Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.