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.     

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.     

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.     

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.     

Endogenous opioids and neural cancer: an immunoelectron microscopic study

[Met5]-enkephalin, an endogenous opioid peptide derived from proenkephalin A, participates in tumorigenic events by serving as a natural trophic factor that inhibits cell replication. In order to understand how endogenous opioids function in modulating neoplasia, the present study examined the fine structural association of enkephalin with the cellular components of a tumor cell. Immunoelectron microscopic studies were undertaken using antibodies recognizing [Met5]-enkephalin-like substances, and murine S20Y neuroblastoma cells that are known to be responsive to endogenous opioid modulation. Enkephalin was found throughout the cell body and process. Immunoreactivity was associated with the plasma membrane, outer nuclear envelope, and a variety of organelles. With the exception of aggregates of immunoreactivity subjacent to the inner nuclear envelope, the nucleus was not reactive. These results establish that growth-related enkephalins are localized discretely within neuroblastoma cells. Since neuroblastoma cells produce and secrete enkephalins, and enkephalins interact with receptors to mediate actions on cell replication, this study examined enkephalins involved in two different patterns of traffic; further work will be needed to examine each aspect.     

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.     

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.     

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.     

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 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.     

Endogenous opioid systems and the growth of oligodendrocyte progenitors: Paradoxical increases in oligodendrogenesis as an indirect mechanism of opioid action

Endogenous opioids inhibit nervous system development by inhibiting the proliferation of certain neuronal and glial progenitors. To determine whether opioids affect the growth of preoligodendrocytes, the effects of the endogenous opioid [Met⁵]-enkephalin were examined in preoligodendrocytes in primary mixed-glial and preoligodendrocyte-enriched (>98% pure) cultures. Proliferating preoligodendrocytes in mixedglial or preoligodendrocyte-enriched cultures were continuously treated for a total of 40 h with either basal growth media (controls), 1 μM [Met⁵]-enkephalin, 1 μM [Met⁵]-enkephalin plus the opioid antagonist naloxone (3 μM), or naloxone alone (3 μM), and incubated in [³H]-thymidine (0.2 μCi/ml/4–6 h) after 34–36 h of opioid exposure. Opioid dependent changes in DNA synthesis were assessed autoradiographically in O4-immunoreactive oligodendrocyte progenitors. Naloxone alone significantly decreased the rate of DNA synthesis and number of O4-immunoreactive preoligodendrocytes in mixed-glial cultures. However, naloxone and/or [Met⁵]-enkephalin did not affect DNA synthesis or the number of O4-immunoreactive preoligodendrocytes in cultures enriched in preoligodendrocytes. The results suggest that astrocytes, or perhaps another cell type, play a permissive role in opioid-dependent alterations in preoligodendrocyte proliferation. Endogenous opioids affect the genesis of neural cells by both direct and indirect mechanisms. © 1993 Wiley-Liss, Inc.     

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.     

Postnatal development of functional dopamine, opioid and tachykinin receptors that regulate acetylcholine release from rat neostriatal slices. Effect of 6-hydroxydopamine lesion

In the present work we have studied the postnatal development of functional dopamine, opioid and tachykinin receptors, which regulate cholinergic activity in the neostriatum. The release of endogenous acetylcholine from rat striatal slices was measured using a chemiluminescent method. We have observed that the inhibition mediated by dopamine through D₂ receptors was not detectable until postnatal day 10, whereas the inhibition mediated by opioid receptors was detectable at postnatal day 15 for δ-receptors ([D-Pen², D-Pen⁵]-enkephalin) and at postnatal day 21 for μ-receptors ([D-Ala², Gly(ol)⁵]-enkephalin). Excitatory effect mediated by tachykinins through NK₁ ([Sar⁹, Met(O2)¹¹]-Substance P), NK₂ ([Nle¹⁰]-Neurokinin A_4–10), or NK₃ (senktide) receptors was already detectable at postnatal day 5.

In order to examine the influence of dopamine in the development of tachykinin and opioid systems in the neostriatum, we induced dopamine deficiency by intraventricular injection of 6-hydroxydopamine at postnatal day 3. We observed an increase in senktide-evoked acetylcholine release at postnatal day 30. The effect produced by [Sar⁹, Met(O₂¹¹]-Substance P and [Nle¹⁰]-Neurokinin A_4–10 was not modified. Furthermore, at postnatal day 35, we could observed that the two opioid receptor agonists have no effect.

Our results show that dopamine, tachykinins and opioids are already able to mediate the modulation of acetylcholine release in early stages of development with a different pattern of postnatal development. Furthermore, the integrity of a dopaminergic system plays an important role in the functional development of the neostriatal cholinergic neurons which are differentially modulated by opioids or tachykinins.     

Evidence for the presence of mu- and kappa- but not of delta-opioid sites in the human fetal brain

The presence of multiple opioid binding sites in human fetal brain at 20 weeks gestational age was determined using the following selective tritiated ligands: D-Ala2, N-MePhe4, Gly-ol5-enkephalin (DAGO) for the mu-type, D-Pen2, D-Pen5-enkephalin (DPDP) for the delta-type and U-69,593 for the kappa-type. [3H]DAGO and [3H]U-69,593 each bind to a single class of high-affinity sites in membrane preparations with respective Kd values of 0.96 +/- 0.33 nM and 0.57 +/- 0.20 nM. The binding capacity for [3H]DAGO is 1.38 +/- 0.21 pmol/g tissue whereas [3H]U-69,593 has a binding capacity of 1.04 +/- 0.22 pmol/g tissue. Using DPDP we did not detect any specific high-affinity binding sites in human fetal brain. The non-selective tritiated opioid ethylketazocine (EKC) labels a homogenous class of sites with a Kd of 0.17 +/- 0.01 nM and a binding capacity of 2.15 +/- 0.15 pmol/g tissue, a value that is not statistically different from the total capacity obtained with [3H]DAGO and [3H]U-69,593. Competition studies using selective unlabeled opioids against the binding of [3H]-DAGO, [3H]U-69,593 or [3H]EKC indicate that opioid sites present in human fetal brain possess similar pharmacological characteristics to those found in human and mammalian adult brain. The results of this study present the first evidence for the presence of mu- and kappa-type opioid sites in human fetal brain and this may support the postulated role of the opioid system in the neurobiological development.     

Opioids and migration, chemotaxis, invasion, and adhesion of human cancer cells

This study was designed to examine the role of opioids on cell migration, chemotaxis, invasion, and adhesion, with an emphasis on whether the opioid growth factor (OGF, [Met(5)]-enkephalin) or the opioid antagonist naltrexone (NTX) impacts any or all of these processes. Drug concentrations of OGF and NTX known to depress or stimulate, respectively, cell proliferation and growth were analyzed. Three different human cancers (pancreatic, colon, and squamous cell carcinoma of the head and neck), represented by seven different cancer cell lines (PANC-1, MIA PaCa-2, BxPC-3, CAL-27, SCC-1, HCT-116, and HT-29), were evaluated. In addition, the influence of a variety of other natural and synthetic opioids on cell motility, invasion, and adhesion was assessed. Positive and negative controls were included for comparison. OGF and NTX at concentrations of 10(-4) to 10(-6)M, and dynorphin A1-8, beta-endorphin, endomorphin-1, endomorphin-2, leucine enkephalin, [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2), MePhe(4), Glycol(5)]-enkephalin (DAMGO), morphine, and U69,593 at concentrations of 10(-6)M, did not alter cell migration, chemotaxis, or invasion of any cancer cell line. OGF and NTX at a concentration of 10(-6)M, and incubation for 24 or 72h, did not change adhesion of these cancer cells to collagen I, collagen IV, fibronectin, laminin, or vitronectin. Moreover, all other opioids tested at 10(-6)M concentrations and for 24h had no effect on adhesion. These results indicate that the inhibitory or stimulatory actions of OGF and NTX, respectively, on cell replication and growth are independent of cell migration, chemotaxis, invasion, and adhesive properties. Moreover, a variety of other exogenous and endogenous opioids, many specific for the micro, delta, or kappa opioid receptors, also did not alter these biological processes, consonant with previous observations of a lack of effects of these compounds and their receptors on the biology of cancer cells.     

Opioids and the apoptotic pathway in human cancer cells

This study was designed to examine the role of opioids in cell survival, with an emphasis on the mechanism of opioid growth factor (OGF, [Met(5)]-enkephalin)-dependent growth inhibition. Using three human cancer cell lines: MIA PaCa-2 pancreatic adenocarcinoma, HT-29 colon adenocarcinoma, and CAL-27 squamous cell carcinoma of the head and neck, and OGF and the opioid antagonist naltrexone (NTX) at a dosage (10(-6)M) selected because it is known to repress or increase, respectively, cell replication, the effects on apoptosis (TUNEL, Annexin V) and necrosis (trypan blue) were investigated on days 2, 5, and 7 of exposure. In addition, the influence of a variety of other natural and synthetic opioids on apoptosis and necrosis was examined at a dosage of 10(-6)M. OGF, NTX, naloxone, [D-Pen(2,5)]-enkephalin, [Leu(5)]-enkephalin, dynorphin A1-8, beta-endorphin, endomorphin-1 and -2, and methadone at concentrations of 10(-6)M did not alter cell viability of any cancer cell line. Exposure of cultures to [D-Ala(2),MePhe(4),Glycol(5)]-enkephalin (DAMGO), morphine, or etorphine at 10(-6)M significantly increased the number of adherent cells positively stained for TUNEL and Annexin V, as well as the number of necrotic cells in the supernatant, from control levels at all time points studied. The effects of DAMGO, morphine, and etorphine on apoptosis/necrosis were not fully blocked by concomitant administration of naloxone. Despite the increase in cell death in some opioid-treated groups, the number of apoptotic and necrotic adherent cells, and the number of necrotic cells in the supernatant, was no more than 1-2% of the total cell population. These results indicate that the inhibitory (OGF) or stimulatory (NTX) action on cell growth in tissue culture is not due to alterations in apoptotic or necrotic pathways. Moreover, although some opioids increased cell death, and dose-effect relationships need to be established, this activity was not of great magnitude and supports the previously reported lack of growth inhibition of many of these compounds.     

Homeostasis of ocular surface epithelium in the rat is regulated by opioid growth factor

Endogenous opioid peptides serve as growth factors in developing, renewing, and neoplastic cells and tissues. This study examined the hypothesis that opioids serve to modulate the homeostatic renewal of ocular surface epithelium in the rat. DNA synthesis in the epithelium of the central (CC) and peripheral (PC) cornea, limbus (LM), and conjunctiva (CN) was investigated using adult male rats. Animals received an injection of opioid growth factor (OGF), [Met⁵]-enkephalin, OGF and naloxone (NAL), NAL alone, naltrexone (NTX), or an equivalent volume of sterile water (CO) and sacrificed 4 h later (i.e. 16:00 h). [³H]thymidine was administered 1 h before sacrifice. With the exception of NTX (20 mg/kg), all compounds were given at 10 mg/kg. Examination of 5 time points over an 18-h period revealed no variation in DNA synthesis within a region of ocular surface basal epithelium (BE). OGF depressed DNA synthesis of the BE by 25, 48, and 50% in the PC, LM, and CN, respectively; little labeling was recorded in the BE of the CC. Exposure to OGF-NAL or NAL alone did not alter DNA synthesis of the BE. Complete blockade of OGF-ζ receptor interaction by administration of the potent opioid antagonist, NTX, increased the number of epithelial cells in the PC, LM, and CN undergoing DNA synthesis by 30 to 72%. The effects of OGF and NTX on DNA synthesis of BE also were observed in an organ culture setting. Utilizing immunocytochemistry, OGF and its receptor ζ were associated with both the basal and the suprabasal cells of the ocular surface epithelium. These results indicate that an endogenous opioid peptide, OGF, and its receptor are present and govern homeostatic cellular renewal processes in ocular surface epithelium. OGF regulates DNA synthesis in a direct manner, and does so by a tonic, inhibitory, and receptor-mediated mechanism.     

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.     

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.     

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.