Extracellular ATP regulation of human erythrocytes in health and disease

LAURI N, LEAL DENIS MF, ALVAREZ CL, ALVAREZ HA , HERLAX V, CHARA O, SCHWARZBAUM PJ.

Ciudad de Mexico

Workshop; Latinamerican workshop and conference on systems biology; 2017

Severalstimuli trigger the release of ATP from human erytrocytes (rbcs). ATP exit canbe modulated by mechanical stress, purinergic signaling, adhesion and/orchanges in cell volume (Vr). In vivo,intravascular increases of rbcs-derived extracellular ATP (ATPe) may regulate vasodilationand inflammation. Westudied the effects of peptides on the regulation of rbcs-derived ATPe. We used1- the tetradecapeptide mastoparan 7 (MST7), which may activate cell swelling (i.e.,Vr increase) and/or an increase of cAMP concentration. Both processes mayactivate ATP exit; 2- Beta amyloid peptides Ab1-40and Ab1-42,which accumulate in the blood of healthy individuals and, more importantly, ofAlzheimer patients. Ageing of Ab (i.e.peptide incubation in the absence of cells) provoke the irreversible transitionof these peptides from its monomeric form to oligomers and fibers, with unknownconsequences on ATPe regulation of rbcs.  MethodsATPewas assessed continuously by real time luminiscence using theluciferase-luciferin system. Results were expressed as [ATPe] at every timepoint of a kinetic curve (i.e., ATPe kinetics), with [ATPe] expressed aspmol/106 cells or nM/40 μl. Alternatively, increases in [ATPe] wereevaluated as the difference between [ATPe] at 1 min post stimulus and the basal[ATPe], and are indicated as ΔATP1 (nM/3 106 cells).Vrwas followed by fluorescence microscopy using BCECF loaded rbcs. Rbcswere attached on coverslips coated with 0.01% poly-D-lysine. ForMST7 exposed rbcs, the dynamic regulation of Vr and ATPe was analyzed by meansof a data-driven mathematical model. Studiesusing AbAb1-40and Ab1-42 wereallowed to age for 0.25-72h before use. MST7 was used as a positive control ofATP release. The effects of 1 µM Abpeptides on ATP release was acute, thus allowing to calculate ΔATP1values. Inrbcs, Ab1-40 agedfor 23hs and 72hs, causing a xxx and xxxx nM/(3 106cells) increaseof ΔATP1 with respect to untreated rbcs (controls).Onthe other hand, exposure of cells to Ab1-42 agedfor 0.25-23hs triggered a xxx-xxx nM/(3 106cells) increase of ΔATP1.For Ab1-42 ageing≥48hs, increased ΔATP1 by xxxx-xxxx, approx. xxxx-xxx% higher thatMST7 induce ATP release.Currentexperiments are aimed at identifying the plasma membrane conduits responsiblefor Ab dependent ATPrelease of rbcs. Studiesusing MST7MST7triggered ATP release and swelling. Since swelling is a well-known inducer ofATP release, and extracellular (ATPe), interacting with purinergic receptors,can affect Vr, we explored the reciprocal regulation between Vr and ATPe.Kineticsof ATPe and Vr was assessed in the absence and presence of blockers of ATP conduits,swelling and purinergic receptors.MST7promoted acute, strongly correlated changes in [ATPe] and Vr of rbcs. WhereasMST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling ina hyperosmotic medium + MST7 reduced [ATPe] by 40%. InMST7 treated rbcs, pre-incubation of rbcs with 10 mMof either carbenoxolone or probenecid, two inhibitors of the ATP conduitpannexin 1, reduced [ATPe] by 40-50% andswelling by 40-60%, while in the presence of 80 U/ml apyrase, an ATPescavenger, cell swelling was prevented.Pre-exposureto 10 mM NF110, a blocker of ATP-P2Xreceptors mediating sodium influx, reduced MST7-dependent [ATPe] by 48%, andswelling by 80%, whereas in sodium free medium swelling decreased by 92%.Resultswere 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 ATPpermeability mediated by two kinetically different conduits, both of which wereactivated by swelling and inactivated by time. The accumulated ATPe activatedP2X receptors, followed by sodium influx, resulting in cell swelling, which inturn further activated ATP release. This sequence of events constitutes apositive feedback loop underlying ATP-induced ATP release of rbcs. GeneralconclusionsResultswith MST7 show that swelling and ATPe may regulate reciprocally in a dynamicmanner, with purinergic signaling acting as a bridge connecting the dynamics ofVr and ATPe. This means that even slight increases of Vr may strongly activateATP release. Abpeptides, on the other hand, can activate ATP release to different extends.Thefact that the magnitude of ATP release increased with ageing of the peptidessuggests a role of their oligomerization state in controlling ATPe homeostasis.Preliminaryresults show that Ab peptidesmay also cause rbcs swelling, a result which ?as with MST7- may anticipate aclosed connection between changes in Vr and ATPe. Withgrants from PIP 11220150100459 CONICET, UBACYT 20020130100027BA, PICT 0327ANPCyT.