KAPPA-OPIOID BINDING-SITES ON A MURINE LYMPHOMA CELL-LINE

As a first step in determining whether any subset of lymphocytes expresses opioid receptors, membranes prepared from mouse lymphoma cell lines were screened for [H-3]naloxone binding sites. Membranes from the R1.1 cell line specifically bound [H-3]naloxone. The Hill coefficient for [H-3]naloxone binding was 0.93 +/- 0.18, and nonlinear regression analysis indicated that a one-site model was the best fit of the [H-3]naloxone saturation binding data. Low concentrations of kappa-selective opioids, but neither mu nor delta opioids, inhibited [H-3]naloxone binding. Saturation binding studies with the kappa-selective compound [H-3]U69,593 revealed a single binding site with a K(D) value of 0.204 +/- 0.039 nM and a B(max) value of 31.7 +/- 3.1 fmol/mg of membrane protein. The Hill coefficient for [H-3]U69,593 binding was 1.03 +/- 0.11, indicative of a single site. Time courses for the association and dissociation of [H-3]U69,593 binding at 25-degrees-C exhibited properties consistent with a single class of binding sites. Low concentrations of kappa-selective opioids, including dynorphin peptides, inhibited [H-3]U69,593 binding, while high concentrations of mu opioids were needed to inhibit binding, and the delta-selective ligands were ineffective at concentrations up to 10 muM. Stereoselectivity of the binding site was demonstrated by the finding that the K(i) value for (-)-pentazocine in inhibiting [H-3]U69,593 binding was 25 times less than for the (+)-isomer. Based on its high affinity for U69,593, alpha-neo-endorphin, and dynorphin B, the kappa opioid binding site on R1.1 cell membranes belongs to the kappa1b subtype. As observed with brain kappa opioid binding sites, sodium inhibited [H-3]U69,593 binding to R1.1 cell membranes in a concentration-dependent manner. These data demonstrate that the murine lymphoma cell line R1.1 expresses kappa opioid binding sites that are very similar to brain kappa opioid binding sites.