Desensitization of P2Y(2) receptor-activated transepithelial anion secretion

Desensitization of P2Y(2) receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epitheli al diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y(2) receptors, freshly excised or cultured murin e gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure s hort-circuit current (I-sc), an index of electrogenic anion secretion. Lumi nal treatment with nucleotide receptor agonists increased the I-sc with a p otency profile of ATP = UTP > 21-methylthio-ATP >> alpha,beta-methylene-ATP . RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. T he desensitization of anion secretion required a 10-min preincubation with the P2Y(2) receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximate to 10(-6) M). Approximately 40% of the anion secre tory response was unaffected by maximal desensitizing concentrations of UTP . Recovery from UTP-induced desensitization was rapid (<10 min) at preincub ation concentrations less than the EC50 (1.9 x 10(--6) M) but required prog ressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensiti zed with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was u naffected by preincubation with a desensitizing concentration of UTP. It wa s concluded that 1) desensitization of transepithelial anion secretion stim ulated by the P2Y(2) receptor agonist UTP is time and concentration depende nt; 2) recovery from desensitization is prolonged (>90 min) at UTP concentr ations >10(-5) M; and 3) UTP-induced desensitization occurs before the oper ation of the anion secretory mechanism.