Increase of Intracellular Ca2+ by Purinergic Receptors in Cultured Rat Lacrimal Gland Myoepithelial Cells
Increase of Intracellular Ca2+ by Purinergic Receptors in Cultured Rat Lacrimal Gland Myoepithelial Cells
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DOI：
10.1167/iovs.11-7809
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发表时间：
2011-12-01
影响因子：
4.4
通讯作者：
Dartt, Darlene A.
中科院分区：
文献类型：
--
作者：
Ohtomo, Kaori;Shatos, Marie A.;Dartt, Darlene A.
目的.目的：分离培养大鼠泪腺肌上皮细胞（myoepithelial cells，MEC），鉴定其嘌呤能受体亚型。对大鼠泪腺进行胶原酶消化，并使MEC生长。对从MEC分离的RNA进行嘌呤能受体P2 X（7）、P2 Y（1）、P2 Y（11）和P2 Y（13）的RT-PCR。免疫荧光实验用针对MEC标记物和P2 X（7）、P2 Y（1）、P2 Y（11）和P2 Y（13）嘌呤能受体的抗体进行。使用Western印迹分析技术分离来自MEC的蛋白质。此外，将细胞与Fura 2四乙酰氧基甲酯孵育，并测定细胞内[Ca 2 +]（[Ca 2 +]（i））对P2嘌呤能激动剂的响应。MEC表达MEC蛋白α-平滑肌肌动蛋白、波形蛋白、α-辅肌动蛋白和腺苷酸环化酶II。RT-PCR、Western印迹和免疫荧光技术证实了嘌呤能受体P2 X（7）、P2 Y（1）、P2 Y（11）和P2 Y（13）的存在。嘌呤能激动剂ATP、苯甲酰苯甲酰基ATP（BzATP）、α，β亚甲基ATP、UTP、2-甲硫基-ATP（MeSATP）和ATP γ S增加[Ca 2 +]（i）。由于BzATP与P2 X（7）受体结合，因此研究了该受体的特异性特征。P2 X（7）受体抑制剂和细胞外Mg 2+或Ca 2+的去除都不会影响BzATP刺激的[Ca 2 +] i增加。重复应用BzATP使这种反应脱敏。P2 Y（1）、P2 Y（11）和P2 Y（13）的抑制剂均能降低BzATP刺激的[Ca 2 +]（i）升高，其中P2 Y（1）抑制剂最有效。MEC可以从大鼠泪腺中分离，并且它们表达P2 X（7）、P2 Y（1）、P2 Y（11）和P2 Y（13）嘌呤能受体。令人惊讶的是，BzATP结合P2 Y（1）受体，其主要负责BzATP刺激的[Ca 2 +] i增加。（Invest Ophthalmol维斯科学。2011; 52：9503-9515）DOI：10.1167/iovs.11-7809
PURPOSE. To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs.METHODS. Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) 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.RESULTS. MECs expressed the MEC proteins alpha-smooth muscle actin, vimentin, alpha-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), alpha,beta methylene ATP, UTP, 2-methylthio-ATP (MeSATP), and ATP gamma S increased [Ca2+](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) 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 P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca2+](i) with the P2Y(1) inhibitor most effective.CONCLUSIONS. MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca2+] i. (Invest Ophthalmol Vis Sci. 2011; 52:9503-9515) DOI: 10.1167/iovs.11-7809