Dynamic recycling of extracellular ATP in human epithelial intestinal cells.

Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. The function of these cells is strongly influenced by purinergic signalling activated by extracellular ATP (eATP) and other nucleotides. The activity of several ecto-enzymes determines the dynamic regulation of eATP. In pathological contexts, eATP may act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from pathogens present in the intestinal lumen. In this study, we characterized the dynamics of eATP on polarized and non-polarized Caco-2 cells. eATP was quantified by luminometry using the luciferin-luciferase reaction. Results show that non-polarized Caco-2 cells triggered a strong but transient release of intracellular ATP after hypotonic stimuli, leading to low micromolar eATP accumulation. Subsequent eATP hydrolysis mainly determined eATP decay, though this effect could be counterbalanced by eATP synthesis by ecto-kinases kinetically characterized in this study. In polarized Caco-2 cells, eATP showed a faster turnover at the apical vs the basolateral side. To quantify the extent to which different processes contribute to eATP regulation, we created a data-driven mathematical model of the metabolism of extracellular nucleotides. Model simulations showed that eATP recycling by ecto-AK is more efficient a low micromolar eADP concentrations and is favored by the low eADPase activity of Caco-2 cells. Simulations also indicated that a transient eATP increase could be observed upon the addition of non-adenine nucleotides due the high ecto-NDPK activity in these cells. Model parameters showed that ecto-kinases are asymmetrically distributed upon polarization, with the apical side having activity levels generally greater in comparison with the basolateral side or the non-polarized cells. Finally, experiments using human intestinal epithelial cells confirmed the presence of functional ecto-kinases promoting eATP synthesis. The adaptive value of eATP regulation and purinergic signalling in the intestine is discussed.

Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells.

Pannexin 1 (PANX1) was proposed to drive ATP release from red blood cells (RBCs) in response to stress conditions. Stomatin, a membrane protein regulating mechanosensitive channels, has been proposed to modulate PANX1 activity in non-erythroid cells. To determine whether stomatin modulates PANX1 activity in an erythroid context, we have (i) assessed the in situ stomatin-PANX1 interaction in RBCs, (ii) measured PANX1-stimulated activity in RBCs expressing stomatin or from OverHydrated Hereditary Stomatocytosis (OHSt) patients lacking stomatin, and in erythroid K562 cells invalidated for stomatin. Proximity Ligation Assay coupled with flow imaging shows 27.09% and 6.13% positive events in control and OHSt RBCs, respectively. The uptake of dyes 5(6)-Carboxyfluorescein (CF) and TO-PRO-3 was used to evaluate PANX1 activity. RBC permeability for CF is 34% and 11.8% in control and OHSt RBCs, respectively. PANX1 permeability for TO-PRO-3 is 35.72% and 18.42% in K562 stom+ and stom- clones, respectively. These results suggest an interaction between PANX1 and stomatin in human RBCs and show a significant defect in PANX1 activity in the absence of stomatin. Based on these results, we propose that stomatin plays a major role in opening the PANX1 pore by being involved in a caspase-independent lifting of autoinhibition.

Interactive Dynamics of Cell Volume and Cell Death in Human Erythrocytes Exposed to α-Hemolysin from Escherichia coli.

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (Pf) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced Pf in control- and aquaporin 1-null red blood cells, although the relative Pf decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Human erythrocytes release ATP by a novel pathway involving VDAC oligomerization independent of pannexin-1.

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24-59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.

Dynamic regulation of extracellular ATP in Escherichia coli.

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (ß-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration.