Characterization of P2X7 Purinergic Receptors and Their Function in Rat Lacrimal Gland

PURPOSE. To characterize the effects of P2X(7) purinergic receptors on lacrimal gland function. METHODS. P2X(7) purinergic receptors were identified by RTPCR, Western blot analysis, and immunofluorescence techniques. Rat lacrimal gland acini were isolated by collagenase digestion. Acini were incubated with the fluorescent indicator molecule fura 2, and [Ca(2+)](i) was measured by a fluorescence imaging system. Protein secretion was measured with a fluorescence assay system. Activation of ERK 1/2 was determined by Western blot analysis with an antibody against phosphorylated (active) ERK 1/2. RESULTS. P2X(7) receptors were present in the lacrimal gland by RT-PCR and Western blot analysis. These receptors were located in the membranes of acinar and ductal cells and the cytoplasm of acinar cells. Activation of P2X(7) receptors with (benzoylbenzoyl) adenosine 5'-triphosphate increased [Ca(2+)](i), peroxidase secretion, and ERK 1/2 activation, each of which was inhibited by the P2X(7) receptor inhibitors Brilliant Blue G or A 438079. CONCLUSIONS. P2X(7) purinergic receptors are present in rat lacrimal gland and when stimulated increase [Ca(2+)](i), protein secretion, and ERK 1/2 activation. (Invest Ophthalmol Vis Sci. 2009; 50: 5681-5689) DOI: 10.1167/iovs.09-3670