'Depletion' of ATP by 2-deoxyglucose: secretion by electroporated human neutrophils is not restored by readdition of ATP.
Studies of intracellular signal transduction are facilitated by the use of permeabilized cell systems, which permit the ready manipulation of the cytosol. These model systems have helped to define the roles that small solutes, particularly Ca2+ and nucleotides, play in stimulus-response coupling. In circumstances where the full depletion of intracellular ATP contents is required, some investigators have resorted to prior treatment with metabolic toxins, with the expectation that the role of ATP in signal transduction could then be more unambiguously studied. However, in the work reported here, we found that treatment with 2-deoxyglucose (2-DOG) irreversibly altered the cells: when poisoned human neutrophils were then permeabilized, the cells failed to degranulate well in response to Ca2+, and their sensitivity to Ca2+ could not be recovered by the readdition of ATP. Inhibition of secretion by 2-DOG was most pronounced when low concentrations of Ca2+ were used as the stimulus. Preincubation of the cells with only 1 mM 2-DOG for 10 min at 37 degrees C (prior to washing and permeabilizing the cells) was sufficient for maximal inhibition. Even without preincubation, high concentrations of 2-DOG directly inhibited secretion. The refractory nature of poisoned cells was not restored by the presence of Mg2+ and/or ATP. The protein kinase C agonist phorbol myristate acetate also did not restore sensitivity of secretion to Ca2+. Addition of ATP and/or GTP to the permeabilization medium (to maximize penetration of the nucleotides) failed to restore sensitivity; tracer studies demonstrated that these conditions were adequate for repletion of the nucleotide pool. These data indicate that human neutrophils poisoned with 2-DOG were irreversibly altered, such that restoration of the putative deficiency (ATP) was without effect. Experiments in which such preincubation measures are employed should be viewed with caution.
Smolen
J E
JE
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor.
Kuczynski
B
B
Koh
E K
EK
Balazovich
K J
KJ
Woronoff
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000328:Adult; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects; D002463:Cell Membrane Permeability; D003847:Deoxyglucose / Q000494:pharmacology*; D004594:Electrophysiology; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction
D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D002118:Calcium