Extracellular ATP induces cell death in human intestinal epithelial cells.

BACKGROUND: Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP. METHODS: We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique. RESULTS: ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells. GENERAL SIGNIFICANCE: The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.

Lectin histochemistry of microglia in superior colliculus of the developing opossum

The development of microglia in the opossum superior colliculus (SC) has been studied by lectin histochemistry (Griffonia simplicifolia B4 isolectin, GsI/B4). Prior to the end of neurogenesis (by postconceptional day 26, PcD 26), there are virtually no GsI/B4+ cells in the SC parenchyma although rare roundish elements are found at the tectal and, in larger numbers, the tegmental border of the aqueduct. The appearance of microglia in the SC follows a ventrodorsal gradient, correlating with the direction of neurogenesis, cytomorphological differentiation and growth of the vascular network rather than with a leptomeningeal source, and without forecasting value for astroglial differentiation. In the superficial layers (sSC), relatively few but moderately ramified cells rather than macrophages coexist with regressive changes in retinocollicular axons (by PcD 39-53). By the end of and soon after this period, there is a striking increase in the number of fairly ramiried GsI/B4+ cells within the SC proper. Macrophages also become abundant but remain restricted to the vicinity of the aqueductal ependyma and are fewer at the tectal than at the tegmental aspect. These supraependymal macrophages as well as ramified parenchymal cells maintain the ability to divide at a low rate throughout maturation. The ingress via the aqueduct and cell proliferation may contribute to the complement of SC microglia but the major immediate source remains unknown.