Endogenous opioid systems regulate growth of neural tumor cells in culture

Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer. Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, [Met5]-enkephalin was the most potent compound to influence cell replication. With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner. [Met5]-Enkephalin depressed both DNA synthesis and mitosis. [Met5]-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide. Immunocytochemistry showed [Met5]-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells. Binding of [3H]-[Met5]-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein. [Met5]-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma. These results indicate that [Met5]-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth. Since cancer cells produce [Met5]-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism.     

Demonstration and characterization of zeta (zeta), 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, [Met5]enkephalin, was examined to ascertain the zeta (zeta) 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 Kd of 1.6 nM and a binding capacity (Bmax) of 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]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. Cell density (log, confluence, postconfluence) did not alter the Kd or Bmax. This study serves as the first demonstration and characterization of the zeta (zeta) opioid receptor in tissue culture cells. The homogeneous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the zeta receptor.     

Endogenous opioid systems and neural cancer: Transmission and scanning electron microscopic studies of murine neuroblastoma in tissue culture

Endogenous opioid systems participate in carcinogenic events. To understand further the action of opioids on growth, S20Y neuroblastoma cells in tissue culture were exposed to i) [Met5]-enkephalin, a naturally occurring opioid pentapeptide, at a concentration (10(-6) M) that inhibits cell replication by 66% of control levels, ii) [Met5]-enkephalin (10(-6) M) and the opioid antagonist naloxone (10(-6) M) which blocks opioid agonist action, or iii) naltrexone (10(-6) M), a potent antagonist that disrupts endogenous opioid-opioid receptor interaction and increased cell number 76% above control values. The morphology of cells exposed to these agents for 2-4 days were similar to controls (i.e., exposed to sterile water) as determined by scanning and transmission electron microscopy. These results support the hypothesis that endogenous opioid systems act as trophic factors as they regulate growth; their effects on cell growth and survival, however, do not alter the basic ultrastructural morphology of the cells. Moreover, these data strengthen the validity of paradigms and therapeutic regimens that utilize opioid agonists and antagonists to modulate the relationship of endogenous opioid-opioid receptor interactions in neural cancer.     

Endogenous opioids regulate cell proliferation in the retina of developing rat

The role of endogenous opioids and opioid receptors (endogenous opioid systems) in modulating cell proliferation in the developing mammalian retina was examined in 1-day-old rats. In contrast to a labeling index (LI) of 35.8% in control animals, administration of the opioid peptide [Met5]-enkephalin (100 micrograms/kg) significantly reduced (10.6%) the proportion of cells incorporating [3H]thymidine; concomitant injection of 1 mg/kg naloxone blocked the inhibitory effects of [Met5]-enkephalin on cell division. Naloxone (1 mg/kg) alone did not alter the LI. The interruption of endogenous opioid-opioid receptor interaction by naltrexone (50 mg/kg), a potent opioid antagonist, was accompanied by a significant increase (6.4%) in the LI relative to control levels. Immunocytochemical experiments revealed the presence of enkephalin-like immunoreactivity, with staining of the cortical cytoplasm of proliferating and differentiating retinal cells recorded; no immunoreactivity was noted in the adult retina. In vitro autoradiography using 125I-[Met5]-enkephalin indicated that [Met5]-enkephalin binding sites were localized to the developing retina; no binding of the radiolabeled ligand was recorded in the adult retina. These results demonstrate the presence of growth-related endogenous opioids and opioid receptors in the developing mammalian retina, but not in adult retina, and suggest that endogenous opioids serve as natural inhibitory trophic factors that tonically regulate cell proliferation.     

Opioids and differentiation in human cancer cells

This study was designed to examine the role of opioids on cell differentiation, with an emphasis on the mechanism of opioid growth factor (OGF, [Met5]-enkephalin)-dependent growth inhibition. Three human cancer cell lines (SK-N-SH neuroblastoma and SCC-1 and CAL-27 squamous cell carcinoma of the head and neck), along with OGF and the opioid antagonist naltrexone (NTX) at a dosage (10(-6) M) known to repress or increase, respectively, cell replication, were utilized. The effects on differentiation (neurite formation, process lengths, betaIII-tubulin, involucrin) were investigated in cells exposed to OGF or NTX for up to 6 days. In addition, the influence of a variety of other natural and synthetic opioids on differentiation was examined. OGF, NTX, naloxone, [D-Pen2,5]-enkephalin, dynorphin A1-8, beta-endorphin, endomorphin-1 and -2, [D-Ala2, MePhe4, Glycol5]-enkephalin (DAMGO), morphine, and U69,593 at concentrations of 10(-6) M did not alter cell differentiation of any cancer cell line. In NTX-treated SK-N-SH cells, cellular area was increased 23%, and nuclear area was decreased 17%, from control levels; no changes in cell or nuclear area were recorded in OGF-exposed cells. F-actin concentration was increased 40% from control values in SK-N-SH cells subjected to NTX, whereas alpha-tubulin was decreased 53% in OGF-treated cells. These results indicate that the inhibitory or stimulatory actions of OGF and NTX, respectively, on cell growth in tissue culture are not due to alterations in differentiation pathways. However, exposure to OGF and NTX modified some aspects of cell structure, but this was independent of differentiation. The absence of effects on cancer cell differentiation by a variety of other opioids supports the previously reported lack of growth effects of these compounds.     

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.     

Zeta (zeta), a growth-related opioid receptor in developing rat cerebellum: identification and characterization.

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [3H][Met5]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (Kd) of 2.2 nM and a binding capacity (Bmax) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na+, Mg2+, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met5]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met5] enkephalin was the most potent displacer of [3H][Met5]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [3H][Met5]-enkephalin was recorded, mu and kappa receptors were also found in the developing rat cerebellum, while mu, delta, and kappa receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met5]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (zeta), in the developing nervous system.     

Proenkephalin-A derived peptides do not modulate cardiovascular effects of epinephrine on the isolated rat atrial preparations

The catecholamines and the opioid peptides are found to be co-localized in the adrenomedullary chromaffin cells. They are co-secreted from the chromaffin granules in response to various stimuli. The stress-induced released of epinephrine is known to exert its effect on the cardiovascular system resulting in the changes in heart rate and blood pressure. However, the role of the co-released proenkephalin-A derived peptides has not been extensively characterized. Previous work from several investigators suggested that the peptides modulate cardiac functions of the catecholamines. There is considerable conflicting results among these reports. Results from the isolated rat atrial preparation indicated that enkephalins attenuated the increase in atrial rate induced by norepinephrine through restriction of the calcium fluxes. Nonetheless, others reported insensitivity of the enkephalins in similar or different test systems. We further re-examined these discrepancies using the isolated rat atrial preparation to investigate the opioid peptide modulatory effect on the cardiovascular changes induced by exogenous epinephrine. Alterations in rate and force of contraction resulting from epinephrine and the peptides were both studied in parallel. The opioid peptides used in this study were [Met5]-enkephalin (ME), [Leu5]-enkephalin (LE), FMRFamide, [Met5]-enkephalyl-Arg6-Phe7 (MEAP), peptide E, and the non-selective opioid agonist, etorphine. We report here that none of the opioid peptides were effective in alleviating or attenuating the increase in heart rate and developed tension caused by epinephrine. The peptides did not affect the basal beating rate nor the force of contraction. Thus, the present results clearly demonstrate the insensitivity of the enkephalins in modulating the cardiac effects of epinephrine. They further indirectly support the prejunctional synaptic nerve endings as the potential peripheral site of action of the peptides.     

Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro.

To determine whether one or more opioid receptor types might be preferentially involved in gliogenesis, primary mixed glial cultures derived from mouse cerebra were continuously treated with varying concentrations of opioid agonists selective for mu (mu), i.e., DAGO ([D-Ala2, MePhe4, Gly(ol)5]enkephalin), delta (delta), i.e., DPDPE ([D-PEN2,D-PEN5]enkephalin), or kappa (kappa), i.e., U69,593, opioid receptor types. In addition, a group of cultures was treated with [Met5]-enkephalin, an agonist for delta opioid receptors as well as putative zeta (zeta) opioid receptors. Opioid-dependent changes in growth were assessed by examining alterations in (1) the number of cells in mixed glial cultures at 3, 6, and 8 days in vitro (DIV), (2) [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes at 6 DIV, and (3) the area and form factor of GFAP-immunoreactive, flat (type 1) astrocytes. DPDPE at 10(-8) or 10(-10) M, as well as [Met5]-enkephalin at 10(-6), 10(-8), or 10(-10) M, significantly reduced the total number of glial cells in culture; but this effect was not observed with DAGO or U69,593 (both at 10(-6), 10(-8), or 10(-10) M). Equimolar concentrations (i.e., 10(-6) M) of [Met5]enkephalin or U69,593, but not DPDPE or DAGO, suppressed the rate of [3H]thymidine incorporation by GFAP-immunoreactive, flat (type 1) astrocytes. DAGO had no effect on growth, although in previous studies morphine was found to inhibit glial numbers and astrocyte DNA synthesis. [Met5]enkephalin (10(-6) M) was the only agonist to significantly influence astrocyte area. Collectively, these results indicate that delta (and perhaps mu) opioid receptor agonists reduce the total number of cells in mixed glial cultures; while [Met5]enkephalin-responsive (and perhaps kappa-responsive) opioid receptors mediate DNA synthesis in astrocytes. This implies that delta opioid receptors, as well as [Met5]enkephalin-sensitive, non-delta opioid receptors, mediate opioid-dependent regulation of astrocyte and astrocyte progenitor growth. These data support the concept that opioid-dependent changes in central nervous system growth are the result of endogenous opioid peptides acting through multiple opioid receptor types.     

Regulation of human natural cytotoxicity by enkephalins and selective opiate agonists

This study reports a bidirectional effect of the enkephalins and selective opiate receptor agonists on human natural killer (NK) cell activity. Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and enriched for T lymphocytes and large granular lymphocytes (LGL) by passage over nylon wool columns. Nylon wool nonadherent cell populations were preincubated for 18 h in the presence of fetal bovine serum with and without methionine-enkephalin, leucine-enkephalin, dynorphin (fragment 1-13), [D-Ser2]-leucine-enkephalin-Thr (DSLET), and [D-Ala2,N Me-Phe4, Gly-ol5]-enkephalin (DAGO). NK activity was measured by a standard 51Cr release assay with radiolabeled K562 cells. The cytolytic capacity of low NK responder populations was enhanced by the endogenous opioids while the NK activity of high responder populations was suppressed. These results suggest an immunoregulatory action of opioid peptides on NK activity. This possibility was confirmed using a serum-free system in conjunction with recombinant interferon-alpha. In addition, the classic opioid receptor antagonist naloxone displayed both antagonist and direct immunomodulatory properties, which may indicate the presence of lymphocyte derived opioid peptides in the culture system.     

Immunoelectron microscopic localization of the opioid growth factor receptor (OGFr) and OGF in the cornea

This study was conducted to determine the cellular and subcellular location(s) of the opioid growth factor receptor (OGFr), and the opioid growth factor (OGF), [Met(5)]-enkephalin, in the corneal epithelium. Laser scanning confocal microscopy analysis revealed that both OGFr and OGF were colocalized in the paranuclear cytoplasm and cell nuclei in basal, as well as suprabasal, cells of adult rat corneal epithelium. Using a postembedding immunogold procedure for immunoelectron microscopy that included embedding in Unicryl, both single- and double-face labeling studies were performed. Immunogold labeling of OGFr was detected on the outer nuclear envelope, in the paranuclear cytoplasm proximal to the nuclear envelope, perpendicular to the nuclear envelope in a putative nuclear pore complex, and within the nucleus adjacent to heterochromatin. Immunoreactivity for OGF was noted in locations similar to that for OGFr. In addition, aggregates of staining for OGF were found throughout the cytoplasm, including subjacent to the plasma membrane. Double labeling experiments revealed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, extending across the nuclear pore complex, and in the nucleus. Anti-OGFr IgG by itself, but not anti-OGF IgG alone, was associated with the outer nuclear envelope, and uncomplexed OGF immunoreactivity was detected in the cytoplasm in dual labeling experiments. These results based on complementary approaches of confocal microscopy and immunoelectron microscopy, suggest that: (i) OGFr resides on the outer nuclear envelope, (ii) OGF interacts with OGFr at the outer nuclear envelope, (iii) the colocalized receptor and peptide translocates between the cytoplasm and the nucleus at the nuclear pore, and (iv) signal transduction for modulation of cell proliferation necessitates a peptide-receptor complex that interfaces with chromatin in the nucleus.     

Release of endogenous opioid peptides displaces [3H]diprenorphine binding in rat hippocampal slices

Pharmacological depolarization by KCl or veratrine reduced [3H]diprenorphine binding to opioid receptors in the hippocampal slice in a transient, calcium-dependent, and peptide-sensitive manner. These results suggest that endogenous opioid peptides were released from synaptic terminals and competitively displaced [3H]diprenorphine binding to opioid receptors. [3H]diprenorphine binding was significantly reduced by calcium-dependent depolarization throughout the hippocampus as determined by subsequent receptor autoradiography and quantitative densitometry. Displacement of binding was evident at sites in the CA1 and CA3 regions, the dentate gyrus, and the subiculum. The most dramatic reduction was evident in stratum lacunosum moleculare of CA3. Correlating the sites of maximal [3H]diprenorphine displacement with the previously described distribution of the opioid peptides suggests that the perforant path fibers release enkephalins in stratum lacunosum moleculare of CA3 and stratum moleculare of the dentate gyrus, and that mossy fibers may release both dynorphins and enkephalins near stratum pyramidale of CA3 and stratum granulosum. The lack of complete overlap between the distribution of opioid terminals and the sites of displacement indicates that these peptides may diffuse a moderate distance to their sites of action. Radioligand displacement defines the sites of endogenous opioid binding, suggests the likely sources of peptide release, and thus predicts the sites of endogenous opioid action within the hippocampus.     

Opioid growth factor and organ development in rat and human embryos

In addition to neurotransmission, the native opioid peptide, [Met5]enkephalin, is a tonically active inhibitory growth molecule that is termed opioid growth factor (OGF). OGF interacts with the zeta (zeta) opioid receptor to influence cell proliferation and tissue organization. We now identify OGF and the zeta receptor in embryonic derivatives including ectoderm, mesoderm, and endoderm of the rat on gestation day 20. Messenger RNA for preproenkephalin (PPE), the precursor of OGF, was detected in the developing cells, suggesting an autocrine production of this peptide. Acute exposure of the pregnant female to OGF resulted in a decrease in DNA synthesis in cells of organs representing all three germ layers, and did so in a receptor-mediated fashion. The influence of OGF was direct, as evidenced in organ culture studies. Blockade of endogenous opioid interaction using naltrexone (NTX) produced an increase in DNA synthesis, indicating the constitutive and functional nature of opioid activity on growth during prenatal life. Human fetal cells contained OGF and the zeta receptor. These data support the hypothesis that endogenous opioid modulation of organ development is a fundamental principle of mammalian embryogenesis, and that OGF has a profound influence on ontogeny. Irregularities in the role of opioids as growth regulators in relationship to the more than 500,000 newborns suffering from birth defects each year in the US needs to be examined.     

The amide of the naturally occurring opioid [Met]enkephalin-Arg6-Phe7 is a potent analog of the molluscan neuropeptide FMRFamide

The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH₂), the opioid [Met]enkephalin, and two peptides analogous to both were tested on four isolated molluscan muscles. Both FMRFamide and [Met]enkephalin-Arg⁶-Phe⁷-NH₂ (YGG-FMRFamide) excited the ventricle of $\text{Macrocallista nimbosa}$ and inhibited that of $\text{Lampsilis claibornensis}$. These same two peptides also caused contractures of the anterior byssus retractor muscle (ABRM) of $\text{Mytilus edulis}$ and the radula protractor muscle of $\text{Busycon contrarium}$. The effect on the ABRM was a catch contracture, relaxed by 5-hydroxytryptamine. In all cases, the two peptides were equipotent and their actions were identical. Neither [Met]enkephalin, nor the naturally occurring opioid peptide [Met]enkephalin-Arg⁶-Phe⁷ (YGG-FMRF), affected these preparations. In conclusion, these tissues are specifically sensitive to the structure at the C-terminal of FMRFamide, but not to its N-terminal or that of the enkephalins; that is, FMRFamide receptors, but not opioid ones, are present. Nevertheless, the amino acid sequence common to [Met]enkephalin-Arg⁶-Phe⁷ and FMRFamide implies that these two peptides and their naturally occurring congeners are homologous molecules.     

Characterization of a polyclonal antibody to the mu 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 mu 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 (mu opioid) from rat membranes, but was ineffective against the binding of [3H]ethylketocyclazocine (kappa [3H]D-Ala2,D-Leu5-enkephalin (delta opioid) or [3H]phencyclidine (phencyclidine/sigma receptor ligand). The antibody was able to precipitate the Mr 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 X 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.     

Localization of preproenkephalin A mRNA in the neonatal rat retina.

An opioid growth factor, [Met5]-enkephalin, is known to regulate developmental events in the neonatal rat retina. This growth factor interacts with the zeta (zeta) opioid receptor to modulate retinal ontogeny. Both peptide and receptor are present in developing retina, but not in adult retina. We have used in situ hybridization histochemistry to identify and localize preproenkephalin A mRNA in the neonatal rat retina. Preproenkephalin mRNA was localized to the ganglion cell layer, with some radiolabeling found in the neuroblast layer. This result indicates that 1) the mRNA to preproenkephalin A is present during the critical stage of development in the neonatal retina that coincides with the presence of the growth-regulating peptide, [Met5]-enkephalin, and 2) that the source of the opioid growth factor controlling the production of retinal cells appears to be autocrine (i.e., retinal neuroblasts) and paracrine (i.e., ganglion cells) in nature.     

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.     

Endogenous opioid systems regulate cell proliferation in the developing rat brain

The role of endogenous opioid systems in modulating the proliferation of developing cerebellar cells was examined autoradiographically in 6-day-old rats. The blockade of endogenous opioid-opioid receptor interaction by naltrexone, a potent opioid antagonist, was accompanied within 1–2 h by an increased proportion of cells incorporating [³H]thymidine. When high doses of naltrexone (50 mg/kg) were administered this index was still elevated 12 h later; however, when low doses of naltrexone (1 mg/kg) were administered the index of labeled cells was decreased markedly. Injection of methionine-enkephalin, an endogenous opioid peptide, also resulted in a decrease in the proportion of cells incorporating [³H]thymidine. Concomitant injection of 1 mg/kg naloxone, however, blocked the inhibitory effects of methionine-enkephalin on cell division but did not itself affect cell generation. These studies demonstrate that endogenous opioid systems can regulate the proliferation of cell populations in the developing nervous system and do so through an inhibitory mechanism.     

Opioids modulate cell division in the germinal zone of the late embryonic neocortex.

Opioid effects on cell division in the embryonic cerebral cortex were examined using two experimental approaches: (i) the presence of opioid receptors in the embryonic day 16 mouse neocortex was tested using immunohistochemical techniques; (ii) the values of the indices of [3H]thymidine pulse labelled cells and mitotic indices were estimated in the ventricular zone of the embryonic day 16 mouse neocortex 2.5, 4.5 and 8.5 h after administration to pregnant females of selected opioid receptor agonists or the opioid antagonist naloxone. The immunohistochemical study demonstrated that distinct subpopulations of the ventricular zone cells express mu, delta or kappa opioid receptors. Acute exposure of mouse embryos to mu, delta and kappa opioid receptor agonists or naloxone differentially affects the indices of [3H] thymidine pulse labelled cells and mitotic indices indicating changes in the cell cycle composition. Treatment with the mu opioid receptor agonist D-Ala2-MePhe4, Gly-ol5-enkephalin (DAGO), or the partially selective kappa opioid receptor agonist bremazocine, increased the [3H]thymidine labelling and mitotic indices. In contrast, the delta receptor agonist (D-Ser8)-leucine enkephalin-Thr (DSLET) produced a decrease in the labelled cell indices and mitotic indices. Naloxone provided a biphasic effect: a decrease in the values of labelled cell indices 2.5 h after naloxone administration, followed by an increase in the values of the indices at 4.5 and 8.5 h. These results suggest that the endogenous embryonic/maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the late embryonic cortex.     

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.