PII S0196-9781(97)00294-5 Opioid Peptide Receptor Studies. 8. One of the Mouse Brain  NCX Binding Sites is Similar to the Cloned Mouse Opioid  Receptor: Further Evidence for Heterogeneity of  Opioid Receptors HENG XU,* YI-FENG LU,* JOHN S. PARTILLA,* JULIA PINTO,² SYLVIA N. CALDERON,² DOROTA MATECKA,² KENNER C. RICE,² JOSEPHINE LAI,‡ FRANK PORRECA,‡ SUBRAMANIAM ANANTHAN** AND RICHARD B. ROTHMAN* 1 *Clinical Psychopharmacology Section, Division of Intramural Research, NIDA, NIH, PO Box 5180, 5500 Nathan Shock Drive, Baltimore, MD 21224 ²Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, MD 20892 ‡Department of Pharmacology, College of Medicine, 1501 North Campbell Street, University of Arizona, Tucson, AZ 85724 **Organic Chemistry Department, Southern Research Institute, 2000 9th Ave. South, Birmingham, AL 35255 Received 11 July 1997; Accepted 10 September 1997 XU, H., Y-F. LU, J. S. PARTILLA, J. PINTO, S. N. CALDERON, D. MATECKA, K. C. RICE, J. LAI, F. PORRECA, S. ANANTHAN AND R. B. ROTHMAN. Opioid peptide receptor studies. 8. One of the mouse brain  NCX binding sites is similar to the cloned moused opioid  receptor: further evidence for heterogeneity of  opioid receptors. PEPTIDES 19(2) 343–350, 1998.—Quantitative ligand binding studies resolved two subtypes of the  opioid receptor, termed  ncx1 and  ncx2 , in mouse brain membranes depleted of  receptors by pretreatment with the irreversible ligand, BIT. The purpose of the present study was to compare the binding parameters, ligand-selectivity profile and pharmacological properties of the cloned mouse  receptor (MDOR) stably expressed in a cell line to the  ncx binding sites of mouse brain. [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin labeled a single binding site in membranes prepared from MDOR cells under several different assay conditions including BIT-pretreatment. The MDOR had high affinity for  agonists and antagonists. [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin labeled two binding sites in mouse brain membranes depleted of  receptors by pretreatment with BIT: the  ncx1 site (high affinity for DPDPE and deltorphin) and the  ncx2 site (low affinity for DPDPE and deltorphin). Some agents were moderately selective for the delta ncx2 site: [pCl]DPDPE (10.9-fold), JP41 (5.9-fold) and JP45 (3.8-fold). The K i values of 12 opioids at the mouse MDOR were determined. These values were highly correlated with their values at the delta ncx1 site but not the delta ncx2 site. These data suggest that the delta ncx2 site may be distinct from the cloned delta opioid receptor. © 1998 Elsevier Science Inc. Delta receptors Receptor subtypes [D-Ala 2 , D-Len 5 ]enkephalin THE recent molecular cloning of ,  and  1 opioid receptors (5,7,11,38,42) firmly established the heterogeneity of opioid receptors as delineated on the basis of traditional pharmaco- logical and biochemical approaches (1,6,8,14,16,22,24,32–34). The relationship of other postulated opioid receptor subtypes to the three cloned receptors is unclear. In regard to  receptors, a variety of pharmacological data support the existence of  receptor subtypes termed  1 and  2 receptors (10,17,18,36). The recent finding that intracerebroventricular (ICV) adminis- tration of an antisense oligodeoxynucleotide complementary to 1 Requests for reprints should be addressed to Dr. Richard Rothman, Division of Intramural Research, NIDA, NIH, PO Box 5180, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: rrothman@lrp.nida.nih.gov Peptides, Vol. 19, No. 2, pp. 343–350, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 + .00 343