Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.
Abstract
The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.
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Chloramines are membrane penetrating oxidants and react rapidly with sulphhydryl groups of proteins in the cytoskeleton and the extracellular matrix, causing disruption of tight junctions and an almost immediate increase in epithelial permeability.
“Chloramines are membrane penetrating oxidants and react rapidly with sulphhydryl groups of proteins in the cytoskeleton and the extracellular matrix causing disruption of tight junctions and an almost immediate increase in epithelial permeability, as shown in in vitro studies [ (Tatsumi and Fliss 1994;Nakamura et al. 1995;Musch et al. 1999), as cited by Carbonnelle et al. (2002)].”
From Inorganic chloramines: a critical review of the toxicological and epidemiological evidence as a basis for occupational exposure limit setting (2020) · cites this paper as Nakamura et al. 1995
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Bernard, A., Carbonnelle, S., Michel, O., Higuet, S., de Burbure, C., Buchet, J.-P., Hermans, C., Dumont, X., & Doyle, I. (2003) · Occupational and Environmental Medicine · doi:10.1136/oem.60.6.385 · Cited by 256 · see citing works
Carbonnelle, S., Francaux, M., Doyle, I., Dumont, X., Burbure, C. D., Morel, G., Michel, O., & Bernard, A. (2002) · Biomarkers · doi:10.1080/13547500210166612 · Cited by 131 · see citing works
Musch, M. W., Sugi, K., Straus, D., & Chang, E. B. (1999) · Gastroenterology · doi:10.1016/s0016-5085(99)70557-3 · Cited by 100 · see citing works
Wastensson, G., & Eriksson, K. (2020) · Critical Reviews in Toxicology · doi:10.1080/10408444.2020.1744514 · Cited by 30 · see citing works
- Authors
- Nakamura, T. Y., Yamamoto, I., Nishitani, H., Matozaki, T., Suzuki, T., Wakabayashi, S., Shigekawa, M., & Goshima, K.
- Affiliations
- Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
- Venue
- The Journal of cell biology
- Publisher
- Rockefeller University Press
- Published
- 1995-10-15
- Volume
- 131
- Issue
- 2
- Pages
- 509-524
- ISSN
- 0021-9525
- 1540-8140
- PMC
- PMC2199986
- Cited by
- 31
- References
- 0
- Language
- en
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