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

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.