Dynamic regulation of cell volume and extracellular ATP of human erythrocytes

SCHWARZBAUM P J; LEAL DENIS M F; ÁLVAREZ H A; CHARA O

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Sociedad Argentina de Biofísica - Seccional Biofísica de la Sociedad Uruguaya de Biociencias

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) in the absence and presence of blockers of ATP efflux, swelling and P 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 uM 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 uM 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 constitute a positive feedback loop underlying ATP-induced ATP release of rbcs. The systemic consequence of these findings will be analyzed.