Characterizing the presence and sensitivity of the P2X7 receptor in different compartments of the gut.

Purinergic signaling has been established as an important feature of inflammation and homeostasis. The expression of a number of P2 receptor subtypes in the gut has been reported. In this study, using a well-known permeabilization method that is assessed by flow cytometry, we show that lymphocytes and macrophages from the mesenteric lymph nodes (MLN) and the peritoneal cavity exhibit different sensitivities to extracellular ATP. Compared with the macrophages, the lymphocytes are more sensitive to ATP in the MLN compartment, whereas in the peritoneal cavity the macrophages are more sensitive to ATP than the lymphocytes. In addition, we have shown that the epithelial cells from the small bowel are more resistant to the ATP effects than the cells from the colon. These cells, however, become susceptible after exposure to IFN-γ. Furthermore, by examining parameters such as pH manipulation, the exposure to divalent cations and the P2X7 antagonist Brilliant Blue G, and the use of cells from P2X7(-/-) mice, we have shown that the P2X7 receptors are the ATP-activated receptors responsible for the permeabilization phenomenon. In addition, using Western blot analysis, we have demonstrated the changes in the P2X7 receptor expression in immune cells isolated from different sites in the gut and in the gut-associated lymphoid tissues. Our findings suggest the existence of the site-specific modulation of P2X7 receptors on epithelial and immune cells, and we define purinergic signaling as a new regulatory element in the control of inflammation and cell fate in the gut and in the gut-associated lymphoid tissues.

P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation.

Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 >> P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.