Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry...

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Publicado:	2019
Materias:	Adhesion, aggregation ATP-transport E.coli hemolysin Hemolysis adenosine triphosphatase alpha hemolysin apyrase calcium carbenoxolone hemoglobin liposome purinergic P2X receptor animal experiment Article calcium cell level cell aggregation cell isolation cell shape cell structure cell swelling cell volume erythrocyte flow kinetics hemolysis hemolysis assay human human cell hydrolysis impedance male mathematical model membrane potential mesenteric arterial bed nonhuman osmotic stress priority journal protein purification protein secretion rat refraction index regulatory mechanism scanning electron microscopy shear stress
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01674889_v1866_n5_p896_LealDenis http://hdl.handle.net/20.500.12110/paper_01674889_v1866_n5_p896_LealDenis
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_01674889_v1866_n5_p896_LealDenis
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spelling	paper:paper_01674889_v1866_n5_p896_LealDenis2023-06-08T15:16:30Z Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis Adhesion, aggregation ATP-transport E.coli hemolysin Hemolysis adenosine triphosphatase alpha hemolysin apyrase calcium carbenoxolone hemoglobin liposome purinergic P2X receptor animal experiment Article calcium cell level cell aggregation cell isolation cell shape cell structure cell swelling cell volume erythrocyte flow kinetics hemolysis hemolysis assay human human cell hydrolysis impedance male mathematical model membrane potential mesenteric arterial bed nonhuman osmotic stress priority journal protein purification protein secretion rat refraction index regulatory mechanism scanning electron microscopy shear stress Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca 2+ i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity. © 2019 Elsevier B.V. 2019 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01674889_v1866_n5_p896_LealDenis http://hdl.handle.net/20.500.12110/paper_01674889_v1866_n5_p896_LealDenis
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Adhesion, aggregation ATP-transport E.coli hemolysin Hemolysis adenosine triphosphatase alpha hemolysin apyrase calcium carbenoxolone hemoglobin liposome purinergic P2X receptor animal experiment Article calcium cell level cell aggregation cell isolation cell shape cell structure cell swelling cell volume erythrocyte flow kinetics hemolysis hemolysis assay human human cell hydrolysis impedance male mathematical model membrane potential mesenteric arterial bed nonhuman osmotic stress priority journal protein purification protein secretion rat refraction index regulatory mechanism scanning electron microscopy shear stress
spellingShingle	Adhesion, aggregation ATP-transport E.coli hemolysin Hemolysis adenosine triphosphatase alpha hemolysin apyrase calcium carbenoxolone hemoglobin liposome purinergic P2X receptor animal experiment Article calcium cell level cell aggregation cell isolation cell shape cell structure cell swelling cell volume erythrocyte flow kinetics hemolysis hemolysis assay human human cell hydrolysis impedance male mathematical model membrane potential mesenteric arterial bed nonhuman osmotic stress priority journal protein purification protein secretion rat refraction index regulatory mechanism scanning electron microscopy shear stress Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
topic_facet	Adhesion, aggregation ATP-transport E.coli hemolysin Hemolysis adenosine triphosphatase alpha hemolysin apyrase calcium carbenoxolone hemoglobin liposome purinergic P2X receptor animal experiment Article calcium cell level cell aggregation cell isolation cell shape cell structure cell swelling cell volume erythrocyte flow kinetics hemolysis hemolysis assay human human cell hydrolysis impedance male mathematical model membrane potential mesenteric arterial bed nonhuman osmotic stress priority journal protein purification protein secretion rat refraction index regulatory mechanism scanning electron microscopy shear stress
description	Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca 2+ i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity. © 2019 Elsevier B.V.
title	Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
title_short	Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
title_full	Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
title_fullStr	Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
title_full_unstemmed	Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis
title_sort	regulation of extracellular atp of human erythrocytes treated with α-hemolysin. effects of cell volume, morphology, rheology and hemolysis
publishDate	2019
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01674889_v1866_n5_p896_LealDenis http://hdl.handle.net/20.500.12110/paper_01674889_v1866_n5_p896_LealDenis
_version_	1768545000379908096

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis

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