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R.R. Hodges, M.A. Shatos, J. Vrouvlianis, D.A. Dartt; Identification of P2X7 Purinergic Receptors and Their Effects on Function in Rat Lacrimal Gland . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3531.
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To determine the presence and location of P2X7 purinergic receptors and their effects on lacrimal gland functions.
Lacrimal glands were removed from male Sprague–Dawley rats. Western blot analysis was performed to determine the presence of P2X7 receptors on whole lacrimal glands homogenized in buffer containing 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate 150 mM NaCl pH 7.4, 1% Nonidet P–40, 0.5% sodium deoxycholate, and 0.1% SDS. For immunofluorescence (IF) experiments, glands were fixed in 4% formaldehyde and cryopreserved in 30% sucrose. Six–µm sections were cut and placed on slides. Slides were incubated with an antibody directed against P2X7 receptors for 1 h followed by incubation with a secondary antibody conjugated to Cy3. Acini were isolated by collagenase digestion, incubated with the Ca2+ indicator fura 2 for 1 h and intracellular [Ca2+] ([Ca2+]i) was measured after stimulation with UTP, which is more specific for P2Y receptors, ATP, a nonspecific P2X purinergic receptor agonist, or the P2X7 agonist benzyl–ATP (BzATP). Peroxidase, a marker of protein secretion was also measured after stimulation with these agonists using Amplex Red. Activation of p42/p44 MAPK was measured 10 min after addition of BzATP (10–4 M) and western blot analysis was performed using antibodies to phosphorylated (active) and total p42/p44 MAPK.
Western blot analysis using an antibody to P2X7 receptors showed a single band at the correct molecular weight. IF indicated that P2X7 receptors were present on the basolateral membranes of acinar and duct cells. UTP (10–4 M) did not increase the [Ca2+]i, while ATP gave a increase of 49.7 nM above basal at 10–3 M ATP. BzATP caused concentration–dependent increases that were maximum of 150 nM above basal with 10–4 M. BzATP increased peroxidase secretion 1.24 ± 0.05 fold above basal and p42/p44 MAPK activation 2.36 ± 1.35 fold above basal.
We conclude that the lacrimal gland contains functional P2X7 receptors that stimulate an increase in [Ca2+]i, protein secretion, and p42/p44 MAPK activation.
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