Effect of mastoran-7 and alpha-hemolysin on ATP release and cell volume in human erythrocytes. The role of aquaporin 1

LAURI, NATALIA; SCHWARZBAUM, PABLO JULIO; LEAL DENIS, MARÍA F.; OSTUNI, MARIANO ANIBAL; ALVAREZ, CORA LILIA; HERLAX, VANESA

Santiago de Chile

Simposio; SCHCF + ALAF 2020; 2020

Sociedad Chilena de CIencias Fisiológicas

IntroductionHuman erythrocytes (hRBCs) release ATP in response to physiological and pathologicalstimuli. In vitro, exposure of hRBCs to the peptide MST7 (derived from wasp venom)and the toxin HlyA (derived from uropathogenic strains of E. coli), activate intracellularsignaling mechanisms leading to ATP release and cell swelling.ObjectiveWe studied the regulation of cell volume (Vr) and extracellular ATP (ATPe) of hRBCsexposed to MST7 or HlyA. Additionally, we evaluated the effect of HlyA on waterpermeability through aquaporin 1 (AQP1), the main water transport channel in hRBCs.MethodologyhRBCs from healthy donors and AQP1-KOs were exposed to MST7 or HlyA. ATPewas measured by luciferin-luciferase technique. Intracellular content of sodium (Na+i)and potassium (K+i) were measured by capillary electrophoresis and flame photometry.Changes in Vr were measure by fluorescence microscopy, the Coulter countingprinciple and by light scattering.Statistical significance was determined by one-way ANOVA followed by Turkey?Kramer test. The study was approved by Committee for Ethics on Clinical Investigation,FFyB, UBA. EXP-UBA: 0048676/2017.ResultsMST7 induced an increase of [ATPe] (500 %), Na+i (60%) and Vr (10%).Pharmacological inhibition of ATP release reduced swelling by 50%. Pre-treatmentwith P2X inhibitors reduced 48% [ATPe], 80% swelling and blocked Na+uptake. Thetreatment of MST7 in hyperosmotic medium reduced 40% [ATPe] and blockedswelling.HlyA caused shrinking of hRBCs followed by continuous swelling, reaching 10% Vrincreased over control values, while [ATPe] showed a 36-fold increased. Vr changescorrelated with a 5-8 fold increase of [Na+i] and 5-7 fold decrease of [K+i]. HlyAdecreased water permeability of hRBCs, both in WT and KO AQP1-hRBCs.ConclusionMST7- and HlyA- treated hRBCs triggered activation of ATP release, leading to P2Xactivation, followed by Na+uptake, which in turn causes swelling. This increase in Vrcaused further increments of ATP release, thus forming a positive feedback loop.AcknowledgmentFounded by Grants PIP 112 20110100639, CONICET; 200201701001 52BA,UBACyT; PDTS/CIN 2014 193, CONICET; PICT 2016-1041, ANPCyT; ECOS sudMINCyT A15S01 and Laboratory of Excellence GR-Ex ANR-11-LABX-0051.We thank to Dr. Alleva (IQUIFIB ?Prof. Alejandro C. Paladini" CONICET-UBA,Argentina), for their contribution in the analysis and discussion of the results of theinvestigation.