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

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

Simposio; SCHCF + ALACF 2020; 2020

Sociedad Chilena de Ciencias Fisiológicas

IntroductionHuman erythrocytes (hRBCs) release ATP in response to physiological and pathological stimuli. 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 intracellular signaling mechanisms leading to ATP release and cell swelling.ObjectiveWe studied the regulation of cell volume (Vr) and extracellular ATP (ATPe) of hRBCs exposed to MST7 or HlyA. Additionally, we evaluated the effect of HlyA on water permeability through aquaporin 1 (AQP1), the main water transport channel in hRBCs.MethodologyhRBCs from healthy donors and AQP1-KOs were exposed to MST7 or HlyA. ATPe was 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 counting principle 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-treatment with P2X inhibitors reduced 48% [ATPe], 80% swelling and blocked Na+ uptake. The treatment of MST7 in hyperosmotic medium reduced 40% [ATPe] and blocked swelling.HlyA caused shrinking of hRBCs followed by continuous swelling, reaching 10% Vr increased over control values, while [ATPe] showed a 36-fold increased. Vr changes correlated with a 5-8 fold increase of [Na+i] and 5-7 fold decrease of [K+i]. HlyA decreased water permeability of hRBCs, both in WT and KO AQP1-hRBCs.ConclusionMST7- and HlyA- treated hRBCs triggered activation of ATP release, leading to P2X activation, followed by Na+ uptake, which in turn causes swelling. This increase in Vr caused 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 sud-MINCyT 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 the investigation.