Webster Paul, Saito Kaori, Cortez John, Ramirez Christina, Baum Marc M
Oak Crest Institute of Science, 132 W. Chestnut Avenue, Monrovia, California 91016, United States.
Los Angeles (UCLA) Fielding School of Public Health, University of California, Los Angeles, 650 Charles E. Young Dr. South, 16-035 Center for Health Sciences, Los Angeles, California 90095-1772, United States.
ACS Omega. 2020 Aug 13;5(33):20882-20889. doi: 10.1021/acsomega.0c02329. eCollection 2020 Aug 25.
Transporters are specialized integral membrane proteins, which mediate the passage of virtually all molecules through cell membranes. They are expressed in a broad range of human and animal tissues and play important roles in both normal and disease states. For these reasons, they are evaluated when developing and testing drugs. Two major families of drug transporters, the adenosine 5'-triphosphate-binding cassette and solute carrier transporters (SLC), have critical roles in the absorption, distribution, metabolism, and elimination of drugs. The SLC family contains known nucleoside transporters and therefore are important when nucleoside analogs are used as drugs to prevent or treat viral infections. In this study, we wanted to determine if it was possible to locate one member of the SLC family, the human concentrative nucleoside transporter 3 (CNT3) in human vaginal epithelial cells. The CNT3 protein has important roles in drug delivery, subsequent drug tissue distribution, and, hence, efficacy. Vaginal epithelial cells, taken from two human volunteers (one Caucasian and one African American), were labeled for light and electron microscopy, with a commercial antibody to a cytoplasmic domain of CNT3, the protein product of the SLC28A3 gene. Fluorescent secondary antibodies or protein A-gold were used to detect antibody binding. By electron microscopy, gold particle binding was quantified to determine labeling specificity. By light microscopy, positive labeling with anti-CNT3 antibodies was detected on human vaginal epithelial cells, but specificity to any intracellular structure was not easily determined, most likely a result of specimen preparation. Electron microscopy revealed that the CNT3 transporter protein was present predominantly on microvilli located on one side of some human vaginal epithelial cells. Quantification confirmed specific anti-CNT3 labeling over human vaginal epithelial cell microvilli. The CNT3 protein, present in the microvilli of human vaginal epithelial cells, may have a role in redistributing nucleoside homologues delivered to the vaginal tract. Transporter proteins such as CNT3 could shuttle nucleosides and their analogs through the vaginal epithelium to immune cells located in lower cell layers. Outer layers of cells, which are eventually shed from the epithelium, may remove accumulated nucleoside drug analogs from the vaginal tract.
转运蛋白是一类特殊的整合膜蛋白，介导几乎所有分子通过细胞膜。它们在广泛的人类和动物组织中表达，在正常和疾病状态下都发挥着重要作用。基于这些原因，在药物研发和测试过程中会对它们进行评估。两大类药物转运蛋白，即三磷酸腺苷结合盒转运蛋白和溶质载体转运蛋白（SLC），在药物的吸收、分布、代谢和排泄中起着关键作用。SLC家族包含已知的核苷转运蛋白，因此当核苷类似物被用作预防或治疗病毒感染的药物时，它们就显得尤为重要。在本研究中，我们想确定是否有可能在人阴道上皮细胞中定位SLC家族的一个成员，即人浓缩核苷转运蛋白3（CNT3）。CNT3蛋白在药物递送、随后的药物组织分布以及疗效方面都具有重要作用。从两名人类志愿者（一名白种人和一名非裔美国人）获取的阴道上皮细胞，用针对CNT3（SLC28A3基因的蛋白质产物）胞质结构域的商业抗体进行标记，用于光学显微镜和电子显微镜观察。使用荧光二抗或蛋白A金来检测抗体结合情况。通过电子显微镜，对金颗粒结合进行定量以确定标记特异性。通过光学显微镜，在人阴道上皮细胞上检测到抗CNT3抗体的阳性标记，但不易确定其对任何细胞内结构的特异性，这很可能是标本制备的结果。电子显微镜显示，CNT3转运蛋白主要存在于一些人阴道上皮细胞一侧的微绒毛上。定量分析证实了在人阴道上皮细胞微绒毛上存在特异性的抗CNT3标记。存在于人阴道上皮细胞微绒毛中的CNT3蛋白，可能在重新分布递送至阴道的核苷类似物方面发挥作用。像CNT3这样的转运蛋白可以将核苷及其类似物穿梭通过阴道上皮，到达位于较低细胞层的免疫细胞。最终从上皮脱落的外层细胞，可能会从阴道中清除积累的核苷药物类似物。
