Characterization of EP prostanoid receptor subtypes in primary cultures ofbovine ciliary epithelial cells by immunofluorescent microscopy and functional studies

Purpose. To determine the expression and functional coupling of EP prostano id receptor subtypes to second messenger pathways in bovine ciliary epithel ium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were est ablished and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with sub type selective antibodies in both tissue sections and primary cultures of B CE cells. Messenger RNA expression was determined using reverse transcripti on and polymerase chain reaction. The effects of prostanoid agonists on tot al inositol phosphate accumulation and cAMP formation were used to assess f unctional activity. Results. Positive immunoreactivity was obtained in both frozen thin-section s and primary cultures of bovine ciliary process to the prostanoid EP1, EP2 , EP3 and EP4 receptor subtypes. Reverse transcription followed by the poly merase chain reaction yielded products corresponding to each of the prostan oid EP subtypes which was confirmed by restriction enzyme analysis. PGE(2) dose-dependently stimulated the accumulation of total inositol phosphates i n cultured cells with an EC50 value of 100 nM. PGE(2), forskolin and isopro terenol produced dose-dependent increases in cAMP formation with EC50 value s of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP forma tion was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretre ated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subt ype immunofluorescence was detected in the PE cells. EP3 receptor subtype l abeling was observed in both the NPE and the PE cells. PGE(2) produces oppo sing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activatio n. The predominant effect of PGE(2) is on the adenylyl cyclase stimulatory receptors (EP2/EP4).