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 endom etrial adenocarcinoma cells (an in vitro model for the study of steroi d hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, an d proopiomelanocortin. These opioids may act locally, affecting the ut erine tissues. In the present study, we identified and characterized o pioid-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 acid ification improved specific binding by 2- to 4.5-fold. Characterizatio n of opioid binding using different radiolabeled opioids and effecters has shown the existence of a low concentration of delta-sites (K-d, 6 .20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa(1)-site s (K-d, 10.8 nmol/L; 276,000 sites/cell), kappa(2)-sites with high aff inity for ethylketocyclazocine (K-d, similar to 1 nmol/L) and low affi nity for diprenorphine (K-d, similar to 8 nmol/L) at a concentration o f 93,000 sites/cell, and high affinity kappa(d)-sites (K-d, 3.6 nmol/L ; 77,000 sites/cell). In conclusion, our report characterizes opioid s ites in a particular and homogeneous cell type of human endometrium, i .e. in epithelial cells. The coexistence of opioid sites and their end ogenous ligands in the Ishikawa cell line makes these cells a good mod el for the study of autocrine/paracrine interactions of opioids in non neural tissues.