Structure and Function of P2Y₂ Nucleotide Receptors in Cystic Fibrosis (CF) Epithelium

Abstract

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane-conductance regulator (CFTR), a cAMP-dependent chloride transporter expressed in epithelial tissue. A novel treatment being developed for CF utilizes inhaled nucleotides, particularly uridine 5′-triphosphate (UTP), to activate calcium-dependent chloride channels in airway epithelial cells. This therapy is based on earlier studies from the laboratory of Dr. Richard C. Boucher at the University of North Carolina-Chapel Hill, which have shown that luminal administration of UTP¹ and the Na⁺ channel blocker amiloride,² together overcome the ion transport defects in CF airway epithelial cells by promoting increased Cl⁻ secretion and decreased Na⁺ absorption.³ UTP stimulates Cl⁻ secretion in epithelial cells by activating a P2Y₂ (formerly named P_2U) nucleotide receptor subtype^4,5 that is coupled to phospholipase C via G_qα protein resulting in inositol 1,4,5 trisphosphate (IP₃)-dependent increases in the cytoplasmic calcium concentration ([Ca²⁺]_i) that serve to activate calciumdependent chloride channels.⁶ Evidence that activation of this non-CFTR Cl⁻ secretory conductance may be beneficial in CF therapy was provided by studies with CFTR knockout mice in which the presence of a large, basal, non-CFTR Cl⁻ conductance was associated with the lack of respiratory disease in this species.⁴ Early clinical studies involving administration of aerosolized UTP and amiloride indicate that this therapy has a positive effect on mucociliary clearance in CF nasal epithelia.⁷ However, little is known about the mechanisms of nucleotide regulation of epithelial ion transport. It is clear that fundamental information concerning P2Y₂ receptor biology in epithelia will be useful in optimizing nucleotide therapy for CF. We have utilized the CFTR knockout mouse and its normal counterpart to isolate cultures of gallbladder epithelial cells that form tight junctions and represent a good model for studying the roles of P2Y₂ receptors in mediating signal transduction pathways leading to increased Cl⁻ secretion. In addition, we have cloned the human airway epithelial cell P2Y₂ receptor cDNA and expressed the receptor in a heterologous cell system, human 1321N1 astrocytoma cells, that lack any endogenous nucleotide receptors.⁸ In the 1321 N1 cell transfectants, the human airway P2Y₂ receptor is distinguished pharmacologically by its ability to be activated equipotently by the purine nucleotide ATP and the pyrimidine nucleotide, UTP. The investigation of the molecular and pharmacological properties of the P2Y₂ receptor expressed in 1321N1 cells is being undertaken to develop methods to maximize calcium-dependent chloride conductance in CF epithelial cells through stimulation of endogenous P2Y₂ receptors.

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