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Published in final edited form as: J Neurochem. 1992 September ; 59(3): 1145–1152.
Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis Jacob Barg*, Mariana M. Belcheva†, and Carmine J. Coscia† †E. A. Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A. * Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
Abstract
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An opioid receptor agonist, [D-Ala2,Me-Phe4.Gly-ol5]enkephalin (DAMGE), decreased [3H] thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the μ-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-PenThr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The μ-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly μ-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis. whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H] inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.
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Keywords Opioid receptors; Opioid peptides; Fetal rat brain; Cell aggregates; DNA synthesis; Phosphoinositol Maternal drug abuse can have a long-term, complex impact on embryonic brain ontogeny. Minimally, developmental delays are seen for many drugs, including opiates, and the nature of the insult may depend on the state of maturation of susceptible neuronal and glial cells. A body of evidence originating from both in vivo (Lipton and Kater, 1989; Miller, 1991) and in vitro (Ashkenazi et al., 1989: Eccleston et al., 1989) studies supports the notion that neurotransmitters can modulate cell growth during development. Opioids have been shown to decrease thymidine incorporation into DNA transiently and in a naloxone-reversible manner in developing rat brain (Bartolome et al., 1986, 1991; Kornblum et al., 1987; Zagon and McLaughlin, 1987) and in vitro (Coscia et al., 1991; Davila-Garcia and Azmitia, 1989; StieneMartin and Hauser, 1990). Moreover, opioid antagonists have been reported to increase cell
Address correspondence and reprint requests to Dr. C. J. Coscia at Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine. St. Louis. MO 63104−1079. U.S.A..
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proliferation and modulate brain development in vivo (Vertes et al., 1982; Zagon and McLaughlin, 1987; Schmahl et al., 1989). The mechanisms by which opioids may elicit antimitogenic effects is unknown and is the subject of this report. Here we present evidence for the involvement of the inositol lipid signal transduction system in this process.
MATERIALS AND METHODS Thymidine incorporation
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Cell aggregates were prepared from embryonic day 15 Sprague-Dawley rat brain as described (Simantov and Levy, 1986: Barg et al.. 1989b) and grown for various intervals in a chemically defined medium. The animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Cultures were treated with opioid–[D-Ala2,Me-Phe4,Gly-ol5]enkephalin (DAMGE). morphiceptin, etorphine. U69593. [D-Ala2,D-Leu5]-enkephalin (DADLE). cyclic D-Phe-CysTyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP), or naltrexone–at 1 μM 48 h before harvesting and with [3H]thymidine (106 dpm per plate, 25.5 Ci/mmol; Amersham. Arlington Heights, IL, U.S.A.) for the final 23 h. In all experiments the interval indicated represents the total number of days that the brain cell aggregates were maintained. [3H]Thymidine incorporation into cells was determined by filtering cultures on Whatman GF/B filters (diameter, 25 mm), washing with phosphate-buffered saline (20 ml four times each), and measuring the radioactivity on the filter. Thymidine incorporated into DNA was determined by washing aggregates with 10% trichloroacetic acid, 5% trichloroacetic acid, ethanol/ether (1:1 vol/vol), and ether as described (Barg et al.. 1989a). The difference between the two determinations represents [3H]-thymidine incorporated into cells that are of non-DNA origin (external pool). Thymidine incorporation is expressed as femtomoles per seeded cells, i.e., the number of cells that were present in the beginning of the experiments. In all instances, when the same radioactivity data were determined on the basis of protein content (measured on cell harvesting) instead of seeded cells, comparable results were obtained. Protein content, determined by the method of Lowry et al. (1951), was unchanged by the treatment. Inhibitor and activator experiments
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Cultures were treated with pertussis and cholera toxins (List Biological Laboratories. Campbell. CA. U.S.A.), the benzophenanthridine alkaloid chelerythrine (Vipont Laboratories, Fort Collins, CO, U.S.A.). or LiCl in the presence or absence of varying concentrations of naltrexone and I μM DAMGE 48 h before harvesting and with [3H]-thymidine (106 dpm per plate) for the final 23 h. In addition, cultures were exposed to 1 μM DAMGE for varying intervals, and the opioid agonist was removed by washing the aggregates three times with 40 ml of Eagle's medium, whereupon [3H]thymidine was added for the final 23 h of the 7-day culture. Phosphatidylinositol turnover measurements myo-[3H]Inositol (2.5 μCi/ml, 15.5 Ci/mmol; NEN Dupont, Boston, MA, U.S.A.) was added to the cultures 18 h before harvesting. 3H-Inositol phosphate (3H-IP) analyses were performed following the experimental procedure described by Xu and Chuang (1987). Opioid binding in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p] Brain cell aggregates were treated with pertussis toxin (5 ng/ml) for 48 h. Opioid binding assays with [3H]-diprenorphine (31 Ci/mmol) or [3H]etorphine (44 Ci/mmol) were performed in the presence of varying concentrations of Gpp(NH)p, as previously described (Roth et al.. 1981; Barg et al., 1991). Statistical analyses of the data were performed with Student's t test.
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The effect of DAMGE on [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates grown for 7 days is shown in Fig. IA. DAMGE decreased [3H]thymidine incorporation by 40%, and the attenuation was reversed by naltrexone. At the concentrations of DAMGE used, its specificity for μ receptors is marginal (Goldstein and Naidu, 1989). Therefore, more μ-selective agonist and antagonists were tested (Fig. 1A). Morphiceptin blocks DNA synthesis at 1 μM, a concentration at which it retains its μ-receptor specificity. Moreover, the μ-selective antagonist CTOP reversed the effect of both rnorphiceptin and DAMGE. Dosedependence studies indicated that DAMGE inhibited DNA synthesis with an IC50 of 0.376 ± 0.057 μM (Fig. 1 B). Similarly, the opioid agonist etorphine and the κ-selective U69593, both at 1 μM, decreased thymidine incorporation by 35 and 39%. respectively (data not shown). In contrast, DADLE had no significant effect on [3H]thymidine incorporation (data not shown).
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The action of DAMGE proved to depend on the interval that aggregates were maintained in culture, with the greatest inhibition observed in 7-day aggregates (Fig. 1C). DAMGEdependent attenuation of [3H]thymidine incorporation was not observed in ≥ 12-day cultures despite the fact that DNA synthesis could be detected for 21 days and the cells remained viable for 45 days. In addition, exposure of aggregates to DAMGE for an interval of > 1.5 h was sufficient to decrease thymidine incorporation significantly (Fig. 2). Because maximal attenuation was observed at 48 h (Fig. 1A), subsequent studies focused for the most part on long-term effects. Control studies were performed to determine whether opioids influence the amount of radiolabel incorporated into DNA by altering the size of the intra- or extracellular thymidine pool. On isolation of DNA (Nellen et al., 1987) and assessment of its radioactivity (data not shown), a comparable decrease in thymidine incorporation was observed, corroborating the results in Fig. 1A. Moreover, thymidine uptake from the medium was unchanged. Because thymidine uptake is regulated by a permease (Cleaver, 1967), it could be influenced by the pool size of this pyrimidine nucleoside. Therefore, these last two results and the determination of the non-DNA intracellular label (external pool) of [3H]thymidine in all treatments (Fig. 3) tend to exclude the possibility that the opioid effect originates from a change in the extra- and/ or intracellular thymidine pool.
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Most opioid receptor agonists activate the pertussis toxin-sensitive GTP-binding regulatory proteins (G proteins) Gi, and Go (Burns et al., 1983; Kurose et al., 1983; Hescheler et al., 1987). However, we discovered that the DAMGE inhibitory action was not abolished by pertussis toxin (Fig. 4A). Pertussis toxin decreased thymidine incorporation in a concentrationdependent manner, and the inhibition appeared to be additive in the presence of DAMGE at most toxin concentrations. LiCl, an inhibitor of IP phosphatases (Berridge et al.. 1982). also diminished thymidine incorporation dose-dependently, and inclusion of DAMGE elicited an almost maximal inhibition at ≥5 mM LiCl (Fig. 4A). Naltrexone reversal of DAMGE action was effected in the presence of LiCl or pertussis toxin (Fig. 4B). When 1 μM DAMGE was combined with 0.5 mM LiCl, the inhibition (69%) was intermediate between that of I μM DAMGE alone (30 −40%) and that in the presence of 5 mM LiCl (88%), as seen in Fig. 4A. Naltrexone blocked the effect of DAMGE in the presence of 0.5 mM LiCl, restoring the rate of thymidine incorporation to that exhibited by untreated cultures. Moreover, naltrexone partially offset the 10 mM LiCl effects (Fig. 4B). These results raise the possibility that this antagonist blocks the actions ofendogenous opioid peptides (Simantov and Höllt, 1991). A partial reversal of thymidine incorporation by naltrexone (from 78 to 160 fmol per seeded cells) was evident in brain cell aggregates treated with pertussis toxin and DAMGE.
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The possible involvement of Gs in the inhibition induced by DAMGE was tested using cholera toxin, which increases the activity of this stimulatory G protein. In contrast to pertussis, cholera toxin (100 ng/ml) slightly enhanced thymidine incorporation in control and DAMGE-treated cultures (Fig. 4C). The same opioid inhibitory effect at all toxin concentrations indicates that its attenuation of thymidine incorporation is mediated predominantly by a mechanism that does not involve Gs.
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Because pertussis toxin did not block opioid receptor-mediated inhibition of thymidine incorporation, it was important to ascertain that this toxin had uncoupled some opioid receptors from sensitive G proteins under the conditions of the experiment. One method of testing receptor coupling to G protein is to measure agonist binding in the presence of GTP analogs. Gpp(NH)p and other analogs will disrupt the ligand-receptor-G protein complex and thereby reduce overall binding. The data in Fig. 5 substantiate this by showing that the binding of the agonist etorphine to control membranes was inhibited by a GTP analog, but binding to membranes from pertussis toxin-treated cultures was unaffected. Binding of the partial agonist [3H]diprenorphine was not influenced in either experiment. We chose etorphine and diprenorphine, which have a high affinity for μ-, δ-, and κ-opioid receptors, because at this stage in development there may be fewer μ-opioid receptors coupled to pertussis toxin-sensitive G proteins than in adult brain. The decreased sensitivity of agonist binding in the presence of GTP analog implies that some G proteins were uncoupled from opioid receptors by pertussis toxin under the conditions adopted here (Hsia et al., 1984). The question of whether protein kinase C (PKC) is involved in opioid agonist-mediated inhibition of thymidine incorporation was addressed by including a PKC inhibitor along with a μ-receptor agonist. Chelerythrine, which is selective for PKC (Herbert et al., 1990), decreased thymidine incorporation into DNA in 7-day brain cell aggregates dose-dependently (Fig. 6). When added together with chelerythrine, DAMGE failed to alter thymidine incorporation except at the 10−7 M level of the PKC inhibitor. Moreover, the effect of DAMGE was not additive, which is consistent with a common mechanism of DNA synthesis blockade.
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To implicate the inositol lipid signal transduction system further, the effect of DAMGE on phosphoinositol turnover was studied in 7-, 10-, and 14-day brain cell aggregates. Untreated cultures generated a basal level of [3H]inositol trisphosphate ([3H]IP3), reflecting the composite of stimulatory and inhibitory activities of endogenous neurotransmitters that operate via this system. DAMGE decreased the formation of [3H]IP3 in 7- and 10-day brain cell aggregates by 48 and 36%, respectively (Fig. 7A and B). The decline in [3H]IP3 content was naltrexone reversible. In 14-day cultures neither DAMGE nor naltrexone had a significant effect on IP3 turnover (Fig. 7C). DAMGE also failed to inhibit DNA synthesis in cultures of this age (Fig. 1C). A glutamate-stimulated accumulation of [3H]-inositol monophosphate and [3H]inositol bisphosphate was also detected in 7-day aggregates (Fig. 8). Short-term treatment of the glutamate-stimulated aggregates with DAMGE reversed the effect in these experiments. Naltrexone blocked the opioid action. Long-term DAMGE exposure (48 h) also resulted in the inhibition of glutamate-stimulated IP turnover (data not shown). The IC50, for long-term DAMGE blockade of IP turnover was 0.1 18 ± 0.0 16 μM which is comparable in potency to its inhibitory effect on thymidine incorporation.
DISCUSSION These findings confirm that (a) DAMGE inhibits thymidine incorporation into DNA in a concentration- and culture age-dependent manner; (b) as shown by the comparable action of morphiceptin, and its reversibility by CTOP, this effect of DAMGE is most likely mediated
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via the μ-opioid receptor; (c) DAMGE does not appear to use cholera or pertussis toxinsensitive G protein as the transducer in this process; and (d) DAMGE inhibits basal [3H]IP3 formation and glutamate-stimulated [3H]inositol mono-phosphate and [3H]inositol bisphosphate accumulation by a mechanism that is reversed by naltrexone. In addition, DAMGE inhibits DNA synthesis and IP turnover with comparable potency, and the two effects are temporally associated. There is precedence for opioid receptor utilization of phosphoinositol as a second messenger. Recently, κ-opioid stimulation of phosphoinositide turnover in slices of discrete regions of adult rat brain has been reported (Periyasamy and Hoss, 1990). It is also possible that the opioid receptor may interact with another receptor that transmits the signal by the phosphoinositol system (Ross et al., 1990). For example, although direct inhibition of phosphatidylinositol turnover was not demonstrable in cultured neurons from chick embryo cerebral hemispheres, both carbachol- and bradykinin-induced stimulation of phosphoinositol release was blocked by chronic exposure to opioids (Mangoura and Dawson, 1991). There is also recent electrophysiological evidence that a μ-opioid agonist modulates the N-methyl-D-aspartate receptor-mediated glutamate response via PKC in dorsal horn neurons (Chen and Huang, 1991). Finally, the N-methyl-D-aspartate receptor has been implicated as an important organizational cue of neuronal pathways, and Ca2+ transport is involved in the process (Miller, 1991).
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It has been known for some time that like muscarinic receptors, certain growth factors activate phosphoinositide-specific phospholipase C via a mechanism utilizing G protein (Chuang, 1989). This action can be abolished by pertussis toxin (Seuwen et al., 1988; Moriarty et al., 1990). However, not all G protein-coupled phospholipase C systems are blocked by pertussis toxin (Birnbaumer, 1990). Here we show that inactivation of Gi by pertussis toxin decreases thymidine incorporation, but it makes only a minor contribution, if any, to the inhibitory effect induced by DAMGE. More important is that it does not block the opioid action. Cholera toxin potentiates thymi-dine incorporation but also does not reverse the inhibition of thymidine incorporation by DAMGE, indicating that they are acting via different signaling systems. Cyclic nucleotides, specifically cyclic AMP (Leof et al., 1982; Johnson et al., 1988), are thought to play a mitogenic role. Addition of cholera toxin to 3T3 cells results in increased cyclic AMP levels and stimulates thymidine incorporation (Leof et al.. 1982), as seen in this study. Treatment of T-lymphocytes with isobutylmethylxanthine (an inhibitor of phosphodiesterase) elevated cyclic AMP levels but decreased cell proliferation. These studies suggest that modulation of cyclic AMP levels in both directions may be relevant to the complex proliferative processes that occur in cells. In addition, toxins that act directly on G proteins, i.e., pertussis toxin, can also inhibit cell growth (Murayama and Ui, 1987; Seuwen et al., 1988; Crouch et al., 1990). Nevertheless, the findings reported here indicate that pertussis and cholera toxin-sensitive G protein signaling systems do not participate in the DAMGE inhibition of DNA synthesis. If preceptor opioids act through a G protein-coupled phosphoinositol turnover to modulate thymidine incorporation, most likely the G protein is insensitive to pertussis toxin. The antimitogenicity of Li+ and its role in blocking phosphoinositol turnover have been established (Berridge et al., 1982). Although Li+ was originally adopted as an assay tool because of its ability to inhibit inositol monophosphatases, it is now clear that inositol di- and triphosphatases are also attenuated by this cation (Chuang, 1989). In the present experiments LiCl inhibits thymidine incorporation (Fig. 4A), possibly by acting on an IP phosphatase (Berridge et al., 1982; Chuang, 1989; Cantley et al., 1991). The blockade achieved with low Li+ levels and DAMGE was reversed by naltrexone to control levels. Moreover, the partial reversal of the effect of high Li+ levels by naltrexone in the absence of DAMGE argues for an independent action of this antagonist. A possible explanation for these phenomena is that
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naltrexone blocks a tonic inhibition elicited by endogenous enkephalins in aggregate cultures. The presence of enkephalins in fetal brain cell aggregates has been demonstrated (Simantov and Hollt, 1991). The fact that naltrexone alone slightly potentiated thymidine incorporation (Fig. 1A) is in agreement with this hypothesis. PKC is included among the signal generators that trigger gene activation, leading to DNA synthesis and cell proliferation (Berridge, 1987). Evidence that attenuation of thymidine incorporation entails inhibition of PKC was gained by combining opioids with a PKC inhibitor. Chelerythrine, a recently discovered, selective PKC inhibitor (Herbert et al., 1990), suppressed thymidine incorporation into DNA in a dose-dependent manner. DAMGE did not have an additive effect on the action of chelerythrine on thymidine incorporation. If chelerythrine and DAMGE were acting in a totally independent manner, additive effects of the opioid would have been expected, as seen in the cholera and pertussis toxin experiments. If DAMGE were acting exclusively via PKC, opioid inhibition would not be predicted under conditions of maximal kinase blockade by chelerythrine as seen here. Although other, more complex interpretations of these data are possible, the results observed here are also consistent with a common signaling mechanism.
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Although previous in vivo studies have focused on the growth effects of different but mostly μ-selective opioids (Vertes et al., 1982; Bartolome et al., 1986, 1991; Schmahl et al., 1989), few attempts have been made to examine the mechanism of action of the opioid receptor involved. A reason for this is the myriad of possible mechanisms by which opioids can act and the fact that it is more difficult to implicate a signaling system than to exclude it or to obtain correlative data. For example, one means by which long-term opioid effects may be mediated is through the modulation of G protein expression via opioid receptor down-regulation (Vogel et al., 1990), whereas for short-term effects, Ca2+ channel modulation is possible (Hescheler et al., 1987). The results of this investigation are consistent with the notion of the direct or indirect involvement of the phosphoinositol signal transduction system in mediating μ-opioid inhibition of thymidine incorporation. Because μ-opioid modulation of phosphoinositol turnover has not been reported for adult rat brain, this action may represent a function unique to the neonatal brain. Therefore, the data gained here support our theory that the brain transiently expresses fetal and/or neonatal isoforms of the μ-opioid receptor (Bem et al., 1991).
Acknowledgment This work was supported in part by grant DA 05412 from the National Institute of Drug Abuse.
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Abbreviations used CTOP, cyclic D-Phe-Cys-Tyr-D-Trp-orn-Thr-Pen-Thr amide; DADLE, [D-Ala2,D-Leu5] enkephalin; DAMGE, [D-Ala2,Me-Phe4,Gly-ol5]enkephalin; Gpp(NH)p, 5'guanylylimidodiphosphate; G protein, GTP-binding regulatory protein; IP, inositol phosphate; IP3, inositol trisphosphate; PKC, protein kinase C.
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Johnson KW, Davis BH, Smith KA. cAMP antagonizes interleukin 2-promoted T-cell cycle progression at a discrete point in early G1. Proc. Natl. Acad. Sci. USA 1988;85:6072–6076. [PubMed: 2842759] Kornblum HI, Loughlin SE, Leslie FM. Effects of morphine on DNA synthesis in neonatal rat brain. Dev. Brain Res 1987;31:45–52. Kurose H, Katada T, Amano T, Ui M. Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via α-adrenergic, cholinergic, and opiate receptors in neuroblastoma × glioma hybrid cells. J. Biol. Chem 1983;258:4870–4875. [PubMed: 6300102] Leof EB, Wharton W, O'Keefe E, Pledger WJ. Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1. J. Cell. Biochem 1982;19:93–103. [PubMed: 6181084] Lipton SA, Kater SB. Neurotransmitter regulation of neuronal outgrowth, plasticity and survival. Trends Neurosci 1989;12:265–270. [PubMed: 2475939] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem 1951;193:265–275. [PubMed: 14907713] Mangoura D, Dawson G. Chronic opioid treatment attenuates carbachol-mediated polyphosphoinositide hydrolysis in chick embryo neuronal cultures. Brain Res 1991;548:273–278. [PubMed: 1651142] Miller RJ. The revenge of the kainate receptor. Trends Neurosci 1991;14:477–479. [PubMed: 1726760] Moriarty TM, Padrell E, Carty DJ, Omri G, Lau EM, Lyengar R. Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway. Nature 1990;343:79–42. [PubMed: 2104959] Murayama T, Ui M. Possible involvement of a GTP-binding protein, the substrate of islet-activating protein in receptor-mediated signaling responsible for cell proliferation. J. Biol. Chem 1987;262:12463–12467. [PubMed: 3040750] Nellen W, Datta S, Reymond C, Sivertsen A, Mann S, Crowley T, Firtel RA. Molecular biology in Dictyostelium: tools and applications. Methods Cell Biol 1987;28:67–100. [PubMed: 3600419] Periyasamy S, Hoss W. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain. Life Sci 1990;47:219–225. [PubMed: 1975082] Ross CA, Bredt D, Snyder SH. Messenger molecules in the cerebellum. Trends Neurosci 1990;13:216– 222. [PubMed: 1694327] Roth BL, Laskowski MB, Coscia CJ. Evidence for distinct subcellular sites of opiate receptors: demonstration of opiate receptors in smooth microsomal fractions isolated from rat brain. J. Biol. Chem 1981;256:10117–10121. Schmahl W, Funk R, Miaskowski U, Plendl J. Long-lasting effects of naltrexone, an opioid receptor antagonist, on cell proliferation in developing rat forebrain. Bruin Res 1989;486:297–300. Seuwen K, Magnaldo I, Pouyssegur J. Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT1b receptors coupled to a Gi-protein. Nature 1988;335:254–256. [PubMed: 3045568] Simantov R, Höllt V. Regulation of proenkephalin A gene expression in aggregated fetal rat brain cells. Cell. Mol. Neurobiol 1991;11:245–251. [PubMed: 2029727] Simantov R, Levy R. Plasticity in the phenotypic expression of brain opioid receptors: differential response of forebrain and hindbrain cultures to chemical depolarization. Dev. Brain Res 1986;26:301–307. Stiene-Martin A, Hauser KF. Opioid-dependent growth of glial cultures: suppression of astrocyte DNA synthesis by Met-enkephalin. Life Sci 1990;46:91–98. [PubMed: 2299973] Vertes Z, Melegh G, Vertes M, Kovacs S. Effect of naloxone and d-Met2-Pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain. Life Sci 1982;31:119–126. [PubMed: 7121198] Vogel Z, Barg J, Attali B, Simantov R. Differential effect of μ, δ and κ ligands on G protein α subunits in cultured brain cells. J. Neurosci. Res 1990;27:106–111. [PubMed: 2174976] Xu J, Chuang D-M. Muscarinic acetylcholine receptor-mediated phosphoinositide turnover in cultured cerebellar granule cells: desensitization by receptor agonists. J. Phurrnacol. Exp. Ther 1987;242:238– 244. Zagon IS, McLaughlin PJ. Endogenous opioid systems regulate cell proliferation in the developing rat brain. Brain Res 1987;412:68–72. [PubMed: 3607463]
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FIG. 1.
[3H]Thymidine incorporation into DNA of rat whole-brain cell aggregates. A: Effect of 1 μM DAMGE, morphiceptin (MORPHICEP), CTOP, and naltrexone (NALT) on [3H] thymidine (106 dpm per plate) incorporation into DNA of 7-day brain cell aggregates. CONT, control. B: Dose-dependent DAMGE inhibition of [3H]thymidine incorporation into DNA of 7-day brain cell aggregates. C: Effect of DAMGE on [3H]thymidine incorporation into DNA of rat whole-brain cell aggregates as a function of days in culture. Data are mean ± SE (bars) values from three to five experiments performed in duplicate. *p < 0.05, **p < 0.01, compared with the control.
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FIG. 2.
Preincubation time dependence of DAMGE inhibition of [3H]thymidine incorporation into DNA of rat brain cell aggregates. Cultures were preincubated for the indicated periods with 1 μM DAMGE and washed three times with 40 ml of Eagle's medium. For the last 23 h [3H] thymidine was added, and incorporation was determined for the 7-day culture. Data are mean ± SE (bars) values from three experiments. *p < 0.05 for significance of difference from other time intervals.
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FIG. 3.
[3H]Thymidine incorporation into DNA and non-DNA cell compartments of rat fetal brain cell aggregates. Samples were divided into two aliquots, and [3H]thymidine incorporation into each was determined as described in Materials and Methods. Total [3H]thymidine incorporation into cells was estimated after washing intact aggregates with phosphate-buffered saline (4 × 20 ml). The difference between total incorporation into the cell and the amount incorporated into DNA represents the intracellular thymidine and is referred to as the external pool. Data are mean ± SE (bars) values from three experiments. *p < 0.05 for significance of difference from other treatments.
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FIG. 4.
Modulation of [3H]thymidine incorporation into DNA of rat brain cell aggregates. A Concentration-dependent effects of LiCl or pertussis toxin on DAMGE (1 μM) inhibition of [3H]thymidine (106 dpm per plate) incorporation into DNA of whole-brain cell aggregates. Cultures were exposed to opioids, toxins, or LiCl 48 h before harvesting and to [3H]thymidine for the final 23 h and analyzed after 7 days of culture. B: Dose-dependent naltrexone reversal of the inhibition of [3H]thymidine incorporation into DNA by 10 mM LiCl, 1 μM DAMGE in the presence of 0.5 mM LiCl, or 5 ng/ml of pertussis toxin in 7-day whole-brain cell aggregates. C: Dose-dependent cholera toxin potentiation of [3H]thymidine incorporation into DNA of control or DAMGE (1 μM)-treated 7-day whole-brain cell aggregates. Data are mean ± SE J Neurochem. Author manuscript; available in PMC 2008 October 23.
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(bars) values from three to seven experiments. *p < 0.05, **p < 0.01 for significance of difference from the controls (cells untreated by the agent listed in the abscissa of each panel).
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FIG. 5.
Concentration dependence of Gpp(NH)p inhibition of opioid agonist and partial agonist specific binding to opioid receptors in rat brain cell aggregates. Membranes from pertussis toxin-treated (5 ng/ml, 48 h) or control cells were incubated with 1 nM [3H]diprenorphine or [3H]etorphine in the presence or absence of indicated concentrations of Gpp(NH)p. Specific binding of cells untreated with Gpp(NH)p was > 1,300 dpm per tube. Data are mean ± SE (bars) values from three experiments. *p < 0.05, **p < 0.01 for significance of difference from the pertussis toxin-treated aggregates.
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FIG. 6.
Effect of the PKC inhibitor chelerythrine and DAMGE (1 μM) on [3H]thymidine incorporation into DNA in 7-day brain cell aggregates. Aggregates were exposed to chelerythrine and/or opioid 48 h before harvesting and to [3H]thymidine for the final 23 h of 7-day cultures. Data are mean ± SEM (bars) values from three to six experiments. *p < 0.05 for significance of difference from cultures treated only with chelerythrine (control). #p < 0.05 for significance of difference for cultures not treated with chelerythrine.
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FIG. 7.
Effect of opioid agonist and antagonist on [3H]IP3 accumulation in whole-brain cell aggregates. A: Effect of DAMGE and/or naltrexone (NALT; 1 μM) on [3H]IP3 accumulation in 7-day cultures. B: Effect of DAMGE and/or naltrexone (1 μM) on [3H]IP3 accumulation in 10-day cultures. C: Effect of DAMGE and/or naltrexone (1 μM) on [3H]IP3 accumulation in 14-day rat fetal brain cell aggregates. Data are mean ± SE (bars) values from three to seven experiments. *p < 0.05 for difference from control (CONT).
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Effect of opioid agonist and antagonist on glutamate-promoted (A) [3H]inositol monophosphate ([3H]IP2) and (B) [3H]-inositol bisphosphate ([3H]IP2) accumulation in whole-brain cell aggregates. Aggregates (7 days) were preincubated with 1 μM DAMGE with or without 1 μM naltrexone for 0−60 min and then treated with 40 μM glutamate for 0−60 (A) or 20 (B) min. The cells were harvested, and 3H-IP accumulation was measured as described in Materials and Methods. Data are mean ± SE (bars) values from three to seven experiments. Zero-time values are significantly different from those of all other time points: *p < 0.05. Values for glutamate-stimulated IP accumulation in the presence of DAMGE are significantly different from those obtained in its absence: *p < 0.05.
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Published in final edited form as: J Neurochem. 1992 September ; 59(3): 1145–1152.
Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis Jacob Barg*, Mariana M. Belcheva†, and Carmine J. Coscia† †E. A. Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A. * Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
Abstract
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An opioid receptor agonist, [D-Ala2,Me-Phe4.Gly-ol5]enkephalin (DAMGE), decreased [3H] thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the μ-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-PenThr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The μ-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly μ-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis. whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H] inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.
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Keywords Opioid receptors; Opioid peptides; Fetal rat brain; Cell aggregates; DNA synthesis; Phosphoinositol Maternal drug abuse can have a long-term, complex impact on embryonic brain ontogeny. Minimally, developmental delays are seen for many drugs, including opiates, and the nature of the insult may depend on the state of maturation of susceptible neuronal and glial cells. A body of evidence originating from both in vivo (Lipton and Kater, 1989; Miller, 1991) and in vitro (Ashkenazi et al., 1989: Eccleston et al., 1989) studies supports the notion that neurotransmitters can modulate cell growth during development. Opioids have been shown to decrease thymidine incorporation into DNA transiently and in a naloxone-reversible manner in developing rat brain (Bartolome et al., 1986, 1991; Kornblum et al., 1987; Zagon and McLaughlin, 1987) and in vitro (Coscia et al., 1991; Davila-Garcia and Azmitia, 1989; StieneMartin and Hauser, 1990). Moreover, opioid antagonists have been reported to increase cell
Address correspondence and reprint requests to Dr. C. J. Coscia at Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine. St. Louis. MO 63104−1079. U.S.A..
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proliferation and modulate brain development in vivo (Vertes et al., 1982; Zagon and McLaughlin, 1987; Schmahl et al., 1989). The mechanisms by which opioids may elicit antimitogenic effects is unknown and is the subject of this report. Here we present evidence for the involvement of the inositol lipid signal transduction system in this process.
MATERIALS AND METHODS Thymidine incorporation
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Cell aggregates were prepared from embryonic day 15 Sprague-Dawley rat brain as described (Simantov and Levy, 1986: Barg et al.. 1989b) and grown for various intervals in a chemically defined medium. The animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Cultures were treated with opioid–[D-Ala2,Me-Phe4,Gly-ol5]enkephalin (DAMGE). morphiceptin, etorphine. U69593. [D-Ala2,D-Leu5]-enkephalin (DADLE). cyclic D-Phe-CysTyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP), or naltrexone–at 1 μM 48 h before harvesting and with [3H]thymidine (106 dpm per plate, 25.5 Ci/mmol; Amersham. Arlington Heights, IL, U.S.A.) for the final 23 h. In all experiments the interval indicated represents the total number of days that the brain cell aggregates were maintained. [3H]Thymidine incorporation into cells was determined by filtering cultures on Whatman GF/B filters (diameter, 25 mm), washing with phosphate-buffered saline (20 ml four times each), and measuring the radioactivity on the filter. Thymidine incorporated into DNA was determined by washing aggregates with 10% trichloroacetic acid, 5% trichloroacetic acid, ethanol/ether (1:1 vol/vol), and ether as described (Barg et al.. 1989a). The difference between the two determinations represents [3H]-thymidine incorporated into cells that are of non-DNA origin (external pool). Thymidine incorporation is expressed as femtomoles per seeded cells, i.e., the number of cells that were present in the beginning of the experiments. In all instances, when the same radioactivity data were determined on the basis of protein content (measured on cell harvesting) instead of seeded cells, comparable results were obtained. Protein content, determined by the method of Lowry et al. (1951), was unchanged by the treatment. Inhibitor and activator experiments
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Cultures were treated with pertussis and cholera toxins (List Biological Laboratories. Campbell. CA. U.S.A.), the benzophenanthridine alkaloid chelerythrine (Vipont Laboratories, Fort Collins, CO, U.S.A.). or LiCl in the presence or absence of varying concentrations of naltrexone and I μM DAMGE 48 h before harvesting and with [3H]-thymidine (106 dpm per plate) for the final 23 h. In addition, cultures were exposed to 1 μM DAMGE for varying intervals, and the opioid agonist was removed by washing the aggregates three times with 40 ml of Eagle's medium, whereupon [3H]thymidine was added for the final 23 h of the 7-day culture. Phosphatidylinositol turnover measurements myo-[3H]Inositol (2.5 μCi/ml, 15.5 Ci/mmol; NEN Dupont, Boston, MA, U.S.A.) was added to the cultures 18 h before harvesting. 3H-Inositol phosphate (3H-IP) analyses were performed following the experimental procedure described by Xu and Chuang (1987). Opioid binding in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p] Brain cell aggregates were treated with pertussis toxin (5 ng/ml) for 48 h. Opioid binding assays with [3H]-diprenorphine (31 Ci/mmol) or [3H]etorphine (44 Ci/mmol) were performed in the presence of varying concentrations of Gpp(NH)p, as previously described (Roth et al.. 1981; Barg et al., 1991). Statistical analyses of the data were performed with Student's t test.
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The effect of DAMGE on [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates grown for 7 days is shown in Fig. IA. DAMGE decreased [3H]thymidine incorporation by 40%, and the attenuation was reversed by naltrexone. At the concentrations of DAMGE used, its specificity for μ receptors is marginal (Goldstein and Naidu, 1989). Therefore, more μ-selective agonist and antagonists were tested (Fig. 1A). Morphiceptin blocks DNA synthesis at 1 μM, a concentration at which it retains its μ-receptor specificity. Moreover, the μ-selective antagonist CTOP reversed the effect of both rnorphiceptin and DAMGE. Dosedependence studies indicated that DAMGE inhibited DNA synthesis with an IC50 of 0.376 ± 0.057 μM (Fig. 1 B). Similarly, the opioid agonist etorphine and the κ-selective U69593, both at 1 μM, decreased thymidine incorporation by 35 and 39%. respectively (data not shown). In contrast, DADLE had no significant effect on [3H]thymidine incorporation (data not shown).
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The action of DAMGE proved to depend on the interval that aggregates were maintained in culture, with the greatest inhibition observed in 7-day aggregates (Fig. 1C). DAMGEdependent attenuation of [3H]thymidine incorporation was not observed in ≥ 12-day cultures despite the fact that DNA synthesis could be detected for 21 days and the cells remained viable for 45 days. In addition, exposure of aggregates to DAMGE for an interval of > 1.5 h was sufficient to decrease thymidine incorporation significantly (Fig. 2). Because maximal attenuation was observed at 48 h (Fig. 1A), subsequent studies focused for the most part on long-term effects. Control studies were performed to determine whether opioids influence the amount of radiolabel incorporated into DNA by altering the size of the intra- or extracellular thymidine pool. On isolation of DNA (Nellen et al., 1987) and assessment of its radioactivity (data not shown), a comparable decrease in thymidine incorporation was observed, corroborating the results in Fig. 1A. Moreover, thymidine uptake from the medium was unchanged. Because thymidine uptake is regulated by a permease (Cleaver, 1967), it could be influenced by the pool size of this pyrimidine nucleoside. Therefore, these last two results and the determination of the non-DNA intracellular label (external pool) of [3H]thymidine in all treatments (Fig. 3) tend to exclude the possibility that the opioid effect originates from a change in the extra- and/ or intracellular thymidine pool.
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Most opioid receptor agonists activate the pertussis toxin-sensitive GTP-binding regulatory proteins (G proteins) Gi, and Go (Burns et al., 1983; Kurose et al., 1983; Hescheler et al., 1987). However, we discovered that the DAMGE inhibitory action was not abolished by pertussis toxin (Fig. 4A). Pertussis toxin decreased thymidine incorporation in a concentrationdependent manner, and the inhibition appeared to be additive in the presence of DAMGE at most toxin concentrations. LiCl, an inhibitor of IP phosphatases (Berridge et al.. 1982). also diminished thymidine incorporation dose-dependently, and inclusion of DAMGE elicited an almost maximal inhibition at ≥5 mM LiCl (Fig. 4A). Naltrexone reversal of DAMGE action was effected in the presence of LiCl or pertussis toxin (Fig. 4B). When 1 μM DAMGE was combined with 0.5 mM LiCl, the inhibition (69%) was intermediate between that of I μM DAMGE alone (30 −40%) and that in the presence of 5 mM LiCl (88%), as seen in Fig. 4A. Naltrexone blocked the effect of DAMGE in the presence of 0.5 mM LiCl, restoring the rate of thymidine incorporation to that exhibited by untreated cultures. Moreover, naltrexone partially offset the 10 mM LiCl effects (Fig. 4B). These results raise the possibility that this antagonist blocks the actions ofendogenous opioid peptides (Simantov and Höllt, 1991). A partial reversal of thymidine incorporation by naltrexone (from 78 to 160 fmol per seeded cells) was evident in brain cell aggregates treated with pertussis toxin and DAMGE.
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The possible involvement of Gs in the inhibition induced by DAMGE was tested using cholera toxin, which increases the activity of this stimulatory G protein. In contrast to pertussis, cholera toxin (100 ng/ml) slightly enhanced thymidine incorporation in control and DAMGE-treated cultures (Fig. 4C). The same opioid inhibitory effect at all toxin concentrations indicates that its attenuation of thymidine incorporation is mediated predominantly by a mechanism that does not involve Gs.
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Because pertussis toxin did not block opioid receptor-mediated inhibition of thymidine incorporation, it was important to ascertain that this toxin had uncoupled some opioid receptors from sensitive G proteins under the conditions of the experiment. One method of testing receptor coupling to G protein is to measure agonist binding in the presence of GTP analogs. Gpp(NH)p and other analogs will disrupt the ligand-receptor-G protein complex and thereby reduce overall binding. The data in Fig. 5 substantiate this by showing that the binding of the agonist etorphine to control membranes was inhibited by a GTP analog, but binding to membranes from pertussis toxin-treated cultures was unaffected. Binding of the partial agonist [3H]diprenorphine was not influenced in either experiment. We chose etorphine and diprenorphine, which have a high affinity for μ-, δ-, and κ-opioid receptors, because at this stage in development there may be fewer μ-opioid receptors coupled to pertussis toxin-sensitive G proteins than in adult brain. The decreased sensitivity of agonist binding in the presence of GTP analog implies that some G proteins were uncoupled from opioid receptors by pertussis toxin under the conditions adopted here (Hsia et al., 1984). The question of whether protein kinase C (PKC) is involved in opioid agonist-mediated inhibition of thymidine incorporation was addressed by including a PKC inhibitor along with a μ-receptor agonist. Chelerythrine, which is selective for PKC (Herbert et al., 1990), decreased thymidine incorporation into DNA in 7-day brain cell aggregates dose-dependently (Fig. 6). When added together with chelerythrine, DAMGE failed to alter thymidine incorporation except at the 10−7 M level of the PKC inhibitor. Moreover, the effect of DAMGE was not additive, which is consistent with a common mechanism of DNA synthesis blockade.
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To implicate the inositol lipid signal transduction system further, the effect of DAMGE on phosphoinositol turnover was studied in 7-, 10-, and 14-day brain cell aggregates. Untreated cultures generated a basal level of [3H]inositol trisphosphate ([3H]IP3), reflecting the composite of stimulatory and inhibitory activities of endogenous neurotransmitters that operate via this system. DAMGE decreased the formation of [3H]IP3 in 7- and 10-day brain cell aggregates by 48 and 36%, respectively (Fig. 7A and B). The decline in [3H]IP3 content was naltrexone reversible. In 14-day cultures neither DAMGE nor naltrexone had a significant effect on IP3 turnover (Fig. 7C). DAMGE also failed to inhibit DNA synthesis in cultures of this age (Fig. 1C). A glutamate-stimulated accumulation of [3H]-inositol monophosphate and [3H]inositol bisphosphate was also detected in 7-day aggregates (Fig. 8). Short-term treatment of the glutamate-stimulated aggregates with DAMGE reversed the effect in these experiments. Naltrexone blocked the opioid action. Long-term DAMGE exposure (48 h) also resulted in the inhibition of glutamate-stimulated IP turnover (data not shown). The IC50, for long-term DAMGE blockade of IP turnover was 0.1 18 ± 0.0 16 μM which is comparable in potency to its inhibitory effect on thymidine incorporation.
DISCUSSION These findings confirm that (a) DAMGE inhibits thymidine incorporation into DNA in a concentration- and culture age-dependent manner; (b) as shown by the comparable action of morphiceptin, and its reversibility by CTOP, this effect of DAMGE is most likely mediated
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via the μ-opioid receptor; (c) DAMGE does not appear to use cholera or pertussis toxinsensitive G protein as the transducer in this process; and (d) DAMGE inhibits basal [3H]IP3 formation and glutamate-stimulated [3H]inositol mono-phosphate and [3H]inositol bisphosphate accumulation by a mechanism that is reversed by naltrexone. In addition, DAMGE inhibits DNA synthesis and IP turnover with comparable potency, and the two effects are temporally associated. There is precedence for opioid receptor utilization of phosphoinositol as a second messenger. Recently, κ-opioid stimulation of phosphoinositide turnover in slices of discrete regions of adult rat brain has been reported (Periyasamy and Hoss, 1990). It is also possible that the opioid receptor may interact with another receptor that transmits the signal by the phosphoinositol system (Ross et al., 1990). For example, although direct inhibition of phosphatidylinositol turnover was not demonstrable in cultured neurons from chick embryo cerebral hemispheres, both carbachol- and bradykinin-induced stimulation of phosphoinositol release was blocked by chronic exposure to opioids (Mangoura and Dawson, 1991). There is also recent electrophysiological evidence that a μ-opioid agonist modulates the N-methyl-D-aspartate receptor-mediated glutamate response via PKC in dorsal horn neurons (Chen and Huang, 1991). Finally, the N-methyl-D-aspartate receptor has been implicated as an important organizational cue of neuronal pathways, and Ca2+ transport is involved in the process (Miller, 1991).
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It has been known for some time that like muscarinic receptors, certain growth factors activate phosphoinositide-specific phospholipase C via a mechanism utilizing G protein (Chuang, 1989). This action can be abolished by pertussis toxin (Seuwen et al., 1988; Moriarty et al., 1990). However, not all G protein-coupled phospholipase C systems are blocked by pertussis toxin (Birnbaumer, 1990). Here we show that inactivation of Gi by pertussis toxin decreases thymidine incorporation, but it makes only a minor contribution, if any, to the inhibitory effect induced by DAMGE. More important is that it does not block the opioid action. Cholera toxin potentiates thymi-dine incorporation but also does not reverse the inhibition of thymidine incorporation by DAMGE, indicating that they are acting via different signaling systems. Cyclic nucleotides, specifically cyclic AMP (Leof et al., 1982; Johnson et al., 1988), are thought to play a mitogenic role. Addition of cholera toxin to 3T3 cells results in increased cyclic AMP levels and stimulates thymidine incorporation (Leof et al.. 1982), as seen in this study. Treatment of T-lymphocytes with isobutylmethylxanthine (an inhibitor of phosphodiesterase) elevated cyclic AMP levels but decreased cell proliferation. These studies suggest that modulation of cyclic AMP levels in both directions may be relevant to the complex proliferative processes that occur in cells. In addition, toxins that act directly on G proteins, i.e., pertussis toxin, can also inhibit cell growth (Murayama and Ui, 1987; Seuwen et al., 1988; Crouch et al., 1990). Nevertheless, the findings reported here indicate that pertussis and cholera toxin-sensitive G protein signaling systems do not participate in the DAMGE inhibition of DNA synthesis. If preceptor opioids act through a G protein-coupled phosphoinositol turnover to modulate thymidine incorporation, most likely the G protein is insensitive to pertussis toxin. The antimitogenicity of Li+ and its role in blocking phosphoinositol turnover have been established (Berridge et al., 1982). Although Li+ was originally adopted as an assay tool because of its ability to inhibit inositol monophosphatases, it is now clear that inositol di- and triphosphatases are also attenuated by this cation (Chuang, 1989). In the present experiments LiCl inhibits thymidine incorporation (Fig. 4A), possibly by acting on an IP phosphatase (Berridge et al., 1982; Chuang, 1989; Cantley et al., 1991). The blockade achieved with low Li+ levels and DAMGE was reversed by naltrexone to control levels. Moreover, the partial reversal of the effect of high Li+ levels by naltrexone in the absence of DAMGE argues for an independent action of this antagonist. A possible explanation for these phenomena is that
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naltrexone blocks a tonic inhibition elicited by endogenous enkephalins in aggregate cultures. The presence of enkephalins in fetal brain cell aggregates has been demonstrated (Simantov and Hollt, 1991). The fact that naltrexone alone slightly potentiated thymidine incorporation (Fig. 1A) is in agreement with this hypothesis. PKC is included among the signal generators that trigger gene activation, leading to DNA synthesis and cell proliferation (Berridge, 1987). Evidence that attenuation of thymidine incorporation entails inhibition of PKC was gained by combining opioids with a PKC inhibitor. Chelerythrine, a recently discovered, selective PKC inhibitor (Herbert et al., 1990), suppressed thymidine incorporation into DNA in a dose-dependent manner. DAMGE did not have an additive effect on the action of chelerythrine on thymidine incorporation. If chelerythrine and DAMGE were acting in a totally independent manner, additive effects of the opioid would have been expected, as seen in the cholera and pertussis toxin experiments. If DAMGE were acting exclusively via PKC, opioid inhibition would not be predicted under conditions of maximal kinase blockade by chelerythrine as seen here. Although other, more complex interpretations of these data are possible, the results observed here are also consistent with a common signaling mechanism.
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Although previous in vivo studies have focused on the growth effects of different but mostly μ-selective opioids (Vertes et al., 1982; Bartolome et al., 1986, 1991; Schmahl et al., 1989), few attempts have been made to examine the mechanism of action of the opioid receptor involved. A reason for this is the myriad of possible mechanisms by which opioids can act and the fact that it is more difficult to implicate a signaling system than to exclude it or to obtain correlative data. For example, one means by which long-term opioid effects may be mediated is through the modulation of G protein expression via opioid receptor down-regulation (Vogel et al., 1990), whereas for short-term effects, Ca2+ channel modulation is possible (Hescheler et al., 1987). The results of this investigation are consistent with the notion of the direct or indirect involvement of the phosphoinositol signal transduction system in mediating μ-opioid inhibition of thymidine incorporation. Because μ-opioid modulation of phosphoinositol turnover has not been reported for adult rat brain, this action may represent a function unique to the neonatal brain. Therefore, the data gained here support our theory that the brain transiently expresses fetal and/or neonatal isoforms of the μ-opioid receptor (Bem et al., 1991).
Acknowledgment This work was supported in part by grant DA 05412 from the National Institute of Drug Abuse.
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Abbreviations used CTOP, cyclic D-Phe-Cys-Tyr-D-Trp-orn-Thr-Pen-Thr amide; DADLE, [D-Ala2,D-Leu5] enkephalin; DAMGE, [D-Ala2,Me-Phe4,Gly-ol5]enkephalin; Gpp(NH)p, 5'guanylylimidodiphosphate; G protein, GTP-binding regulatory protein; IP, inositol phosphate; IP3, inositol trisphosphate; PKC, protein kinase C.
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FIG. 1.
[3H]Thymidine incorporation into DNA of rat whole-brain cell aggregates. A: Effect of 1 μM DAMGE, morphiceptin (MORPHICEP), CTOP, and naltrexone (NALT) on [3H] thymidine (106 dpm per plate) incorporation into DNA of 7-day brain cell aggregates. CONT, control. B: Dose-dependent DAMGE inhibition of [3H]thymidine incorporation into DNA of 7-day brain cell aggregates. C: Effect of DAMGE on [3H]thymidine incorporation into DNA of rat whole-brain cell aggregates as a function of days in culture. Data are mean ± SE (bars) values from three to five experiments performed in duplicate. *p < 0.05, **p < 0.01, compared with the control.
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FIG. 2.
Preincubation time dependence of DAMGE inhibition of [3H]thymidine incorporation into DNA of rat brain cell aggregates. Cultures were preincubated for the indicated periods with 1 μM DAMGE and washed three times with 40 ml of Eagle's medium. For the last 23 h [3H] thymidine was added, and incorporation was determined for the 7-day culture. Data are mean ± SE (bars) values from three experiments. *p < 0.05 for significance of difference from other time intervals.
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FIG. 3.
[3H]Thymidine incorporation into DNA and non-DNA cell compartments of rat fetal brain cell aggregates. Samples were divided into two aliquots, and [3H]thymidine incorporation into each was determined as described in Materials and Methods. Total [3H]thymidine incorporation into cells was estimated after washing intact aggregates with phosphate-buffered saline (4 × 20 ml). The difference between total incorporation into the cell and the amount incorporated into DNA represents the intracellular thymidine and is referred to as the external pool. Data are mean ± SE (bars) values from three experiments. *p < 0.05 for significance of difference from other treatments.
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FIG. 4.
Modulation of [3H]thymidine incorporation into DNA of rat brain cell aggregates. A Concentration-dependent effects of LiCl or pertussis toxin on DAMGE (1 μM) inhibition of [3H]thymidine (106 dpm per plate) incorporation into DNA of whole-brain cell aggregates. Cultures were exposed to opioids, toxins, or LiCl 48 h before harvesting and to [3H]thymidine for the final 23 h and analyzed after 7 days of culture. B: Dose-dependent naltrexone reversal of the inhibition of [3H]thymidine incorporation into DNA by 10 mM LiCl, 1 μM DAMGE in the presence of 0.5 mM LiCl, or 5 ng/ml of pertussis toxin in 7-day whole-brain cell aggregates. C: Dose-dependent cholera toxin potentiation of [3H]thymidine incorporation into DNA of control or DAMGE (1 μM)-treated 7-day whole-brain cell aggregates. Data are mean ± SE J Neurochem. Author manuscript; available in PMC 2008 October 23.
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(bars) values from three to seven experiments. *p < 0.05, **p < 0.01 for significance of difference from the controls (cells untreated by the agent listed in the abscissa of each panel).
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FIG. 5.
Concentration dependence of Gpp(NH)p inhibition of opioid agonist and partial agonist specific binding to opioid receptors in rat brain cell aggregates. Membranes from pertussis toxin-treated (5 ng/ml, 48 h) or control cells were incubated with 1 nM [3H]diprenorphine or [3H]etorphine in the presence or absence of indicated concentrations of Gpp(NH)p. Specific binding of cells untreated with Gpp(NH)p was > 1,300 dpm per tube. Data are mean ± SE (bars) values from three experiments. *p < 0.05, **p < 0.01 for significance of difference from the pertussis toxin-treated aggregates.
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FIG. 6.
Effect of the PKC inhibitor chelerythrine and DAMGE (1 μM) on [3H]thymidine incorporation into DNA in 7-day brain cell aggregates. Aggregates were exposed to chelerythrine and/or opioid 48 h before harvesting and to [3H]thymidine for the final 23 h of 7-day cultures. Data are mean ± SEM (bars) values from three to six experiments. *p < 0.05 for significance of difference from cultures treated only with chelerythrine (control). #p < 0.05 for significance of difference for cultures not treated with chelerythrine.
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FIG. 7.
Effect of opioid agonist and antagonist on [3H]IP3 accumulation in whole-brain cell aggregates. A: Effect of DAMGE and/or naltrexone (NALT; 1 μM) on [3H]IP3 accumulation in 7-day cultures. B: Effect of DAMGE and/or naltrexone (1 μM) on [3H]IP3 accumulation in 10-day cultures. C: Effect of DAMGE and/or naltrexone (1 μM) on [3H]IP3 accumulation in 14-day rat fetal brain cell aggregates. Data are mean ± SE (bars) values from three to seven experiments. *p < 0.05 for difference from control (CONT).
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Effect of opioid agonist and antagonist on glutamate-promoted (A) [3H]inositol monophosphate ([3H]IP2) and (B) [3H]-inositol bisphosphate ([3H]IP2) accumulation in whole-brain cell aggregates. Aggregates (7 days) were preincubated with 1 μM DAMGE with or without 1 μM naltrexone for 0−60 min and then treated with 40 μM glutamate for 0−60 (A) or 20 (B) min. The cells were harvested, and 3H-IP accumulation was measured as described in Materials and Methods. Data are mean ± SE (bars) values from three to seven experiments. Zero-time values are significantly different from those of all other time points: *p < 0.05. Values for glutamate-stimulated IP accumulation in the presence of DAMGE are significantly different from those obtained in its absence: *p < 0.05.
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