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The Influence of Exogenous ATP on Functional Responses of Murine Bone Marrow Granulocytes
Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology  (IF),  Pub Date : 2020-01-01, DOI: 10.1134/s1990747819060096
D. A. Serov, M. E. Astashev, I. V. Tikhonova, V. G. Safronova

Abstract — Extracellular ATP is a potent inflammatory mediator and regulator of neutrophil activity in an inflammatory site. The role of ATP in the regulation of bone marrow granulocyte functions remains unexplored. The aim of this work was to study effects of ATP on the cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ), generation of reactive oxygen species (ROS) and adhesion of bone marrow granulocytes. Changes of [Ca 2+ ] i were estimated with Fura-2 AM ratiometric probe. To reveal cells with calcium activity in a population we developed an automated procedure based on the calculation of skewness and kurtosis of the [Ca 2+ ] i value distribution. ROS generation was estimated by luminol-dependent chemiluminescence. Cell adhesion was evaluated by a standard absorbance (492 nm) method. The population of bone marrow granulocytes contained cells that generated calcium spikes in response to 10 μM ATP (35.2 ± 4.0%). The automated scanning allowed us to identify four groups of cells: those responding to ATP (23.4 ± 4.9%) or to the addition of Hanks’ solution (16.9 ± 2.5%) or to both ATP and Hanks’ solution (6.7 ± 1.5%), and cells not responding to either stimulus (53.1 ± 8.2%). ATP increased the adhesion of granulocytes, while apyrase, which hydrolyzes ATP, did not significantly change the adhesion. ATP did not influence the respiratory burst initiated by the bacterial peptide N-formyl-MLF (fMLF). Apyrase significantly enhanced the response to fMLF, and cytochalasin D inhibited this effect. In summary, exogenous ATP enhances adhesion and generation of calcium spikes in the population of bone marrow granulocytes. Endogenous ATP likely reduces respiratory burst through a mechanism involving the cytoskeleton. The studied granulocyte population contains cells with different ATP sensitivity.