DETACHMENT OF CULTURED-CELLS FROM THE SUBSTRATUM INDUCED BY THE NEUTROPHIL-DERIVED OXIDANT NH2CL - SYNERGISTIC ROLE OF PHOSPHOTYROSINE AND INTRACELLULAR CA2+ CONCENTRATION

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not t he non-membrane-permeating chloramine, taurine-NHCl) induced detachmen t of fetal mouse cardiac myocytes and other cell types (fibroblasts, e pithelial cells, and endothelial cells) from the culture dish, concomi tant with cell shrinkage (''peeling off''). Stimulated human neutrophi ls also induced peeling off of cultured mouse cardiac myocytes when th e latter were pretreated with inhibitors of . OH and elastase. Immunof luorescence 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)-conta ining proteins. Decrease in the content of the p-Tyr-containing protei ns of the NH2Cl-treated cells was analyzed by immunoblotting technique s. Coating of fibronectin on the culture dish prevented both NH2Cl-ind uced 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 dephosphor ylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling o ff was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+](i)) in mouse cardiac myocytes and WI-38 fibroblasts. The absen ce 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 tha t an increase in [Ca2+](i) is also necessary for peeling off, Depletio n of microsomal and cytosolic Ca2+ by incubation with the microsomal C a2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquino (BHQ) pl us EGTA prevented both NH2Cl-induced increases in [Ca2+](i) and peelin g off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+](i) increment. A combination o f p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinas e) and an increase in [Ca2+](i) by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphoryla tion and increased [Ca2+](i) in NH2Cl-induced peeling off.