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.     

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.     

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.     

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.     

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.     

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.     

Endogenous opioid systems and the regulation of dendritic growth and spine formation

The role of endogenous opioid systems (endogenous opioids and opioid receptors) in neuronal development was examined in 10- and 21-day-old rats by utilizing an opioid antagonist (naltrexone) paradigm. Throughout the first 3 weeks of life, Sprague-Dawley rats were given daily subcutaneous injections of either 50 mg/kg naltrexone, a dosage that invoked a complete (24 hours/day) receptor blockade, or 1 mg/kg naltrexone, a dosage which intermittently blocked (4-6 hours/day) opioid receptors and exacerbated opioid action; animals injected with sterile water served as controls. Pyramidal cells from the frontoparietal cortex (layer III) and hippocampal field CA1, and cerebellar Purkinje cells, were impregnated by using the Golgi-Kopsch method; total and mean dendrite segment length, branch frequency, and spine concentration were analyzed morphometrically. Perturbations of endogenous opioid systems caused region-dependent alterations in dendrite complexity and/or spine concentration in all brain areas. Continuous opioid receptor blockade resulted in dramatic increases in dendrite and/or spine elaboration compared to controls at 10 days in all brain regions; however, these increases were only evident in the hippocampus at 21 days. With intermittent blockade, dendrite and/or spine growth were often subnormal, being predominant at day 21. Our results indicate that endogenous opioid systems are critical regulators of neuronal differentiation, and they control growth through an inhibitory mechanism. Considering previous findings demonstrating that neurobehavioral ontogeny is dependent on endogenous opioid-opioid receptor interactions, the present results suggest an opioid-dependent, structure-function relationship between neuronal and behavioral maturation.     

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 neural development: Ultrastructural studies in the cerebellar cortex of infant and weanling rats

Endogenous opioid systems participate in regulating the development of the nervous system. Opioid antagonists like naltrexone (NTX) perturb the relationship between endogenous opioids and opioid receptors quite effectively and reveal the function(s) of endogenous opioid systems during neuro-ontogeny. In this study, the effects of NTX, as well as the repercussions of modulation of endogenous opioid systems during critical stages of neural ontogeny, have been examined at the electron microscopic level of resolution in infant (10 day) and weanling (21 day) rats. Preweaning rats were subjected to complete (50 mg/kg NTX) or intermittent (1 mg/kg NTX) daily receptor blockade. Extensive ultrastructural examinations were conducted on the external germinal (granule), molecular, Purkinje, internal granule, and medullary layers of the cerebellar cortex. The NTX groups had striking similarities in morphology to that of controls at postnatal days 10 and 21. 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 further strengthen the validity of paradigms utilizing opioid antagonists to explore the relationship of endogenous opioid-opioid receptor interactions and neural morphogenesis.     

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.     

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.     

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.     

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.     

Opioid antagonist-induced modulation of cerebral and hippocampal development: histological and morphometric studies

The role of endogenous opioid systems in preweaning cerebral and hippocampal development was explored in rats utilizing naltrexone, a potent opioid antagonist. Sprague-Dawley rats were given daily injections (s.c.) of either 1 or 50 mg/kg naltrexone to invoke a temporary or complete blockade, respectively, of opioid receptors throughout the first 3 weeks of postnatal life; animals injected with sterile water served as controls. At weaning (Day 21), macroscopic, morphometric, and histological assessments were undertaken. In general, 50 mg/kg naltrexone had a stimulatory action on brain development, whereas 1 mg/kg naltrexone had an inhibitory influence. In most cases, both males and females were affected comparably. Opioid antagonist action was especially directed at cellular and tissue differentiation, with marked changes in macroscopic and areal dimensions and histotypic organization observed in the cerebrum. A prominent effect on the cerebrum of the 1 mg/kg naltrexone group was a substantial increase in packing density of the neural cells, reflecting a reduced area for accommodating neural elements. Changes in the hippocampus were largely restricted to the 1 mg/kg group. However, the number of granule cells was increased in the dentate gyrus of the 50 mg/kg group, suggesting that opioid receptor blockade affects cell types undergoing postnatal proliferation. Cellular elements derived prior to naltrexone treatment (e.g., pyramidal neurons) were capable of being influenced in only differentiative capacity. Our results show that endogenous opioids are natural trophic factors in brain development and provide evidence for the crucial role of endogenous opioid-opioid receptor interaction in neuro-ontogeny.     

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 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.     

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.     

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.     

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.