Gorodeski G I, Burfeind P, Gan S U, Pal D, Abdul-Karim F W
Department of Reproductive Biology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Am J Physiol. 1998 Sep;275(3):C758-65. doi: 10.1152/ajpcell.1998.275.3.C758.
Extracellular ATP stimulates acute changes in paracellular permeability across cultures of human uterine cervical epithelial cells [G. I. Gorodeski, D. E. Peterson, B. J. De Santis, and U. Hopfer. Am. J. Physiol. 270 (Cell Physiol. 39): C1715-C1725, 1996]. In this paper, we characterize mRNA for a P2Y2 nucleotide receptor in human cervical cells. Using oligonucleotide primers based on the sequence of human airway epithelium P2Y2 receptor, a single 632-bp cDNA band was identified in RT-PCR experiments in extracts of human endocervical and ectocervical tissues and in lysates of human cervical CaSki cells, but not in 3T3 fibroblasts. The nucleotide sequence was homologous to the corresponding human airway epithelium P2Y2 receptor. Northern blot analyses revealed hybridization of the P2Y2 receptor probe to a 2.0-kb mRNA fragment, as well as to 2.2-, 3. 0-, and 4.6-kb species, indicating that human cervical cells express P2Y2 receptor mRNA. Incubation of CaSki cells in retinoid-free medium abolished the ATP-induced changes in permeability and decreased the expression of the P2Y2 receptor mRNA; treatment with retinoids restored the responses to ATP and upregulated the P2Y2 receptor mRNA, suggesting that the receptor mediates ATP-related changes in permeability. Treatment with actinomycin D decreased the expression of the P2Y2 receptor RNA, but the ratio density of the receptor RNA relative to glyceraldehyde-3-phosphate dehydrogenase RNA remained unchanged, suggesting that retinoids upregulate transcription of the receptor mRNA. We conclude that retinoid-dependent modulation of the P2Y2 receptor expression, and hence of the responses to ATP, may be an important mechanism for the regulation of secretion of cervical mucus in vivo.
细胞外ATP可刺激人子宫颈上皮细胞培养物的细胞旁通透性发生急性变化[G.I.戈罗德斯基因、D.E.彼得森、B.J.德桑蒂斯和U.霍普费尔。《美国生理学杂志》270(细胞生理学39):C1715 - C1725，1996]。在本文中，我们对人宫颈细胞中P2Y2核苷酸受体的mRNA进行了表征。基于人气道上皮P2Y2受体的序列使用寡核苷酸引物，在人宫颈内膜和宫颈外膜组织提取物以及人宫颈CaSki细胞裂解物的逆转录聚合酶链反应实验中鉴定出一条单一的632碱基对的cDNA条带，但在3T3成纤维细胞中未鉴定出。核苷酸序列与人相应的气道上皮P2Y2受体同源。Northern印迹分析显示P2Y2受体探针与一个2.0千碱基的mRNA片段以及2.2、3.0和4.6千碱基的片段杂交，表明人宫颈细胞表达P2Y2受体mRNA。将CaSki细胞在无类视黄醇培养基中孵育消除了ATP诱导的通透性变化，并降低了P2Y2受体mRNA的表达；用类视黄醇处理恢复了对ATP的反应并上调了P2Y2受体mRNA，表明该受体介导了与ATP相关的通透性变化。用放线菌素D处理降低了P2Y2受体RNA的表达，但受体RNA相对于甘油醛-3-磷酸脱氢酶RNA的密度比值保持不变，表明类视黄醇上调了受体mRNA的转录。我们得出结论，类视黄醇对P2Y2受体表达的依赖性调节以及因此对ATP的反应调节可能是体内宫颈黏液分泌调节的重要机制。
