Identification of P2X₃ and P2X₇ purinergic receptors activated by ATP in rat lacrimal gland.

Robin R Hodges, Joanna Vrouvlianis, Rachel Scott, Darlene A Dartt

Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. robin.hodges@schepens.harvard.edu

Investigative ophthalmology & visual science 2011 May

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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.

Citation

Robin R Hodges, Joanna Vrouvlianis, Rachel Scott, Darlene A Dartt. Identification of P2X₃ and P2X₇ purinergic receptors activated by ATP in rat lacrimal gland. Investigative ophthalmology & visual science. 2011 May;52(6):3254-63