S68 duodenum and, subsequently, the secretion of HCO3−, which in turn activates IAP, thereby decreasing ATP concentrations and P2Y signaling. Immunofluorescence studies using anti-P2Y1 receptor, anti-P2Y2 receptor, anti- P2Y4 receptor, and anti-P2Y6 receptor antibodies showed that P2Y1 receptors are expressed on the brush border membranes of the duodenum (Fig. 4a). P2Y2 and P2Y4 expression were not observed (Fig. 4b, c), while that of P2Y6 had a diffuse pattern of expression (Fig. 4d). The receptors were also pharmacologically characterized such that the activation of P2Y1 with ATP triggered an increase in HCO3− secretion, which was reduced by the addition of P2Y1 antagonists. P2Y receptors in other tissues and cells Clara-like cells are epithelial cells present in the intra pulmonary airways of various animal species and reside near pulmonary neuroepithelial bodies (NEBs). They demonstrate pluripotent stem cell-like features and are mainly involved in airway epithelial regeneration following lung injury [32–34]. NEBs are clusters of pulmonary neuroendocrine cells and have been suggested to have a role in various physiological processes in the lungs, with a significant role in airway oxygen sensing [35, 36]. Purinergic signaling in NEBs has only been indirectly demonstrated by ATP accumulation in secretory vesicles of rat NEBs [37]. In order to understand the possible cross talk between NEBs and Clara-like cells, the hypothesis that ATP acts as a paracrine transmitter in the NEB microenviron- ment was tested. Calcium imaging, pharmacology, and Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 immunohistochemistry (using Alomone Labs antibodies) were all employed to identify and localize purinoceptors [33]. Clara-like cells were shown to express functional P2Y2 receptors. The study further shows that the two cell types are indeed coupled by the release of ATP by NEB cells and the subsequent activation of purinoceptors (P2Y2) localized on the membranes of Clara-like cells. This signaling pathway may be essential for normal airway function, for the regeneration of airway epithelial following injury, and also perhaps for the pathogenesis of small cell carcinomas [33]. P2Y receptors in disease states Hepatocytes express receptors for high-density lipoprotein (HDL) which are responsible for carrying cholesterol from peripheral tissues to the liver for elimination. Studies show that HDL levels are inversely proportional to the risk of developing coronary artery disease [38]. In hepatocytes, HDL undergoes endocytosis in a P2Y13-dependent manner [38–40]. The signaling pathway leading from P2Y13 activa- tion to the internalization of HDL was examined. The study shows that P2Y13 stimulation specifically induced RhoA activation and its effector ROCK I, leading to HDL endocytosis through cytoskeleton reorganization. P2Y13 silencing via shRNA, which was demonstrated by both a decrease in mRNA levels and protein levels (as shown by Western blot analysis using anti-P2Y13 receptor antibody), prevented the membrane translocation of ROCK I induced by known P2Y13 agonists. Deciphering new molecular targets involved in HDL endocytosis may be important for Fig. 4 Expression of P2Y receptors in rat duodenal epithelium. Immunohistochemical staining was done using anti-P2Y1 receptor antibody (#APR-009), anti-P2Y2 receptor antibody (#APR-010), and anti-P2Y6 receptor antibody (#APR-011). a P2Y1 is expressed along the brush border membranes of villous cells. P2Y2 and P2Y4 receptors are not expressed (b, c). d P2Y6 receptor is diffusely expressed in the villous cells and interstitium. Adapted from refer- ence 31 with permission of John Wiley and Sons