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), leading 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-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.
Lang
F
F
Department for Physiology, University of Tübingen, Germany. florian.lang@uni-tuebingen.de
Madlung
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J
Bock
J
J
Lükewille
U
U
Kaltenbach
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Lang
K S
KS
Belka
C
C
Wagner
C A
CA
Lang
H J
HJ
Gulbins
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E
Lepple-Wienhues
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002087:Butyrates / Q000494:pharmacology; D048429:Cell Size; D003600:Cytosol / Q000737:chemistry; D053938:DNA Fragmentation / Q000187:drug effects; D005434:Flow Cytometry; D005452:Fluoresceins; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019169:Jurkat Cells; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000502:physiology; D013450:Sulfones / Q000494:pharmacology; D013601:T-Lymphocytes / Q000378:metabolism*; D019014:fas Receptor / Q000187:drug effects / Q000502:physiology*
D002087:Butyrates; D005452:Fluoresceins; D006146:Guanidines; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; D019014:fas Receptor; D000643:Ammonium Chloride; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000255:Adenosine Triphosphate