ATP release in human erythrocytes infected by Plasmodium falciparum

ALVAREZ CL, DE SOUZA SILVA L, FERREIRA-DA SILVACT, PINHEIRO AAS, CARUSO-NEVES C, MUANIS PERSECHINI P, SCHWARZBAUM PJ Y SCHACHTER J

Rio de Janeiro

Congreso; XVIII International Congress for tropical Medicine and Malaria; 2012

IFTM, XVIII ICTMM, SBMT, FioCruz

In human erythrocytes (RBCs) various stimuli induce increases in cAMP that trigger the non-lytic release of ATP, with pannexin 1 being one of the main proteins mediating such a release. Previous reports showed that the resulting extracellular ATP (ATPe) can act on purinergic receptors of endothelial cells to promote the relaxation of the vascular caliber in the microcirculation. RBCs are one of the main targets of the malaria parasite Plasmodium falciparum, and patients infected with these parasites exhibit altered microvascular responsiveness of unknown origin. Still it is not clear whether these circulatory problems of malaria patients are linked to an altered release of ATP by infected RBCs, and whether the potential changes in ATPe accumulation might affect the infectious process and the host response. We thus evaluate the kinetics of cAMP induced ATP release by quantifying the kinetics of ATPe concentration from RBCs at various stages of infection (ring, trophozoite and schizont stages) with Plasmodium falciparum. A mixture, here called 3V containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP dependent ATP release. Addition of 3V to healthy RBCs induced an acute increase of ATPe to a maximum value, which remained constant thereafter, as previously shown by some of us. Using parasited RBCs (parasitemias ranging from 2,5 to 12,5%) at different stages produced the release of ATP with as similar kinetics as in healthy cells, but the amplitude of the response was significantly increased. All parasited RBCs produce an approx. two fold increase in ATPe release as compared with controls, with schizont cells showing the highest values (2.5 fold) . In separate experiments using 3V induced parasited RBCs, a positive correlation was found between ATPe concentration and parasitemia in the ring and trophozoite stages (p=0,012 and 0,0012, respectively); but not in the case of schizont cells (p=0,1923). To study the mechanism enabling ATP efflux in parasited RBCs we used mefloquine and carbenoxolone , two well known blockers of pannexin 1. When a pure population of trophozoites was incubated with either mefloquine (100 nM) or carbenoxolone (100 µM) 10 min before 3V addition, ATP release was inhibited by 71 and 65% respectively. This is at variance with healthy RBCs, where carbenoxolone and mefloquine at the same doses are able to inhibit 100% of the 3V induced response. These preliminary data suggest that parasite invasion will enhance pannexin1-mediated ATP efflux of adrenergically stimulated RBCs, with potential consequences on microsvascular resistance. We are currently evaluating potential changes in ATPe degradation by RBCs ectonucleotidases, which could partially counteract the observed enhancements of ATP release. With grants from CAPES-MINCYT (197/11), UBA, CONICET and ANPCyT (PICT 0151).