Chem. 280, 11152C11164 [PubMed] [Google Scholar] 34. binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, and model systems, that DOR and MOR antagonize each other’s ligand binding ability and function on NK cells by increasing the physical association between them to form heterodimers. Furthermore, we test whether an opioid antagonist reduces protein levels of the targeted receptor and thereby increases levels of opposing receptor monomer and homodimer PF 429242 and their ligand binding ability and functions. Additionally, we test whether ethanol increases opioid receptor heterodimerization to suppress functions in NK cells. Because NK cells participate in cell-mediated immune response to tumor cells, we also decided the effectiveness of the combination treatment of opioid agonists and antagonists in prevention of NMU-induced mammary tumor growth. EXPERIMENTAL PROCEDURES Alcohol Feeding with or without Opioid Agonist and/or Antagonist Treatments in Animals Male Fischer-344 rats, 150C175 g body weight, were maintained in a controlled environment, given free choice of water, and fed a liquid diet containing alcohol (8.7% v/v) or pair-fed an isocaloric liquid diet (Bio-Serv, Frenchtown, NJ). The ethanol treatment regimen used has been shown to maintain blood alcohol levels within the range of 115C123 mg/dl between days 10 and 30 (20). We used pair-fed rats as our control rats to calorie-match the alcohol-fed group. Furthermore, we have previously shown that pair-fed animals and by determining their effects around the levels of the cytotoxic factors of NK cells in the spleen as well as the ability to inhibit NMU-induced mammary tumor growth of these opiodergic agents. In this study, 50-day-old virgin female Fischer rats were injected with a dose of NMU (50 mg/kg body weight). Nine weeks after NMU injection, animals were implanted with naltrexone pellets (100 mg, 60 days release) or placebo pellets under the skin. Seven days after naltrexone pellet implantation, DPDPE (100 g/kg body weight) was injected daily i.p. until animals were sacrificed at 16 weeks. Animals were palpated every week to check for tumor growth. Tumor length and width were measured with a calibrator. At the end of this treatment, animals were sacrificed; tumors were collected, PAK2 and slices of tumors were put in formalin and processed for histology staining. Animal surgery and care were performed in accordance with institutional guidelines and complied with National Institutes of Health policy. Opioid Agonist and Antagonist Treatments in RNK16 Cells For experiments, we used RNK16 cells, a Fisher 344 rat-derived rat natural killer cell line. These cells were maintained in PF 429242 RPMI 1640 media made up of 10% fetal bovine serum (FBS) and 50 m -mercaptoethanol. During experimentation, 1 106 cells/well were plated in a 12-well plate for 24 h. Cells were starved with serum-free media for 1 h and then treated with naltrexone (10 ng/ml) PF 429242 or naltrindole (50 m). These treatments were repeated at 24-h intervals for a period of 72 h. Cultures were additionally treated with [d-Ala2,studies, we used the rat-derived NK cell line RNK16 cells (1C4 106). Naltrexone (10 ng/ml, Sigma) and DPDPE (10 nm) were used as MOR antagonist and DOR agonist, respectively, for studies. Immunoprecipitation of Spleen and RNK16 Lysates by DOR or MOR Antibodies Spleen and RNK16 lysates were immunoprecipitated by anti-MOR or DOR antibodies (rabbit polyclonal, R&D Antibodies, Las Vegas, NV). 10 g of either antibody was coupled to protein A/G resin and then cross-linked with disuccinimidyl suberate using cross-link immunoprecipitation kit (Pierce) to eliminate the co-elution of antibody with antigen during the elution step. The lysate (500 g) was then immunoprecipitated with antibody cross-linked resin. Antigen (DOR or MOR) was eluted by elution buffer and subsequently used for SDS-PAGE. This antigen was free from any antibody contamination. Detection of DOR and MOR Protein Levels by Western Blot After immunoprecipitation and subsequent elution, DOR or MOR were detected by 4C20% gradient SDS-PAGE, transferred to PVDF membrane, followed by probing with anti-DOR or anti-MOR antibodies (1:1000; rabbit polyclonal, Millipore). Receptor bands were identified by clean blot immunoprecipitation detection reagent (HRP, Pierce) followed by chemiluminescent detection. Detection of Cytotoxic Factors by Western Blot For protein analysis, 2 106 RNK16 cells or splenocytes were subjected to standard SDS-PAGE. Proteins were transferred to PVDF membranes following standard procedures. PF 429242 Protein blots were probed with Granzyme-B mAb, Perforin polyclonal Ab, and IFN- polyclonal antibody (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:1000), and actin mAb (Oncogene, San Diego) to normalize values. After chemiluminescence detection (ECL plus, Amersham Biosciences), densitometric analyses were performed using Scion Image software. Immunohistochemistry Cell cultures were fixed in 4% paraformaldehyde for 30 min and then in 70% ethanol for an additional 30 min..