Anthony T L, Pierce K L, Regan J W
Department of Pharmacology & Toxicology, The University of Arizona, College of Pharmacy, Tucson, Arizona 85721-0207, USA.
Curr Eye Res. 2000 May;20(5):394-404.
To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium.
Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity.
Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP(1 ), EP(2), EP(3) and EP(4) receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE(2) dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC( 50) value of 100 nM. PGE(2), forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC(50) values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP(3) receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin.
This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP(1) and EP(4) receptor subtypes were found primarily in the NPE cells, whereas, EP(2) receptor subtype immunofluorescence was detected in the PE cells. EP(3) receptor subtype labeling was observed in both the NPE and the PE cells. PGE(2) produces opposing effects on adenylyl cyclase through EP(2)/EP(4) and EP(3) receptor activation. The predominant effect of PGE(2) is on the adenylyl cyclase stimulatory receptors (EP(2)/EP(4)).
确定前列腺素E（PGE）受体亚型在牛睫状体上皮细胞中与第二信使途径的表达及功能偶联。
建立牛睫状体上皮（BCE）细胞原代培养物，并培养传代至四代。在组织切片和BCE细胞原代培养物中，使用亚型选择性抗体通过间接免疫荧光显微镜检查EP受体蛋白表达。采用逆转录和聚合酶链反应测定信使核糖核酸表达。用前列腺素激动剂对总肌醇磷酸积累和环磷酸腺苷（cAMP）形成的影响来评估功能活性。
在牛睫状体的冰冻薄片和原代培养物中，对前列腺素EP（1）、EP（2）、EP（3）和EP（4）受体亚型均获得了阳性免疫反应性。逆转录后进行聚合酶链反应产生了与每种前列腺素EP亚型相对应的产物，经限制性酶切分析证实。PGE（2）以剂量依赖性方式刺激培养细胞中总肌醇磷酸的积累，半数有效浓度（EC）（50）值为100 nM。PGE（2）、福斯可林和异丙肾上腺素使cAMP形成呈剂量依赖性增加，EC（50）值分别为100、300和200 nM。在培养的BCE细胞中，EP（3）受体激动剂舒前列素减弱了异丙肾上腺素刺激的cAMP形成。舒前列素引起的抑制作用在用百日咳毒素预处理的细胞中被逆转。
本研究证明牛睫状体上皮中存在功能性前列腺素EP受体亚型。EP（1）和EP（4）受体亚型主要存在于非色素上皮（NPE）细胞中，而在色素上皮（PE）细胞中检测到EP（2）受体亚型免疫荧光。在NPE和PE细胞中均观察到EP（3）受体亚型标记。PGE（2）通过激活EP（2）/EP（4）和EP（3）受体对腺苷酸环化酶产生相反的作用。PGE（2）的主要作用是作用于腺苷酸环化酶刺激受体（EP（2）/EP（4））。
