离子型嘌呤能受体(P2XR)被ATP和ATP类似物激活。ATP可以通过ATP-可渗透通道如pannexin半通道释放。激活后,P2XRs变得对Ca2+具有渗透性,结膜杯状细胞(CGC)中粘蛋白分泌的有效刺激物。这项研究的目的是研究P2XRs在CGCs中的存在和功能。我们还检查了pannexin半通道的存在。用杯状细胞标记抗细胞角蛋白7抗体和针对P2X1-7受体和pannexin1-3的特异性抗体对大鼠第一代CGC进行染色。通过RT-PCR使用对P2XR和膜联蛋白特异的引物测定mRNA表达。使用与免疫荧光(IF)显微术相同的抗体,用蛋白质印迹(WB)鉴定蛋白质。为了研究受体功能,CGC与Fura2-AM孵育,暴露于激动剂和拮抗剂,和细胞内[Ca2+]([Ca2+]i)测量。在敲除P2X4和P2X7受体表达后也测量[Ca2]i,以及利用P2XR特定特性时。最后,添加几种P2XR激动剂后测量粘蛋白分泌。所有P2XRs和pannexins用中频显微镜观察,并用RT-PCR和WB进行鉴定。当用ATP(10-7-10-4M)刺激时,[Ca2+]i显著增加。苏拉明,10-4M的非选择性P2XR拮抗剂不会降低ATP诱导的峰[Ca2]i。强效的P2X7激动剂,BzATP(10-7-10-4M)增加[Ca2+]i,虽然程度低于ATP。当测量[Ca2]i时,在10-5或10-6M重复施用ATP的作用,30-60s后反应“脱敏”。P2X4特异性拮抗剂5-BDBD降低了P2X4激动剂,2MeSATP,-诱导[Ca2+]i增加。此外,针对P2X4R的siRNA,但不是P2X7R,降低激动剂诱导的峰[Ca2+]i。ATP(10-5M),BzATP(10-4M)和2MeSATP(10-5M)诱导粘蛋白分泌。我们得出的结论是,所有7种P2XR都存在于培养的大鼠CGC中。在P2XR中,只有同三聚体P2X4R的激活似乎增加[Ca2]i并诱导粘蛋白分泌。CGC中的P2X4R为保护性粘蛋白分泌提供了新的治疗靶标。
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 Ca2+, 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 [Ca2+] ([Ca2+]i) measured. [Ca2+]i 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. [Ca2+]i was significantly increased when stimulated with ATP (10-7-10-4 M). Suramin, a non-selective P2XR antagonist at 10-4 M did not reduce ATP-induced peak [Ca2+]i. The potent P2X7 agonist, BzATP (10-7-10-4 M) increased the [Ca2+]i, although to a lesser extent than ATP. When measuring [Ca2+]i the effect of repeated applications of ATP at 10-5 or 10-6 M the response \"desensitized\" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca2+]i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca2+]i. ATP (10-5 M), BzATP (10-4 M) and 2MeSATP (10-5 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 [Ca2+]i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.