title: Identification of P2X₃ and P2X₇ purinergic receptors activated by ATP in rat lacrimal gland.
abstract: PURPOSE. To identify the type of purinergic receptors activated by adenosine triphosphate (ATP) in rat lacrimal gland and to determine their role in protein secretion. METHODS. Purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Acini from rat lacrimal gland were isolated by collagenase digestion. Acini were incubated with the fluorescence indicator fura-2 tetra-acetoxylmethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined. Protein secretion was measured by fluorescence assay. RESULTS. The authors previously showed that P2X(7)receptors were functional in the lacrimal gland. In this study, they show that P2X(1-4) and P2X(6)receptors were identified in the lacrimal gland by RT-PCR, Western blot, and immunofluorescence analyses. P2X(5) receptors were not detected. ATP increased [Ca(2+)](i) and protein secretion in a concentration-dependent manner. Removal of extracellular Ca(2+) significantly reduced the ATP-stimulated increase in [Ca(2+)](i). Repeated applications of ATP caused desensitization of the [Ca(2+)](i) response. Incubation with the P2X(1) receptor inhibitor NF023 did not alter ATP-stimulated [Ca(2+)](i). Incubation with zinc, which potentiates P2X(2) and P2X(4) receptor responses, or lowering the pH to 6.8, which potentiates P2X(2) receptor responses, did not alter the ATP-stimulated [Ca(2+)](i). P2X(3) receptor inhibitors A-317491 and TNP-ATP significantly decreased ATP-stimulated [Ca(2+)](i) and protein secretion, whereas the P2X(3) receptor agonist α,β methylene ATP significantly increased them. The P2X(7) receptor inhibitor A438079 had no effect on ATP-stimulated [Ca(2+)](i) at 10(-6) M but did have an effect at 10(-4) M. CONCLUSIONS. Purinergic receptors P2X(1-4) and P2X(6) are present in the lacrimal gland. ATP uses P2X(3) and P2X(7) receptors to stimulate an increase in [Ca(2+)](i) and protein secretion.
authors.affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. robin.hodges@schepens.harvard.edu
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Joanna
authors.initials: J
authors.lastname: Vrouvlianis
authors.forename: Rachel
authors.initials: R
authors.lastname: Scott
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect; D016257:Fura-2 / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D058474:Receptors, Purinergic P2X1 / Q000378:metabolism; D058477:Receptors, Purinergic P2X3 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013498:Suramin / Q000031:analogs & derivatives / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: C105374:NF023; D058920:Purinergic P2X Receptor Antagonists; D058474:Receptors, Purinergic P2X1; D058477:Receptors, Purinergic P2X3; D058486:Receptors, Purinergic P2X7; D013498:Suramin; D000255:Adenosine Triphosphate; D015032:Zinc; D002118:Calcium; D016257:Fura-2