Search

As embryonic stem cell-derived neural progenitors (NPs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of functional mu opioid receptors (MOR) and kappa opioid receptors (KOR) in mouse embryonic stem cells and NPs. Here, MOR and KOR immunoreactivity was detected in NP-derived oligodendrocytes during three stages of their maturation in vitro. Moreover, we examined the modulation of retinoic acid-induced NP differentiation to astrocytes and neurons by mu, [D-ala(2), mephe(4), gly-ol(5)] enkephalin, or kappa, U69, 593, opioids. Both opioid agonists inhibited NP-derived neurogenesis and astrogenesis via their corresponding receptors as determined by immunocytochemistry. By administering selective inhibitors, we found that opioid inhibition of NP-derived astrogenesis was driven via extracellular-signal regulated kinase (ERK), while the p38 mitogen-activated protein kinase pathway was implicated in opioid attenuation of neurogenesis. In addition, mu and kappa opioids stimulated oligodendrogenesis from NP-derived NG2(+) oligodendrocyte progenitors via both ERK and p38 signaling pathways. Accordingly, both opioids induced ERK phosphorylation in NG2(+) cells. These results indicate that small molecules, such as MOR and KOR agonists may play a modulatory role in NP terminal differentiation.

GTP binding regulatory protein (G protein)-coupled receptors can activate MAPK pathways via G protein-dependent and -independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and beta-arrestin 2 pathways in kappa opioid receptor-induced, extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non-nitrogenous agonist, C(2)-methoxymethyl salvinorin B (MOM-Sal-B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gbetagamma subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of beta-arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM-Sal-B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM-Sal-B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gbetagamma subunits or beta-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome.

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.

Endorphins, enkephalins, and synthetic or naturally occurring exogenous opiates have been shown to affect the proliferation of neural cells (for reviews, see McDowell and Kitchen, 1987; Hammer and Hauser, 1992; Zagon and Slotkin, 1992; Leslie, 1993). Despite their presence at early stages of brain development (Rius et al., 1991), little is known about the actions of dynorphins during this period. During gestation, dynorphin immunoreactivity rises to 25 fmol/brain, but a 100-fold increase is then seen in the subsequent postnatal period wherein glial cell proliferation is predominant. Fetal brain cell aggregates represent a useful in vitro model for the study of neural cell development, providing the possibility of probing either proliferating or differentiating neuronal and glial cells (Guentert-Lauber and Honegger, 1985; Guentert-Lauber et al., 1985). These cultures can be maintained for more than a year (Choi et al., 1993) and neural cells exhibit a developmental pattern similar to brain in vivo, with regard to opioid and other receptor systems (Vogel et al., 1976; Barg et al., 1989; Guentert-Lauber and Honegger, 1985). The aggregate system was shown to be a suitable model to explore the role of opioid agonists in the modulation of neural cell proliferation and differentiation (Barg et al., 1992a, 1993a,c). Recently, we have shown that both κ- and μ-opioid receptors affect DNA synthesis modulation elicited by synthetic opiates and opioid peptide analogues (Barg et al., 1992a, 1993a,c). Moreover, β-endorphin proved to be a potent inhibitor of [3H]thymidine incorporation into DNA of 7-day fetal rat brain cell aggregates (IC50 0.7 nM). The action of this endogenous opioid peptide was reversed partially by individual κ- and μ-antagonists (Barg et al., 1993c). Complete blockade of the β-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Such studies suggested the involvement of a discrete κ-subtype of opioid receptor that has affinity for β-endorphin (Barg et al., 1993c). Synthetic κ-agonists {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and {"type":"entrez-nucleotide","attrs":{"text":"U50488","term_id":"1277101","term_text":"U50488"}}U50488 at 1 μM concentrations displayed a similar inhibition in 7-day cultures of rat brain cell aggregates (Barg et al., 1993a). Moreover, in experiments with 21-day brain cell aggregates (Barg et al., 1993a) or spinal cord cultures (Barg et al., 1993b), stimulation of DNA synthesis could be observed that was also blocked by the specific κ-selective antagonist norbinaltorphimine (nor-BNI). The dichotomic neurotrophic effects of opioids appear to be associated with differences in the developmental stage of the aggregates. Among the many questions to be addressed with regard to this process is one on the role of the dynorphins in neurotrophic activities in the developing brain. In this study dynorphins were examined for their ability to modulate [3H]thymidine incorporation into DNA of fetal brain cell aggregates prepared from rat or guinea pig. Because rat brain aggregates express >50% of their total opioid receptor population as μ, whereas guinea pig aggregates synthesize more κ- than μ- or δ-receptors, it was also of interest to investigate neurotrophic effects of endogenous κ-selective opioid peptides, the dynorphins, on DNA synthesis in the guinea pig system.

Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for delta opioid binding using [3H][D-Ala2,D-Leu5]enkephalin and for sigma 1 and sigma 2 binding with 1,3-[3H]di-o-tolylguanidine in the presence and absence of 1 microM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and sigma Bmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/sigma ligands were similar in vitro and in vivo. With successive passages in the murine host, delta opioid and sigma 1 binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, sigma 2 receptor Bmax values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of delta opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for sigma binding was more complex, with the sigma 2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of sigma sites in transformed human meninges, kidney, and colon tissue.

Opioid receptors (ORs) are members of the heptahelical, G protein-coupled receptor (GPCR) family that are targets of endogenous opioid peptides as well as opiate alkaloids, such as morphine. Opioid effects on neural development primarily entail suppression of neural cell proliferation as seen in studies performed in vivo (Johannesson et al., 1972; Vertes et al., 1982; Zagon and McLaughlin, 1983, 1991; Kornblum et al., 1987; Bartolome et al., 1991) and in primary cultures from embryonic brain (Barg et al., 1992, 1993a,b; for review, see Hammer and Hauser, 1992). The effects of opioids on glial cells have proven diverse but often also result in the inhibition of proliferation (Stiene-Martin and Hauser, 1991; Barg et al., 1993a; Gurwell et al., 1996; Hauser et al., 1996). The rat C6 glioma cell line expresses κ-ORs and low levels of a novel type of μ-OR that has been demonstrated by radioligand binding (Albouz et al., 1982; Reggiani et al., 1987; Barg et al., 1991; Bohn et al., 1998), RT-PCR (Bohn et al., 1998), and RNase protection assays (L.M.B., unpublished data). The C6 cells have been used as a model system to study opioid inhibition of mitogen-stimulated DNA synthesis (Barg et al., 1994). Endothelin-1 stimulates C6 cell proliferation that correlates with increases in DNA synthesis, phosphoinositide (PI) turnover, extracellular signal-regulated kinase (ERK) activity, and Ca2+ influx (MacCumber et al., 1990; Chuang et al., 1991; Couraud et al., 1991; Lin et al., 1992; Barg et al., 1994; Lazarini et al., 1996) similar to the extent of stimulation obtained with the κ-opioid agonist as reported in our preceding article (Bohn et al., 2000). The μ-OR agonists morphine, β-endorphin, and a morphine-mimicking, antiidiotypic antibody (Coscia et al., 1991) inhibit PI turnover, Ca2+ influx, and growth of endothelin-stimulated C6 cells (Barg et al., 1994). In our preceding article we reported that activation of endogenous κ-ORs stimulates C6 cell DNA synthesis by a mechanism that also involves activation of PI turnover and ERK1/2 phosphorylation (Bohn et al., 2000). This raises the question of whether the highly selective, endogenous μ-OR agonist endomorphin-1 (Zadina et al., 1997) attenuates κ-OR-mediated stimulation of DNA synthesis, PI turnover, and ERK1/2 phosphorylation in C6 cells. Internalization of GPCRs may represent a prerequisite event in initiating mitogenic signaling (for review, see Lefkowitz, 1998). Dynamin mediates clathrin-dependent internalization of many receptors by participating in the “pinching off” of endocytotic vesicles from the plasma membrane (Hinshaw and Schmid, 1995; Takei et al., 1995; Wang et al., 1995). A mutation in the dynamin molecule (K44A or K44E) rendering it GTPase-defective creates a dominant suppressor mutant that blocks epidermal growth factor receptor and GPCR endocytosis (Vieira et al., 1996; Zhang et al., 1996; Chu et al., 1997; Daaka et al., 1998). Such suppressors of receptor endocytosis can attenuate GPCR-mediated activation of ERK without interfering with classical second messenger [cyclic AMP, inositol phosphates (IPs), and Ca2+] production (Luttrell et al., 1997a; Daaka et al., 1998; Ignatova et al., 1999). In addition, ERK activation by β2-adrenergic receptors expressed in human embryonic kidney (HEK) 293 cells (Daaka et al., 1998), LPA receptors expressed in Rat-1 cells (Luttrell et al., 1997a), and μ-, κ-, and δ-ORs expressed in HEK293 or COS-7 cells (Ignatova et al., 1999) requires functional dynamin. In this article we show that μ-OR agonists inhibit not only endothelin-stimulated DNA synthesis, but also the κ-OR-mediated stimulation of DNA synthesis, PI turnover, and ERK phosphorylation. Moreover, κ-opioid agonist phosphorylation of ERK is unchanged in C6 cells on overexpression of dynamin K44A, whereas μ-opioid agonist inhibition of κ-opioid mitogenic signaling is attenuated.

An opioid receptor agonist, [D-Ala2, Me-Phe4, Glyol5]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-Pen-Thr 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 in hibited 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.

Although micro, kappa, and delta opioids activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which micro opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The micro-opioid agonists ([d-ala(2), mephe(4), gly-ol(5)] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G(i/o) protein, calmodulin (CaM), and beta-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the micro-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of beta-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of micro-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and micro-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and beta-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala(2),mephe(4),gly-ol(5)] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5'-bromo-2'-deoxy-uridine labeling). PTX and beta-arrestin2 siRNA but not W-7 reversed the micro-opioid inhibition. Unexpectedly, beta-arrestin-2 siRNA diminished both EGF-induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different micro-opioid signaling pathways to ERK and their cellular responses.

Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.

Substantial evidence exists to support the notion that maternal drug abuse elicits, in addition to fetal growth delay, physiological and behavioral deficits in offspring (McDowell and Kitchen, 1987; Hayford et al., 1988; Farrar and Blumer, 1991). Among the drugs abused are opioid agonists, which appear to modify phenotypic expression of opioid receptors during brain ontogeny (Zagon and McLaughlin, 1987; Tempel et al., 1988; Barg et al., 1989a). In addition, opioids act as DNA synthesis modulators during brain development (Vertes et al., 1982; Bartolome et al., 1986, 1991; Kornblum et al., 1987; Schmahl et al., 1989). Opioid regulation of thymidine incorporation in rat brain has proven transient and dependent on developmental age (Kornblum et al., 1987; Barg et al., 1990). Opioid agonists such as morphine, β-endorphin, [d-Ala2,Me-Phe4,Gly-ol5]enkephalin (DAMGE), and Met-enkephalin decrease thymidine incorporation into DNA in vivo and their actions are reversed by the opioid antagonist naloxone, indicating opioid receptor mediation (Bartolome et al., 1986, 1991; Kornblum et al., 1987; Schmahl et al., 1989). Other reports emphasize the effect of opioids on DNA synthesis in vitro (Davila-Garcia and Azmitia, 1989; Eccleston et al., 1989; Barg et al., 1990; Coscia et al., 1991). Alteration of DNA synthesis by opioids in vitro is not restricted to neural systems, as it occurs in nonneural cells present in human lung and breast cancers and in the immune system as well (Gilmore and Weiner, 1989; Kusnecov et al., 1989; Maneckjee and Minna, 1990; Maneckjee et al., 1990; Taub et al., 1991). Protein kinase C (PKC) is included among the signal generators that trigger gene activation, leading to DNA synthesis and cell proliferation (Berridge, 1987; Kikkawa et al., 1989; Huang, 1990; Gschwendt et al., 1991). Moreover, modulators of this enzyme, such as phorbol esters, can decrease or increase DNA synthesis. PKC activators facilitate DNA synthesis and differentiation of neurons. Among the signal transduction systems that activate PKC are the phosphoinositide (PtdIns) systems. Implication of PKC in opioid modulation of thymidine incorporation into DNA has yet to be addressed. Furthermore, it is not known whether opioid abatement of thymidine incorporation involves activation of other receptor systems and/or intercellular signaling. Also unresolved are the target cells and opioid receptor types that mediate thymidine incorporation. Previously we have examined the ability of μ opioids to influence DNA synthesis (Coscia et al., 1991; Barg et al., 1992). In this study, the role of κ opioids is addressed and evidence is gained to suggest the intermediacy of κ-opioid receptors in not only neural (neuronal and glial) but also mixed glial cultures. A pertussis toxin-sensitive GTP-binding regulatory protein (G protein) and PtdIns turnover are also implicated.

Regulation of neural cell division can be mediated by growth factors, hormones, and neurotransmitters, including opioids (Zagon and McLaughlin, 1987; Eccleston et al., 1989; Stiene-Martin and Hauser, 1990, 1991; Barg et al., 1992b, 1993). Morphine and β-endorphin were shown to inhibit [3H]thymidine incorporation into brain DNA during the first postnatal week (Kornblum et al., 1987; Lorber et al., 1990; Bartolome et al., 1991). These effects were age and dose dependent, could be reversed by opioid antagonists, and were expressed in defined brain regions. A monoclonal anti-idiotypic antibody to μ- and δ-opioid receptors and the μ-selective opioid peptide agonists [d-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGE) and morphiceptin also inhibited [3H]thymidine incorporation into DNA of fetal rat brain aggregates. These actions were completely reversed by the μ-opioid antagonists cyclic d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide (CTOP) or naltrexone (Coscia et al., 1991; Barg et al., 1992b). Thymidine incorporation of the same cells was also modulated by the κ agonists U69,593 and U50,488, and this regulation was reversed by the κ antagonist norbinaltorphimine (nor-BNI) (Barg et al., 1993). Both [d-Pen2,d-Pen5]enkephalin (DPDPE) and Met-enkephalin also inhibited thymidine labeling in astrocyte cultures (Stiene-Martin and Hauser, 1990, 1991). In accordance with the inhibition of thymidine incorporation by opioid agonists, the administration of antagonists naloxone and naltrexone to rats potentiated cell proliferation and increased DNA synthesis in both fetal and neonatal brain (Vertes et al., 1982; Zagon and McLaughlin, 1987). Relatively high concentrations (micromolar or greater) of opiate alkaloids or opioid peptides were required to demonstrate their antiproliferative effects in cultured cells (Stiene-Martin and Hauser, 1990, 1991; Barg et al., 1992b, 1993). Here we show that nanomolar levels of β-endorphin can diminish thymidine incorporation. These are concentrations at which growth factors are known to act and at which β-endorphin occurs under physiological conditions.

The relative subcellular distributions of mu-opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1-day-old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of mu-opioid sites were assessed using [3H] [D-Ala2,Me-Phe4,Gly-ol5]enkephalin. P1 LMs possessed 43% of the total mu-opioid binding detected compared to 16% in the adult. Although NaCl inhibited mu binding in LMs to a greater extent than in HMs, age-dependent differences were not observed. P1 LM mu binding possessed greater sensitivity to 5'-guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Go alpha protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Go alpha after one- or two-dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular mu sites than adult LMs.

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.

A study of the onset of cation and guanine nucleotide regulation of delta, mu, and kappa rat brain opioid receptors during postnatal development was undertaken. Site-specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 microM guanosine-5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to delta sites was stimulated by Mn2+, whereas that to mu sites was not affected and kappa binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on delta sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of kappa site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of delta binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of mu and delta receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time-dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations.(ABSTRACT TRUNCATED AT 250 WORDS)

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.