RESUMO
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.
Assuntos
Trifosfato de Adenosina , Células Epiteliais , Humanos , Trifosfato de Adenosina/metabolismo , Células CACO-2 , Células Epiteliais/metabolismo , Proteínas de Transferência de FosfolipídeosRESUMO
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.
Assuntos
Tamanho Celular , Eritrócitos/citologia , Eritrócitos/fisiologia , Proteínas de Escherichia coli/farmacologia , Proteínas Hemolisinas/farmacologia , Modelos Biológicos , Trifosfato de Adenosina/metabolismo , Biomarcadores , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Relação Dose-Resposta a Droga , Proteínas de Escherichia coli/imunologia , Proteínas Hemolisinas/imunologia , Hemólise , Interações Hospedeiro-Patógeno , Humanos , Cinética , PermeabilidadeRESUMO
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⠵M). 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.