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K. Ohtomo, M. A. Shatos, R. R. Hodges, D. A. Dartt; BzATP Activates P2X7 and P2Y Receptors in Lacrimal Gland Myoepithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4180.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the types of purinergic receptors activated by the P2X7 compared to P2Y receptor agonist in rat lacrimal gland myoepithelial cells in culture
Rat lacrimal glands were dissociated by repetitive cycles of digestion in collagenase. Cells were placed in serum-supplemented RPMI-1640 medium until myoepithelial cells appeared, typically within 4 weeks. Cells were evaluated for expression of the myoepithelial cell markers smooth muscle actin (α-SMA), α-actinin and adenyl cyclase II. Immunofluorescence experiments were performed using antibodies to P2X7, P2Y11 and P2Y13 receptors. For intracellular calcium ([Ca2+]i) measurement, myoepithelial cells were seeded onto coverslips and cultured. All cells were loaded with the calcium indicator dye fura2 in a HEPES-buffered solution (119mM NaCl, 4.8mM KCl, 1.2mM MgSO4, 1mM CaCl2, 1.2mM KH2PO4, 25mM NaHCO3, 5.5mM glucose, 10mM HEPES, pH7.50) for 1hr at 37°C. Calcium measurements were obtained using the InCyt Im2TM ratio Imaging System using excitation wavelength of 340 and 380nm. The purinergic receptor agonists ATP (P2Y and P2X agonist), UTP (P2Y agonist), 2’(3’)-O-(4-benzoylbenzoyl) adenosine 5’ -triphosphate (BzATP, P2X7 agonist) were used. KN-62 and Brilliant blue G (BBG) were used as P2X7 receptor inhibitors.
α-SMA, α-actinin and adenyl cyclase II were expressed in the cultured lacrimal gland myoepithelial cells. Immunohistochemistry confirmed the presence of P2X7, P2Y11 and P2Y13 receptors on these cells. ATP increased [Ca2+]i in a concentration-dependent manner with maximum increase of 360±32nM at 10-3M. UTP increased [Ca2+]i in a concentration-dependent manner with maximum increase of 351±7nM at 10-4M. BzATP (10-4M) increased [Ca2+]i in the presence of Mg2+ by 160±54nM and the absence of Mg2+ by 191±9nM. ATP (10-4M) increased [Ca2+]i by 342±25nM in the presence of Mg2+ and by 289±85nM in the absence of Mg2+. The increase in [Ca2+]i was partially inhibited by pretreatment with KN-62 (10-4M) and BBG(10-4M), by 43% and 36% respectively (p≤<0.05). Addition of BzATP desensitized ATP receptor Ca2+ response and ATP desensitized the BzATP Ca2+ response.
We conclude that cultured myoepithelial cells contain P2X7 and P2Y receptors that increase [Ca2+]i. Activation of P2X7 receptors by ATP and BzATP appears to be the minor, whereas BzATP activating P2Y receptors appears to be the major, Ca2+ response.
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