Extracellular ATP hydrolysis in Caco-2 human intestinal cell line.

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5'-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques. In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

[Comparison of 2 serologic methods for the diagnosis of hydatidosis]. / Comparación de dos métodos serológicos para el diagnóstico de hidatidosis.

The sera of 176 patients with epidemiologic antecedents or radiologic and clinical signs of hydatidosis were tested by counter-immunoelectrophoresis (CIE) and enzyme-linked immunoassay (ELISA). A semipurified antigen from cysts of human origin were used for both techniques. The results were compared with those obtained from complementary radiologic studies and were confirmed by examination of excised cysts. Biopsy confirmed the diagnosis of hydatidosis in 65 patients (37%) and revealed the presence of other diseases in the remaining 111 (63%). Of the original 176 patients, 36 (20.4%) were positive by CIE and 62 (35.2%) by ELISA. Both techniques showed an excellent correlation with postsurgical diagnosis; neither produced any false positives, and the ELISA gave false negative results for only three patients (4.6%) with cysts that were infected, infertile, or calcified to some degree. The paper describes standardization of an inexpensive and easy-to-use microELISA.