Dynamic reuglation of cell volumen and extracellular ATP of human erythrocytes

SCHWARZBAUM PJ, LEAL DENIS MF, ALVAREZ HA, LAURI N, CHARA O

Salto

Conferencia; Latin American Cross Talk in Biophysics and Physiology; 2015

Sociedad Argentina de Biofisica

The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs)the release of ATP and swelling.Since swelling is a well-known inducer of ATP release, and extracellular(ATPe), interacting with P (purinergic) receptors, can affect cell volume(Vr), we explored the reciprocal regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of both Vr(by epifluorescence microscopy) and [ATPe] (by online luminometry) inthe absence and presence of blockers of ATP efflux, swelling and Preceptors.MST7 promoted acute, strongly correlated changes in [ATPe] and Vr ofrbcs.Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe],blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by40%.In MST7 treated rbcs, pre-incubation of rbcs with 10 μM of eithercarbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1,reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presenceof 80 U/ml apyrase, an ATPe scavenger, cell swelling was prevented.Pre-exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediatingsodium influx, reduced MST7-dependent [ATPe] by 48%, and swelling by80%, whereas in sodium free medium swelling decreased by 92%.Results were analyzed by means of a mathematical model. The best fitmodel showed that, upon MST7 exposure, ATP efflux required an approx.2000-fold increase of ATP permeability mediated by two kineticallydifferent conduits, both of which were activated by swelling and inactivatedby time. The accumulated ATPe activated P2X receptors, followed bysodium influx, resulting in cell swelling, which in turn further activatedATP release. This sequence of events constitute a positive feedback loopunderlying ATP-induced ATP release of rbcs. The systemic consequence ofthese findings will be analyzed.With grants from UBACYT (20020100100090) and CONICET (PIP639).