Involvement of apical P2Y 2 receptor-regulated CFTR activity in muscarinic stimulation of Cl − reabsorption in rat submandibular gland

Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl − channels in Ca 2+ -activated Cl − reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl − movement evoked by muscarinic stimulation. The Cl − concentration ([Cl − ]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTR inh -172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl − reabsorption. Systemically administered propranolol failed to alter the [Cl − ], suggesting little involvement of a β-adrenergic pathway in the Cl − movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl − ], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y 2 -receptor agonist, UTP, decreased the salivary [Cl − ] almost equipotently. CFTR inh -172 and suramin each prevented these effects, whereas 2′,3′- O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X 7 agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y 2 receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl − reabsorption occurs through basal CFTR activity and that this is regulated by P2Y 2 receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.