The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation.
The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.
D019328:Buthionine Sulfoximine; D002467:Cell Nucleus / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D003510:Cyclohexanes / Q000494:pharmacology*; D003512:Cyclohexanones / Q000494:pharmacology*; D005978:Glutathione / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects*; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors
D003510:Cyclohexanes; D003512:Cyclohexanones; D007166:Immunosuppressive Agents; D008717:Methionine Sulfoximine; C028215:2-cyclohexen-1-one; D019328:Buthionine Sulfoximine; D005978:Glutathione