Effect of Loss of P2Y2 Receptor Gene Expression on Nucleotide Regulation of Murine Epithelial Cl−Transport

Abstract

Extracellular nucleotides are believed to be important regulators of ion transport in epithelial tissues as a result of their ability to activate cell surface receptors. Although numerous receptors that bind nucleotides have been identified, the complexity of this receptor family, combined with the lack of pharmacological agents specific for these receptors, has made the assignment of particular receptors and ligands to physiological responses difficult. Because ATP and UTP appear equipotent and equieffective in regulating ion transport in many epithelia, we tested the hypothesis that the P2Y 2 receptor (P2Y 2 -R) subtype mediates these responses in mouse epithelia, with gene targeting techniques. Mice with the P2Y 2 -R locus targeted and inactivated (P2Y 2 -R(؊/؊)) were generated, airways (trachea), gallbladder, and intestines (jejunum) excised, and Cl ؊ secretory responses to luminal nucleotide additions measured in Ussing chambers. Comparison of P2Y 2 -R(؉/؉) with P2Y 2 -R(؊/؊) mice revealed that P2Y 2 -R mediated most (>85-95%) nucleotide-stimulated Cl ؊ secretion in trachea, about 50% of nucleotide responses in the gallbladder, and none of the responses in the jejunum. Dose-effect relationships for nucleotides in tissues from P2Y 2 -R(؊/؊) mice suggest that the P2Y 6 -R regulates ion transport in gallbladder and to a lesser extent trachea, whereas P2Y 4 and/or unidentified receptor(s) regulate ion transport in jejunum. We conclude that the P2Y 2 receptor is the dominant P2Y purinoceptor that regulates airway epithelial ion transport, whereas other P2Y receptor subtypes are relatively more important in other nonrespiratory epithelia.Metabotropic (G protein-coupled) P2Y receptors are expressed in epithelia and regulate epithelial ion transport and, consequently, have become potential targets for drug therapy of diseases that reflect abnormal epithelial ion transport (1). Of particular interest in this context is cystic fibrosis (CF), 1 a disease that reflects widespread defects in epithelial ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) (2). However, the spectrum of epithelia affected by this disease (e.g. airway, gallbladder, and intestine), the diversity of P2Y receptor subtypes (3), and the absence of selective reagents to define which P2Y receptor subtypes regulate ion transport have made it difficult to initiate therapeutic programs to target specific purinoceptors in these tissues.The strongest evidence for a role for P2 receptors in the regulation of ion transport emanates from studies of airway epithelia. Recent reports indicate that extracellular triphosphate nucleotides regulate many of the airway ion transport paths, including slowing Na ϩ absorption (4) and stimulating Cl Ϫ and K ϩ secretion (5, 6). Pharmacological studies in the human airway epithelial cell line CF/T43 have demonstrated that UTP and ATP are equipotent and cross-desensitize in promoting phospholipase C activity, leading to the hypothesis that the P2Y 2 receptor (P2Y 2 -R) is the d...

Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

“…The P2Y 2 receptor is of pharmacological interest due to its significant potential as a drug target. P2Y 2 receptor knockout mice were found to exhibit defective chloride secretion in airway epithelium in response to ATP and UTP, which suggests that P2Y 2 receptor agonists could function as a treatment for patients suffering from cystic fibrosis by bypassing the defective chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) [8]. P2Y 2 receptor agonism may possibly also find application in reducing post-ischemic myocardial damage, since cardioprotective effects were observed for the selective P2Y 2 receptor agonist uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768, compound 2 in Fig.…”

The purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) gene associated with essential hypertension in Japanese men

“…Furthermore, a variety of experimental approaches have provided evidence that local ATP release and P2RY2 stimulation may inhibit water reabsorption in the collecting duct. 8 Previous studies in mice have identified important functions for P2RY2 in a variety of processes, including nucleotide-regulated Ca 2 þ signalling in lung fibroblasts and airway epithelial cells, 9 nucleotide-stimulated Cl À secretion in the trachea and gallbladder, 10 neuronal growth, 11 stimulation of K þ secretion in the colon 12 and neutrophil chemotaxis. 13 In 2007, Rieg et al 14 reported that saltresistant hypertension occurs in the P2Y2 receptor (mice homologue of the human P2RY2 gene) knockout mice because the baroreceptor response to variations in salt intake remains intact.…”

Mediation of neurogenic ion transport by acetylcholine, prostanoids and 5-hydroxytryptamine in porcine ileum

“…As PPADS has some selectivity for P 2 -purinoceptors, we cannot rule out the possibility that electrically-evoked mucosal responses are mediated at least in part by ATP acting through P 1 -purinoceptors. However, active intestinal ion transport has been shown to be modulated predominately by P 2 -purinoceptors (Cressman et al, 1999;Christofi et al, 2004). The lack of effect of thiorphan on this response lends further support for the hypothesis that VIP, substance P and other classes of pro-secretory neuropeptides are not substantially involved in mediating this effect.…”