Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines

Abstract

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca 2+] i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E 2, butaprost and 11-deoxy-prostaglandin E 1. The stimulation of cyclic AMP production by prostaglandin E 2 was partially inhibited by the EP 4 receptor antagonist AH23848. Prostaglandin E 2 and 11-deoxy-prostaglandin E 1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F 2α, 17-phenyl-trinor-prostaglandin E 2 or 11-deoxy-prostaglandin E 1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E 1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F 2α and 17-phenyl-trinor-prostaglandin F 2α increased [Ca 2+] i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E 2 increased [Ca 2+] i. The present studies suggest that in ODMC1–2 cells, EP 2 and EP 4 receptors but neither EP 1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP 2 and FP receptors, while EP 1 receptors appear to be absent in this cell line.