Regulation of extracellular ATP in health and disease.

SCHWARZBAUM PJ

Simposio; Reunión Conjunta de Sociedades de Biociencias; 2017

Human red blood cells (rbcs) can release ATP when exposedto adrenergic stimulation, mechanical deformation,cell adhesion, blood flow turbulence, hypoxia andacidosis or, unspecifically, under conditions promotinghemolysis.A reduction of ATP release has been associated with typeII diabetes and cystic fibrosis, while rbcs infection withPlasmodium falciparum correlated with activated ATP release.Both, in physiological and pathological conditions,the resulting extracellular ATP (ATPe), and its metabolicproducts, can activate purinergic receptors which mediatea wide variety of local and systemic responses, suchas changes in cell volume or intravascular pressure.Irrespective of the nature of the stimuli, kinetics of ATPeaccumulation depends not only on ATP exit, but also onATPe hydrolysis by nucleotidases. This is because ATPeconcentration can be downregulated by ectonucleotidasesof intact rbcs, or by intracellular nucleotidases releasedto the extracellular space under hemolysis.Candidate conduits for regulated ?non lytic- ATP releaseinclude several anionic channels and pore forming proteins.In addition, bacterial toxins such as alpha hemolysin(HlyA) were shown to activate ATP exit of rbcs, presumablyby forming pores through which the nucleotidetransverses the plasma membrane.HlyA is an exotoxin secreted by uropathogenic strainsof Escherichia coli, causing lysis of several mammaliancells, including human rbcs. In vivo HlyA has been associatedwith urinary tract infections and septicemia. HlyAis secreted by bacteria in the intravascular milieu, whereit can bind to rbcs and other cells.During this talk we will analyze ATPe regulation of rbcsexposed to HlyA and analogs.We made a quantitative estimate of ATPe kinetics, togetherwith a description of the main processes controllingHlyA-dependent ATP regulation of rbcs.Our results show that, in rbcs:- Exposure to HlyA produced a nonlinear and biphasicincrement of ATPe, caused by simultaneous lytic and nonlytic ATP release. The [ATPe] increase showed an acutephase, mainly driven by non lytic ATP release, and a latephase that is mostly dependent on lytic ATP release andATPe hydrolysis by nucleotidases.- Moreover, HlyA led to slight swelling and increasedsphericity, which was in part responsible for the observedactivation of ATP release.- Both proHlya, the unacylated precursor of HlyA andHlyA-GF(-), an HlyA mutant lacking the segment capableof binding to rbcs glycophorin, showed little or negligibleATP exit.- Blockage of P2X receptors led to significant reductionof HlyA induced hemolysis, causing downregulation ofATPe kinetics.Several experiments are under way to test other processespotentially affecting ATPe regulation of HlyA treatedrbcs. Among these are aggregability and deformability ofrbcs, intravascular pressure, and rbcs adhesion to endothelialcells.With grants from UBACYT (20020130100027BA), PICT2014-0327, CONICET (PIP0459) y ECOS Sud (Francia)-MINCYT(Argentina) A15S01, PDTS-CIN 193