Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leadin g to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-re ceptor regulates NHE activity in Jurkat T-lymphocytes, we performed convent ional BCECF fluorescence measurements and SNARF flow cytometric analysis (F ACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor HOE694, indi cating that they fully depend on NHE activity. Thus they were taken as a me asure of NHE activity. CD95-receptor stimulation caused a cytosolic acidifi cation and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization follow ing osmotic cell shrinkage was almost abolished by CD95-receptor stimulatio n. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck(56)-deficient J-CaM 1.6 cells and restored by retransfection of J-CaM 1.6-cells with Lck(56) CD95- receptor stimulation led within 4 h to a decrease of cellular ATP which cou ld contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inh ibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD 95-receptor stimulation inhibits NHE activity through a mechanism that depe nds directly or indirectly on the activation of the Src-like kinase Lck(56) This effect contributes to CD95-induced cytosolic acidification, DNA fragm entation and cell shrinkage.