McDonald M L, Ardito T, Marks W H, Kashgarian M, Lorber M I
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06510.
Transplantation. 1992 Feb;53(2):460-6. doi: 10.1097/00007890-199202010-00037.
Cyclophilin (CYP), an intracellular protein sharing amino acid sequence identity with the enzyme peptidyl-prolyl cis-trans isomerase has become the leading candidate for the receptor responsible for cyclosporine biological effects. Avid binding of CYP to cyclosporine and immunosuppressive cyclosporine metabolites has been demonstrated, while nonimmunosuppressive cyclosporine metabolites have tended not to bind to cyclophilin. A previous immunohistochemical analysis documented that CYP localized principally to the cytoplasmic cellular compartment, but nuclear staining was observed among some cells. This study was undertaken to more precisely define the ultrastructural distribution of CYP, and to determine whether CYP cellular content was affected by CsA therapy. Untreated Wistar rats or those receiving 7 days of CsA (15 mg/kg/day, i.p.) were anesthetized, perfusion-fixed in situ, and sacrificed. Analyses of lymph node, spleen, thymus, kidney, liver, heart, brain, and ileum used an affinity purified, rabbit anticyclophilin IgG. Transmission electron microscopy was performed after staining with anti-CYP using a horseradish peroxidase/biotin/avidin technique. Quantitative immunofluorescence was measured by confocal microscopy using anti-CYP, with a biotin/avidin/phycoerythrin technique. Cyclophilin localized to the cytoplasmic compartment--however, association with mitochondria endoplasmic reticulum, Golgi, and with the nuclear membrane among lymphocytes, as well as cells from kidney, liver and ileum--was documented. Cyclophilin was not identified within the nucleus proper. Tissues obtained from animals receiving CsA exhibited a generalized increase in CYP content compared with tissues from untreated controls, suggesting the possibility that CsA may exert a regulatory influence upon CYP gene activation. Collectively, the data were consistent with the hypothesis that CYP exerts a central role in cellular metabolism, and that CsA-mediated biologic effects result from the CsA/CYP interaction.
亲环蛋白（CYP）是一种细胞内蛋白质，其氨基酸序列与肽基脯氨酰顺反异构酶相同，已成为负责环孢素生物学效应的受体的主要候选者。已证实CYP与环孢素和免疫抑制性环孢素代谢物有紧密结合，而非免疫抑制性环孢素代谢物往往不与亲环蛋白结合。先前的免疫组织化学分析表明，CYP主要定位于细胞质细胞区室，但在一些细胞中观察到核染色。本研究旨在更精确地确定CYP的超微结构分布，并确定CYP细胞含量是否受环孢素A治疗的影响。对未治疗的Wistar大鼠或接受7天环孢素A（15mg/kg/天，腹腔注射）的大鼠进行麻醉、原位灌注固定并处死。使用亲和纯化的兔抗亲环蛋白IgG对淋巴结、脾脏、胸腺、肾脏、肝脏、心脏、大脑和回肠进行分析。使用辣根过氧化物酶/生物素/抗生物素蛋白技术用抗CYP染色后进行透射电子显微镜检查。使用抗CYP通过共聚焦显微镜测量定量免疫荧光，采用生物素/抗生物素蛋白/藻红蛋白技术。亲环蛋白定位于细胞质区室——然而，已证明其与线粒体、内质网、高尔基体以及淋巴细胞以及肾脏、肝脏和回肠细胞中的核膜有关联。在细胞核内部未发现亲环蛋白。与未治疗对照组的组织相比，接受环孢素A的动物获得的组织中CYP含量普遍增加，这表明环孢素A可能对CYP基因激活发挥调节作用。总体而言，这些数据与以下假设一致，即CYP在细胞代谢中发挥核心作用，并且环孢素A介导的生物学效应是由环孢素A/CYP相互作用产生的。
