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Kaori Ohtomo, Marie A. Shatos, Joanna Vrouvlianis, Dayu Li, Robin R. Hodges, Darlene A. Dartt; Increase of Intracellular Ca2+ by Purinergic Receptors in Cultured Rat Lacrimal Gland Myoepithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(13):9503-9515. doi: 10.1167/iovs.11-7809.
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To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs.
Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X7, P2Y1, P2Y11, and P2Y13 on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X7, P2Y1, P2Y11, and P2Y13 purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca2+] ([Ca2+]i) was determined in response to P2 purinergic agonists.
MECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X7, P2Y1, P2Y11, and P2Y13. The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,β methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca2+]i. As BzATP binds to the P2X7 receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X7 receptors nor removal of extracellular Mg2+ or Ca2+ had an effect on the BzATP-stimulated increase in [Ca2+]i. Repeated applications of BzATP desensitized this response. Inhibitors for P2Y1, P2Y11, and P2Y13 each decreased the BzATP-stimulated increase in [Ca2+]i with the P2Y1 inhibitor most effective.
MECs can be isolated from rat lacrimal glands, and they express P2X7, P2Y1, P2Y11, and P2Y13 purinergic receptors. Surprisingly, BzATP binds the P2Y1 receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca2+]i.
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