Differential lymphocyte growth-modifying effects of oxidants: changes in cytosolic Ca+2

An increase in the concentration of cytosolic Ca+2 ([Ca-2]i) is among the earliest changes seen in mitogen-stimulated lymphocytes and is a consequence of signal transduction which usually results in the initiation of cell cycle progression. However, increased [Ca+2]i has also been correlated with cytotoxicity. We have determined whether modulations of [Ca+2]i are involved in the functional inactivation of cells observed with sublethal concentrations of oxidants. Specifically, [Ca+2]i was measured in mouse splenic lymphocytes that were treated with different oxidants in order to determine if oxidative stress interferes with mitogen-stimulated increases in [Ca+2]i, if oxidants themselves modulated [Ca+2]i, and, if so, whether such Ca+2 modulations by oxidants had stimulatory or inhibitory effects on the response of lymphocytes to mitogens. The oxidants employed were copper phenanthroline (CuP; surface thiol oxidizer), N-ethyl maleimide (NEM; permeant thiol alkylator), hydrogen peroxide (H2O2; generates hydroxyl radical within the cell), and radiation (Cs137; generates hydroxyl radical by radiolysis). Growth of all treated cells was equally inhibited upon stimulation with Con A or PMA/A23187, suggesting that all the oxidants inhibited cell functions required distal in activation to the transduction pathway utilized by Con A but bypassed by PMA/A23187. Doses of CuP, NEM, and radiation which fully inhibited Con A-stimulated proliferation had little effect on resting or mitogen-stimulated changes of [Ca+2]i, but H2O2 doses which fully inhibited proliferation increased [Ca+2]i in unstimulated cells and prevented the increase normally caused by Con A. Both intra- and extracellular Ca+2 contributed to the increased [Ca+2]i seen in unstimulated cells. An elevated [Ca+2]i was sufficient to reduce responsiveness, since pharmacologically increasing the [Ca+2]i with the ionophore A23187 rendered lymphocytes less responsive to Con A. Unlike A23187, H2O2 was unable to synergize with PMA, suggesting that the H2O2-induced increase of [Ca+2]i delivered predominantly negative signals to the cell. The results also suggest that [Ca+2]i utilization by Con A versus PMA-activated lymphocytes must be different. When cells were treated with H2O2 under conditions where intracellular and extracellular Ca+2 were chelated with BAPTA and EGTA, respectively, the response to Con A was restored. Under these conditions, higher concentrations of H2O2 were required to inhibit the response to Con A. Our results indicate that signal transduction may be compromised in cells treated with H2O2, but not in cells treated with CuP, NEM, or radiation.(ABSTRACT TRUNCATED AT 400 WORDS)