Demonstration and characterization of zeta (ζ), a growth-related opioid receptor, in a neuroblastoma cell line

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Me⁵]enkephalin, was examined to ascertain the zeta (ζ) opioid receptor. Specific and saturable binding of [³H]-[Met⁵]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a K_d of 1.6 nM and a binding capacity B_max off 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na⁺, Ca²⁺, and Mg²⁺; GppNHp at concentrations of 100–500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [³H]-[Met⁵]enkphalin winding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin. Cell density (log, confluence, postconfluence) did not alter the K_d or B_max. This study serves as the first demonstration and characterization of the zeta (ζ) opioid receptor in tissue culture cells. The homogenous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the ζ receptor.     

Conserved expression of the opioid growth factor, [Met]enkephalin, and the zeta (ζ) opioid receptor in vertebrate cornea

In addition to neuromodulation, endogenous opioids serve as growth factors. The naturally occurring opioid peptide, [Met⁵]enkephalin, termed opioid growth factor (OGF), has been found to be a potent and tonic inhibitor of processes related to growth and renewal, particularly cell proliferation. OGF mediates its actions through the zeta (ζ) opioid receptor. In order to determine if OGF and/or the ζ receptor are present in human corneal epithelium, immunocytochemistry was utilized. Immunoreactivity with regard to OGF and to the ζ receptor could be detected in the cortical cytoplasm of both basal and suprabasal epithelial cells, but was not associated with the cell nucleus. Investigation of the ubiquity of OGF and ζ receptor in the vertebrate cornea showed that both elements are present in a wide variety of classes of the phylum Chordata, including mammalia, aves, reptilia, amphibia, and osteichthyes. These results suggest that an endogenous opioid system related to growth may have originated as early as 300 million years ago, and that the function of this system in cellular renewal and homeostasis is a requirement of the vertebrate corneal epithelium.     

Production and characterization of polyclonal and monoclonal antibodies to the zeta (ξ) opioid receptor

The opioid growth factor, [Met⁵]-enkephalin, is an inhibitory agent of cell proliferation and maturation that interacts with the zeta (ξ) opioid receptor to modulate growth. In order to learn more about this receptor, polyclonal and monoclonal antibodies were raised against binding subunits identified on two-dimensional gels by ligand blotting. Using Western blotting, the polyclonal antibodies and some of the monoclonal antibodies recognized all 4 binding polypeptides (32, 30, 17, and 16 kDA) in developing rat cerebellum; no reaction was recorded in adult cerebellum. In addition, other monoclonals were able to distinguish only certain subunits (e.g. 17 kDa). The monoclonal antibodies and their F(ab′)₂ fragments, as well as the polyclonal antibodies, blocked the binding of [³H][Met⁵]-enkephalin to preparations of developing cerebellum. Both the polyclonal and monoclonal antibodies immunoprecipitated ξ opioid binding polypeptides from 6-day-old cerebellar homogenates solubilized by the zwitterionic detergent, CHAPS. Immunocytochemistry performed with polyclonal antibodies showed immunoreactivity associated with proliferating and differentiating cerebellar cells, but no specific staining was detected in the adult cerebellum. These results have identified and characterized antibodies to the ξ opioid receptor, and the antibodies were used to localize this receptor; these antibodies will be valuable to further cellular and molecular studies.     

Characterization of zeta (zeta): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [3H]-[Met5]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded a Kd of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na+ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [3H]-[Met5]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met5]enkephalin was the most potent displacer of [3H]-[Met5]enkephalin; other ligands selective for mu, delta, kappa, epsilon, and sigma were not highly competitive. Given the functional significance of [Met5]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met5]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (zeta) (from the Greek 'Zoe', life) related to the proliferation of cells and tissues.     

Characterization of a polyclonal antibody to the μ opioid receptor

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the μ receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [³H]etorphine (universal opioid) and [³H]dihydromorphine (μ opioid) from rat membranes, but was ineffective against the binding of [³H]ethylketocyclazocine (κ opioid), [³H]d-Ala²,d-Leu⁵-enkephalin (δ opioid) or [³H]phencyclidine (phencyclidine/σ receptor ligand). The antibody was able to precipitate the M_r 94 000 component of the ¹²⁵I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma × Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.     

Opioid antagonist modulation of murine neuroblastoma: a profile of cell proliferation and opioid peptides and receptors

The endogenous opioids and their receptors are known to play a major role in neoplasia. In the present study, naltrexone (NTX), a potent opioid antagonist, was utilized to explore the interactions of opioids and opioid receptors in mice with transplanted neuroblastoma (S20Y). Tumors from mice subjected to either intermittent (4–6h/day; 0.1 mg/kg NTX) or complete (24 h/day; 10 mg/kg NTX) opioid receptor blockade exhibited an up-regulation of DADLE and Met-enkephalin binding sites, as well as tissue levels of β-endorphin and Met-enkephalin. Binding affinity to [ -Ala², -Leu⁵]enkephalin (DADLE) or ethylketocyclazocine (EKC), the levels of plasma β-endorphin, and the anatomical location and quantity of Met- and Leu-enkephalin and cytoskeletal components (i.e. tubulin, actin, brain spectrin(240/235)) were similar in NTX and control tumor-bearing animals. Tissue viability of the 0.1 NTX group was increased compared to controls. Both mitotic and labeling indexes were increased during the period of opioid receptor blockade, but decreased in the period subsequent to receptor blockade. NTX treatment produced a 2-fold increase in sensitivity to opioids. Met-enkephalin (10 mg/kg) produced a depression in both mitotic and labeling indexes in tumor-bearing mice that could be reversed by naloxone (10 mg/kg) administration. Thus, the endogenous opioids are trophic agents that inhibit growth by suppressing cell proliferation. The duration of receptor blockade by opioid antagonists modulates these actions, affecting both tumor incidence and survival time. Complete opioid receptor block prevents the interaction of increased levels of putative growth-related peptides with a greater number of opioid receptors, thereby increasing cell proliferation and accelerating tumor growth. With intermittent blockade, an enhanced opioid-receptor interaction occurs during the interval when the opioid antagonist is no longer present, producing an exaggerated inhibitory action on cell proliferation and the repression of tumorigenic events.     

Melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by increasing the release of β-endorphin an endogenous opioid

The occurrence of systematic diurnal variations in pain thresholds has been demonstrated in human. Salivary melatonin levels change following acute pain when other factors that could explain the change have been removed or controlled. Melatonin-induced analgesia is blocked by naloxone or pinealectomy. By using selective radioligands [3H]-DAMGO, [3H]-DPDPE, [3-U69593, and 3H]-nociceptin, we have shown that the bovine pinealocytes contain delta and mu, but not kappa or ORL1 opioid receptor subtypes. In the present study, by using melatonin receptor agonists (6-chloromelatonin or 2-iodo-N-butanoyl-5-methoxytryptamine) or melatonin receptor antagonist (2-phenylmelatonin), we have shown that these agents do not compete with opioid receptor subtypes. However, we observed a time-dependent release of beta-endorphin an endogenous opioid peptide, by melatonin from mouse pituitary cells in culture. Hence, it is suggested that melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by binding to its own receptors and increasing the release of beta-endorphin.     

δ Opioid receptor activation is required to induce LTP of synaptic transmission in the lateral perforant path in vivo

The role of opioid receptors in long-term potentiation (LTP) of the medial (MPP) and lateral (LPP) divisions of the perforant path-granule cell projection was investigated in urethane anesthetized rats. A stimulating electrode was positioned in the dorsomedial or ventrolateral aspect of the angular bundle for selective activation of M MPP and LPP, respectively. A p ush-pull cannula served to focally perfuse artificial cerebrospinal fluid (ACSF) across the perforant path terminal zone, while perforant path evoked potentials were monitored in the dentate hilus. Robust LTP of the excitatory postsynaptic potential (EPSP) initial slope and population spike height was induced by high frequency stimulation (400 Hz, 8 bursts of 8 pulses) applied to the medial or lateral perforant path in rats perfused with standard medium. In the lateral perforant path, a putative proenkephalin system, LTP of the EPSP and population spike was blocked when ACSF containing 100 μM of the opioid receptor antagonist naloxone was present during the tetanus, while perfusion with 0.1 μM naloxone prevented EPSP potentiation but only reduced the magnitude of the population spike increase. Naloxone had no effect on LTP induction in the MPP. Importantly, 0.1 μM ICI 174,864, a selective antagonist of δ opioid receptors, blocked LTP of synaptic transmission in the LPP while leaving the population spike increase intact. The results indicate that LTP of synaptic transmission in the LPP requires activation of δ opioid receptors, while ‘non-δ’ opioid receptors may be involved in augmenting granule cell output. This opioid receptor-dependent LTP illustrates peptidergic regulation of synaptic plasticity in the hippocampus.     

Distribution of μ, δ and κ opioid receptors in the hypothalamus of the rat

The radioautographic distribution of μ, δ and κ opioid binding sites was examined by in vitro radioautography in the rat hypothalamus using the higlly selective ligands [¹²⁵I]-FK 33-824, [¹²⁵I]azidoDTLET and [¹²⁵I]DPDYN, respectively. Levels of μ opioid binding sites varied considerably amongst hypothalamic nuclei. μ Opioid labeling was dense in the medial preoptic area, medial preoptic nucleus, suprachiasmatic nucleus and ventromedial nucleus, whilst the supraoptic nucleus, paraventricular nucleus, arcuate nucleus and dorsomedial nucleus were devoid of labeling. Delta opioid labeling was sparse throughout most of the hypothalamus; however, moderate binding densities were detected in the suprachiasmatic and ventromedial nucleus. κ Opioid labeling was also scant throughout the hypothalamus with the exception of the suprachiasmatic nucleus which was very densely labeled. Our results indicate that the 3 opioid receptors types are differentially distributed within the hypothalamus, although a significant overlap exists. In general, the distribution of hypothalamic opioid receptors correlates well with that of opioid-containing terminal fibers and may represent the anatomical substrate for opioid involvement in the hypothalamic regulation of autonomic, behavioral and neuroendocrine functions.     

Heterogeneous distribution and upregulation of μ, δ and κ opioid receptors in the amygdala

Levels of μ, δ and κ opioid receptors in 4 subnuclei of the rat amygdala were determined by quantitative autoradiography following chronic treatment with naloxone or saline. A different distribution of each receptor subtype was observed, with μ binding greatest in the lateral nucleus (La), δ greatest in the basolateral (B1), and κ greatest in the medial (Me). Levels of all 3 receptors were very low in the central nucleus. Receptor upregulation following chronic naloxone treatment was also anatomically heterogeneous. Increases in μ receptors were statistically significant in the Me, Bl and La, while increases in δ and κ receptors were significant only in the Bl.     

Regulation of human B lymphocyte activation by opioid peptide hormones. Inhibition of IgG production by opioid receptor class ( μ-, κ-, and δ-) selective agonists

Opioid peptides have been reported by many laboratories to modulate in vitro and in vivo cell-mediated and humoral immune responses. However, less attention has been afforded to the class or classes of opioid receptors involved in these immunomodulatory effects. Previous studies by this laboratory indicated that β-endorphin and methionine-enkephalin were potent inhibitors of Staphylococcus aureus, Cowen strain I (SAC)-induced IgG production by human B lymphocytes. Results obtained from the present studies indicate that, at pharmacological concentrations, μ-, δ-, and κ-receptor-selective agonists arc potent inhibitors of SAC-induced IgG-secreting cells (IgG-ISC) by human B lymphocytes. Moreover, the suppression of IgG-ISC formation was reversed by μ-, δ-, and κ-receptor class-selective antagonists, [ Tic]cTAP, ICI 174,864, and nor-BNI, respectively. These findings are in ag showing that more than one class of receptors are involved in opioid peptide-mediated immunoregulation. Additional studies indicated that all three class-selective receptor agonists were found to suppress SAC-induced IL-6 production in intact PBMC cultures. As observed for suppression of IgG-ISC formation, inhibition of IL-6 production was found to be reversed by the appropriate receptor class-selective antagonist. These results support the hypothesis that one mechanism of opioid peptide-mediated inhibition of antibody production is via the down regulation of cytokine synthesis.     

The biology of the opioid growth factor receptor (OGFr)

Opioid peptides act as growth factors in neural and non-neural cells and tissues, in addition to serving for neurotransmission/neuromodulation in the nervous system. The native opioid growth factor (OGF), [Met⁵]-enkephalin, is a tonic inhibitory peptide that plays a role in cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. Assays with radiolabeled OGF have detected specific and saturable binding, with a one-site model of kinetics. Subcellular fractionation studies show that the receptor for OGF (OGFr) is an integral membrane protein associated with the nucleus. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse. OGFr is distinguished by containing a series of imperfect repeats. The molecular and protein structure of OGFr have no resemblance to that of classical opioid receptors, and have no significant homologies to known domains or functional motifs with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it interfaces with OGF. The peptide–receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus. Signal transduction for modulation of DNA activity is dependent on the presence of an appropriate confirmation of peptide and receptor. This report reviews the history of OGF–OGFr, examines emerging insights into the mechanisms of action of opioid peptide–receptor interfacing, and discusses the clinical significance of these observations.     

Chronic food restriction alters μ and κ opioid receptor binding in the parabrachial nucleus of the rat: a quantitative autoradiographic study

Using quantitative autoradiography, it was previously observed that chronic food restriction alters μ and κ receptor binding in several regions of the rat forebrain. The present autoradiographic study was designed to investigate whether food restriction affects regional μ and κ binding in the brainstem. [³H]DAGO (It,) and μ/δ blocked [³H]BMZ (κ) binding were analyzed in 21 brainstem regions. A signficant decrease in μ binding was observed in the external lateral and external medial subnuclei of the parabrachial nucleus while a significant increase in κ binding was observed in the external lateral subnucleus. The possible functional significance of these changes is discussed.     

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.     

Affinity crosslinking of I-labeled human ß-endorphin to cell lines possessing either μ- or δ-type opioid binding sites

The specific labeling of opioid receptor-related polypeptides was compared in two cell lines which differ in their opioid receptor population: SK-N-SH which contains predominantly μ-type opioid receptors, and NG-108-15, which contains exclusively δ-type opioid receptors. Labeling of opioid receptors was achieved by affinity cross-linking of membranes, using ¹²⁵I-labeled human ß-endorphin, followed by solubilization in sodium dodecyl sulphate (SDS), SDS-gel electrophoresis and autoradiography. Different labeling patterns were obtained from these two cell lines. In SK-N-SH cells, 3 major proteins were labeled, corresponding to molecular weights of 92, 65 and 25 kDa, while in the NG-108-15 cells, 53-kDa and 25-kDa polypeptides were the major ones labeled. The radioactivity incorporated into the 92- and 65-kDa peptide bands derived from SK-N-SH cells was displayed by the μ-selective ligand Tyr- -Ala-Gly-MePhe-Gly-ol (DAGO) but not by the δ-selective ligand [ -Pen², -Pen⁵]enkephalin (DPDPE). The radioactivity incorporated into the NG-108-15-derived peptide bands was displaced by the δ-selective ligand, but not by the μ-selective ligand. This confirms our previous finding in mammalian brain which demonstrated that μ- and δ-opioid binding sites can be identified as distinct proteins which differe in molecular size.     

Disruption of morphine‐conditioned place preference by a δ2‐opioid receptor antagonist: study of μ‐opioid and δ‐opioid receptor expression at the synapse

The addictive properties of morphine limit its clinical use. Learned associations that develop between the abused opiate and the environment in which it is consumed are engendered through Pavlovian conditioning processes. Disruption of the learned associations between the opiate and environmental cues may be a therapeutic approach to prevent morphine dependence. Although a role for the δ‐opioid receptor in the regulation of the rewarding properties of morphine has already been shown, in this study we further characterized the role of the δ‐opioid receptor in morphine‐induced conditioned responses by examining the effect of a selective δ2‐opioid receptor antagonist (naltriben), using a conditioned place preference paradigm in rats. Additionally, we used a subcellular fractionation technique to analyze the synaptic localization of μ‐opioid and δ‐opioid receptors in the hippocampus, in order to examine the molecular mechanisms that may underlie this morphine‐induced conditioned behavior. Our data show that the administration of 1 mg/kg naltriben (but not 0.1 mg/kg) prior to morphine was able to block morphine‐induced conditioned place preference. Interestingly, this naltriben‐induced disruption of morphine conditioned place preference was associated with a significant increase in the expression of the δ‐opioid receptor dimer at the postsynaptic density. In addition, we also observed that morphine conditioned place preference was associated with an increase in the expression of the μ‐opoid receptor in the total homogenate. Overall, these results suggest that modulation of the δ‐opioid receptor expression and its synaptic localization may constitute a viable therapeutic approach to disrupt morphine‐induced conditioned responses.     

Neurobiological mechanisms involved in nicotine dependence and reward: Participation of the endogenous opioid system

Nicotine is the primary component of tobacco that maintains the smoking habit and develops addiction. The adaptive changes of nicotinic acetylcholine receptors produced by repeated exposure to nicotine play a crucial role in the establishment of dependence. However, other neurochemical systems also participate in the addictive effects of nicotine including glutamate, cannabinoids, GABA and opioids. This review will cover the involvement of these neurotransmitters in nicotine addictive properties, with a special emphasis on the endogenous opioid system. Thus, endogenous enkephalins and beta-endorphins acting on mu-opioid receptors are involved in nicotine-rewarding effects, whereas opioid peptides derived from prodynorphin participate in nicotine aversive responses. An up-regulation of mu-opioid receptors has been reported after chronic nicotine treatment that could counteract the development of nicotine tolerance, whereas the down-regulation induced on kappa-opioid receptors seems to facilitate nicotine tolerance. Endogenous enkephalins acting on mu-opioid receptors also play a role in the development of physical dependence to nicotine. In agreement with these actions of the endogenous opioid system, the opioid antagonist naltrexone has shown to be effective for smoking cessation in certain sub-populations of smokers.     

Central injection of a σ opioid receptor agonist alters body temperature of cats

The prototype σ opioid receptor agonist Nallyl-normetazocine (SKF 10,047) was injected into the third cerebral ventricle of conscious, unrestrained cats, and their temperature was monitored automatically from the retroperitoneal space. In a cold environment (0°C) a small, but not dose-related, hypothermia occurred after doses of 100–500 μg. This response was not antagonized by naloxone given intraventricularly either 15 min before or 1 hr after the opioid. A smaller hypothermia resulted after 250 μg SKF 10,047 when the environmental temperature was 22°C, whereas hyperthermia developed in a hot environment (34°C). Thus SKF 10,047 appears to allow body temperature to drift, upward in the heat and downward in the cold, a pattern indicative of thermoregulatory depression. These results are similar to those obtained in the first 2–3 hr after pentazocine administration, and they support a previous classification of the initial temperature response to centrally injected pentazocine as due to stimulation of σ opioid receptors.     

Stress-induced analgesia in μ-opioid receptor knockout mice reveals normal function of the δ-opioid receptor system

Stress-induced analgesia (SIA) was examined in wildtype and μ-opioid receptor knockout mice. We used thermal paw withdrawal (TPW) latency following a continuous 3-min swim in 20°C water, and found a significant increase in TPW latency in both wild-type and knockout mice. Pre-treatment prior to the swim with naltrindole, a selective δ-opioid receptor antagonist, blocked the increase in TPW latency in knockout mice. These results demonstrate an intact δ-receptor-mediated function of a physiologically-released endogenous agonist in the μ-opioid receptor knockout mouse. The present findings are in contrast with previous reports that analgesia induced by exogenous delta agonists is reduced in the knockout mice.     

Prenatal exposure to morphine alters brain μ opioid receptor characteristics in rats

Prenatal morphine exposure alters neither the binding capacity nor the affinity of ligand binding to μ opioid receptors of adult male brains. However, males have significantly higherB_max in the hypothalamus than ovariectomized females. In females, prenatal exposure to morphine reduces theB_max of μ opioid receptors 25% in the hypothalamus and preoptic area. Estrogen treatment increases theB_max of μ opioid receptors in the striatum of all ovariectomized females but in the hypothalamus only of morphine-exposed females, thereby eliminating the sex difference observed in control animals.