Desensitization of P2Y2 receptor-activated transepithelial anion secretion.
Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.
Clarke
L L
LL
Dalton Cardiovascular Research Center and Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211, USA. clarkel@missouri.edu
Harline
M C
MC
Otero
M A
MA
Glover
G G
GG
Garrad
R C
RC
Krugh
B
B
Walker
N M
NM
González
F A
FA
Turner
J T
JT
Weisman
G A
GA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001254:Astrocytoma; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D019281:Dimerization; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D005704:Gallbladder / Q000502:physiology*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D014162:Transfection; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology*
D007295:Inositol Phosphates; C546382:P2RY1 protein, human; C546385:P2RY2 protein, human; C546383:P2ry1 protein, mouse; C546386:P2ry2 protein, mouse; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate