INSTITUTO DE FISICA DE LIQUIDOS Y SISTEMAS BIOLOGICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Extracellular ATP regulation of human erythrocytes in health and disease
LAURI, NATALIA; ALVAREZ, HUGO ARIEL; SCHWARZBAUM, PABLO; ALVAREZ, CORA; CHARA, OSVALDO; LEAL DENIS, MARÍA FLORENCIA; HERLAX, VANESA
Ciudad de México
Conferencia; 1st Latin American Workshop and Conference on Systems Biology; 2017
Several stimuli trigger the release of ATP from human erytrocytes (rbcs). ATP exit can be modulated by mechanical stress, purinergic signaling, adhesion and/or changes in cellvolume (Vr). In vivo, intravascular increases of rbcs-derived extracellular ATP (ATPe) may regulate vasodilation and inflammation. We studied the effects of peptides on the regulation of rbcs-derived ATPe. We used 1- the tetradecapeptide mastoparan 7 (MST7), which may activate cell swelling (i.e., Vr increase) and/or an increase of cAMP concentration. Both processes may activate ATP exit; 2- Beta amyloid peptides A1-40 and A1-42, which accumulate in the blood of healthy individuals and, more importantly, of Alzheimer patients. Ageing of A (i.e. peptide incubation in the absence of cells) provoke the irreversible transition of these peptides from its monomeric form to oligomers and fibers, with unknown consequences on ATPe regulation of rbcs.MethodsATPe was assessed continuously by real time luminiscence using the luciferase-luciferin system. Results were expressed as [ATPe] at every time point of a kinetic curve (i.e., ATPe kinetics), with [ATPe] expressed as pmol/106 cells or nM/40 μl. Alternatively, increases in [ATPe] were evaluated as the difference between [ATPe] at 1 min post stimulus and the basal [ATPe], and are indicated as ΔATP1 (nM/3 106 cells).Vr was followed by fluorescence microscopy using BCECF loaded rbcs.Rbcs were attached on coverslips coated with 0.01% poly-D-lysine. For MST7 exposed rbcs, the dynamic regulation of Vr and ATPe was analyzed by means of a data-driven mathematical model.Studies using AA1-40 and A1-42 were allowed to age for 0.25-72h before use. MST7 was used as a positive control of ATP release. The effects of 1 μM A peptides on ATP release was acute, thus allowing to calculate ΔATP1 values.In rbcs, A1-40 aged for 23hs and 72hs, causing a 6.25 and 21.02 nM/(3 106cells) increase of ΔATP1 with respect to untreated rbcs (controls).On the other hand, exposure of cells to A1-42 aged for 0.25-23hs triggered a 15.0-16.7 nM/(3 106cells) increase of ΔATP1. For A1-42 ageing ≥48hs, ΔATP1 increased by 71.6-104 nM/(3 106cells). These values were approx. 2 to 3-fold higher than those obtained by MST7 exposure.Current experiments are aimed at identifying the plasma membrane conduits responsible for A dependent ATP release of rbcs.Studies using MST7MST7 triggered ATP release and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with purinergic receptors, can affect Vr, we explored the reciprocal regulation between Vr and ATPe.Kinetics of ATPe and Vr was assessed in the absence and presence of blockers of ATP conduits, swelling and purinergic receptors.MST7 promoted acute, strongly correlated changes in [ATPe] and Vr of rbcs.Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%.In MST7 treated rbcs, pre-incubation of rbcs with 10 mM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/ml apyrase, an ATPe scavenger, cell swelling was prevented.Pre-exposure to 10 mM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced MST7-dependent [ATPe] by 48%, and swelling by 80%, whereas in sodium free medium swelling decreased by 92%.Results were analyzed by means of a mathematical model. The best fit model showed that, upon MST7 exposure, ATP efflux required an approx. 2000-fold increase of ATP permeability mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. The accumulated ATPe activated P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activated ATP release. This sequence of events constitutes a positive feedback loop underlying ATP-induced ATP release of rbcs.General conclusionsResults with MST7 show that swelling and ATPe may regulate reciprocally in a dynamic manner, with purinergic signaling acting as a bridge connecting the dynamics of Vr and ATPe. This means that even slight increases of Vr may strongly activate ATP release.A peptides, on the other hand, can activate ATP release to different extends.The fact that the magnitude of ATP release increased with ageing of the peptides suggests a role of their oligomerization state in controlling ATPe homeostasis. Preliminary results show that A peptides may also cause rbcs swelling, a result which ?as with MST7- may anticipate a closed connection between changes in Vr and ATPe.