Functional oxytocin receptors in a human endometrial cell line

OBJECTIVE: Our goal was to demonstrate expression and functionality of oxyt ocin receptors in a human endometrial cell line. This cell line could then be used for further investigation of the role of oxytocin in reproductive f unction at the cellular level. STUDY DESIGN: Oxytocin receptor messenger ribonucleic acid expression was d etermined by reverse transcriptase-polymerase chain reaction deoxyribonucle ic acid amplification with ribonucleic acid from confluent Ishikawa cells. Ligand binding to whole cells was evaluated by nonlinear regression analysi s with an iodinated oxytocin antagonist. The coupling of the oxytocin recep tor to signaling pathways was evaluated by measuring oxytocin-stimulated in creases in intracellular calcium concentration. phosphorylation of ERK2 (ex tracellular-regulated protein kinase 2) mitogen-activated protein kinase, a nd prostaglandin E-2 release. RESULTS: Polyacrylamide gel electrophoresis of the reverse transcriptase-po lymerase chain reaction products demonstrated the presence of oxytocin rece ptor messenger ribonucleic acid in Ishikawa cells. Ligand-binding analysis of these cells demonstrated a single class of noninteracting sites, with a B-max (maximal number of binding sites) of 77.7 fmol/mg deoxyribonucleic ac id and an apparent dissociation constant of 8.3 x 10(-11) mol/L. Stimulatio n with 100-nmol/L oxytocin caused a rapid transient increase in intracellul ar free calcium concentration, which was blocked by 1-mu mol/L oxytocin ant agonist. Treatment of cells with oxytocin for 10 minutes resulted in a mark ed increase in the phosphorylation of ERK2, as determined by Western blot a nalysis, and a 5-fold increase in prostaglandin E-2 release. CONCLUSION: This study is the first to demonstrate functional oxytocin rece ptors in an established human endometrial cell line. This cell line will be useful in elucidating the mechanisms of action of oxytocin in the reproduc tive tract at the molecular level.