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

Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. 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. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP₁, EP₂, EP₃ and EP₄ 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. PGE₂ dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC₅₀ value of 100 nM. PGE₂, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC₅₀ values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP₃ receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated 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 subtype immunofluorescence was detected in the PE cells. EP₃ receptor subtype labeling was observed in both the NPE and the PE cells. PGE₂ produces opposing effects on adenylyl cyclase through EP₂/EP₄ and EP₃ receptor activation. The predominant effect of PGE₂ is on the adenylyl cyclase stimulatory receptors (EP₂/EP₄).