice failed to detect obvious pathology. Serological analyses failed to detect the presence of a virus in the affected mice. Therefore, we conclude that (1) a pathogenic condition was present in the respiratory systems of mice judged pathogen free by standard methodologies, (2) the observed condition produced a pattern of injury comparable to those caused by pulmonary toxicants, (3) high-resolution histopathology and advanced imaging techniques can increase the potential for detection of pathological conditions, and (4) apparently healthy animals can have unrecognized infections with the potential for confounding respiratory toxicology studies.
Toxicology and Applied Pharmacology 09/2003; 190(3):286-93. · 4.45 Impact Factor
