Fjærvoll Haakon K, Fjærvoll Ketil A, Yang Menglu, Bair Jeffrey, Utheim Tor P, Dartt Darlene A
Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States; Medical Student Research Program, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States; Medical Student Research Program, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
Exp Eye Res. 2023 Oct;235:109614. doi: 10.1016/j.exer.2023.109614. Epub 2023 Aug 12.
Ionotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca] ([Ca]) measured. [Ca] was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca] was significantly increased when stimulated with ATP (10-10 M). Suramin, a non-selective P2XR antagonist at 10 M did not reduce ATP-induced peak [Ca]. The potent P2X7 agonist, BzATP (10-10 M) increased the [Ca], although to a lesser extent than ATP. When measuring [Ca] the effect of repeated applications of ATP at 10 or 10 M the response "desensitized" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca] increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca]. ATP (10 M), BzATP (10 M) and 2MeSATP (10 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca] and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.
离子型嘌呤能受体（P2XRs）可被三磷酸腺苷（ATP）及其类似物激活。ATP可通过诸如泛连接蛋白半通道等ATP通透通道释放。激活后，P2XRs对钙离子通透，而钙离子是结膜杯状细胞（CGCs）中粘蛋白分泌的强效刺激物。本研究的目的是调查CGCs中P2XRs的存在情况及其功能。我们还检测了泛连接蛋白半通道的存在情况。用杯状细胞标志物抗细胞角蛋白7抗体以及针对P2X1 - 7受体和泛连接蛋白1 - 3的特异性抗体对大鼠原代传代CGCs进行染色。使用针对P2XRs和泛连接蛋白的特异性引物通过逆转录聚合酶链反应（RT - PCR）测定mRNA表达。使用与免疫荧光（IF）显微镜相同的抗体通过蛋白质印迹法（WB）鉴定蛋白质。为研究受体功能，将CGCs与Fura 2 - AM一起孵育，暴露于激动剂和拮抗剂，并测量细胞内钙离子浓度（[Ca]）。在敲低P2X4和P2X7受体表达后以及利用P2XRs的特异性特征时也测量了[Ca]。最后，在添加几种P2XR激动剂后测量粘蛋白分泌。所有P2XRs和泛连接蛋白均通过IF显微镜可视化，并通过RT - PCR和WB鉴定。用10⁻¹⁰ M的ATP刺激时，[Ca]显著增加。10 μM的非选择性P2XR拮抗剂苏拉明并未降低ATP诱导的[Ca]峰值。强效的P2X7激动剂，10⁻¹⁰ M的BzATP增加了[Ca]，尽管增幅小于ATP。在测量[Ca]时，当以10或10⁻⁶ M重复应用ATP时，30 - 60秒后反应“脱敏”。P2X4特异性拮抗剂5 - BDBD降低了P2X4激动剂2MeSATP诱导的[Ca]增加。此外，针对P2X4R而非P2X7R的小干扰RNA（siRNA）降低了激动剂诱导的[Ca]峰值。10 μM的ATP、10 μM的BzATP和10 μM的2MeSATP诱导了粘蛋白分泌。我们得出结论，培养的大鼠CGCs中存在所有七种P2XRs。在P2XRs中，只有同源三聚体P2X4R的激活似乎会增加[Ca]并诱导粘蛋白分泌。CGCs中的P2X4R为保护性粘蛋白分泌提供了一个新的治疗靶点。
