The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.
Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.
Yegutkin
Gennady G
GG
MediCity Laboratory and Department of Medical Microbiology, Turku University and National Public Health Institute, FIN-20520 Turku, Finland. gennady.yegutkin@utu.fi
Mikhailov
Andrey
A
Samburski
Sergei S
SS
Jalkanen
Sirpa
S
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008214:Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011849:Radioactive Tracers; D011983:Receptors, Purinergic / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010710:Phosphates; D011849:Radioactive Tracers; D011983:Receptors, Purinergic; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase