Regulation of extracellular ATP and cell volume

LAURI N, LEAL DENIS MF, ALVAREZ C, LEFEVRE S, GONZÁLEZ-LEBRERO RM, ESPELT, MV, OSTUNI M, HERLAX V, SCHWARZBAUM PJ

Congreso; XLVII Reunión Anual SAB; 2018

α-hemolysin (HlyA) of E. coli irreversibly binds to human erythrocytes (RBCs) andtrigger changes in extracellular ATP and cell volume, ultimately leading tohemolysis.Here we analyzed the capacity of HlyA treated RBCs to release ATP, change shape,adhere, deform and/or aggregate. Cells were treated with HlyA (HlyA-RBCs) or withthe un-acylated protoxin ProHlyA (Pro-RBCs), and compared to control conditions(c-RBCs).HlyA-RBCs showed an acute 1.3-2.2 fold increase of Ca2+i, increased crenationfollowed by swelling, and externalization of phosphatidylserine. Perfusion of HlyARBCsthrough adhesion platforms showed high adhesion to activated HMEC cells atlow flow (0.2 dyn/cm2), although higher flows induced rapid detachment. Exceptfor crenation, none of these changes occur in Pro-RBCs.Deformability changes were analyzed by ektacytometry as a function of osmolarityor shear stress. In osmoscan curves, c- and Pro-RBCs deformed optimally inisosmolar conditions, while with HlyA-RBCs the toxin time-dependently shifted thecurve to the right, suggesting increased sphericity. HlyA-RBCs displayed highlyreduced elongation under shear stress, and very low aggregation in syllectograms.RBCs were then perfused through a rat mesentery, where intravascular levels ofATP and free hemoglobin (to estimate lytic ATP release) were measured inperfusate samples. Results showed that HlyA-RBCs, unlike c- and Pro-RBCs,increased intravascular ATP 83-fold, and hemolysis -56-fold, with parallelincrements in arterial bed pressure. Non-lytic ATP release of HlyA-RBCs was themajor component explaining intravascular ATP.Overall results showed that HlyA-RBCs displayed activated ATP release, high butweak adhesivity, low deformability and aggregability and high sphericity.AcknowldegmentsThis work was supported by Grants PIP 112 20110100639, CONICET; 200201701001 52BA, UBACyT; PICT2014-0327, ANPCyT; PDTS/CIN 2014 193, CONICET; PICT 2016-1041, ANPCyT; ECOS Plus A15S01 and ExANR-11-LABX-0051, Laboratory of Excellence GR.