Demonstration of κ3-Opioid Receptors in the SH-SY5Y Human Neuroblastoma Cell Line

Abstract: In addition to the μ- and δ-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of κ₃ receptors, accounting for 40% of total opioid binding, as measured with [³H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in κ₃ binding. Fully 75% of the increased binding corresponds to κ₃ sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the μ antagonist d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP). Naloxone benzoylhydrazone, a κ₃ agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional κ₃ receptors.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the platelet-derived growth factor receptor

R^? cells are 3T3-like cells derived from mouse embryos in which the insulin-like growth factor I (IGF-I) receptor (IGF-IR) genes have been disrupted by targeted homologous recombination. These cells cannot grow in serum-free medium supplemented by the growth factors that sustain the growth of other 3T3 cell lines, and cannot be transformed by oncogenes that easily transform wild type mouse embryo cells. We have used these cells to study the role of the IGF-IR in the growth and transformation of cells overexpressing the platelet-derived growth factor (PDGF)-b?b? receptor. We report that an overexpressed PDGF-b?b? receptor fails to induce mitogenesis or transformation in cells lacking the IGF-IR, while capable of doing so in cells expressing the IGF-IR. We conclude that the ability of the activated PDGF-b?b? receptor to stimulate cell proliferation and transformation requires a funcitional IGF-IR. © 1995 Wiley-Liss, Inc.     

Study on the molecular mechanism of antinociception induced by ghrelin in acute pain in mice

Ghrelin has been identified as the endogenous ligand for the GHS-R1α (growth hormone secretagogue receptor 1 alpha). Our previous experiments have indicated that ghrelin (i.c.v.) induces antinociceptive effects in acute pain in mice, and the effects were mediated through the central opioid receptors and GHS-R1α. However, which opioid receptor (OR) mediates the antinociceptive effects and the molecular mechanisms are also needed to be further explored. In the present study, the antinociceptive effects of ghrelin (i.c.v.) could be fully antagonized by δ-opioid receptor antagonist NTI. Furthermore, the mRNA and protein levels of δ-opioid peptide PENK and δ-opioid receptor OPRD were increased after i.c.v injection of ghrelin. Thus, it showed that the antinociception of ghrelin was correlated with the GHS-R1α and δ-opioid receptors. To explore which receptor was firstly activated by ghrelin, GHS-R1α antagonist [D-Lys³]-GHRP-6 was co-injection (i.c.v.) with deltorphin II (selective δ-opioid receptor agonist). Finally, the antinociception induced by deltorphin II wasn’t blocked by the co-injection (i.c.v.) of [D-Lys³]-GHRP-6, indicating that the GHS-R1α isn’t on the backward position of δ-opioid receptor. The results suggested that i.c.v. injection of ghrelin initially activated the GHS-R1α, which in turn increased the release of endogenous PENK to activation of OPRD to produce antinociception.     

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory G_i proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-x_L, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.     

Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.     

Retroviruses expressing different levels of the normal epidermal growth factor receptor: biological properties and new bioassay

Two retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFR cDNA into two different retroviral vectors. One DNA (pCO11-EGFR-neo) also contained a linked selectable marker gene (neoR). The other (pCO12-EGFR) only expresses hEGFR. When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium. Compared with pCO11-EGFR-neo, pCO12-EGFR induced EGF-dependent transformation 2-5 times more efficiently and expressed higher numbers of receptors (4 x 10(5) vs. 1 x 10(5) EGF receptors per cell). The results indicate that transforming potential is directly related to the number of EGF receptors. In defined, serum-free medium that contained only very low concentrations of insulin (0.6 microgram/ml) and transferrin (0.6 micrograms/ml), hEGFR-virus infected cells were able to grow with EGF as the only growth factor. Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth. NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus. The dose-dependent growth response to EGF of infected NR6 cells grown in serum-free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type alpha (TGF alpha). This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg-100 ng/ml). Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF specificity of growth stimulation.     

Flat revertants derived from Kirsten murine sarcoma virus-transformed cells produce transforming growth factors

Two flat cellular revertant cell lines, F-2 and C-11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non-transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F-2 and C-11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum-free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F-2 and C-11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.     

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

The human insulinlike growth factor I receptor was overexpressed in NIH 3T3 cells as well as human and rat primary fibroblast strains. The NIH 3T3 cells displayed a ligand-dependent, highly transformed phenotype. When exposed to insulinlike growth factor I or supraphysiologic levels of insulin, NIH 3T3 cells that expressed high levels of receptors formed aggregates in tissue culture dishes, colonies in soft agar, and tumors in nude mice. Expression of 1 million receptors per cell, a 40-fold increase above the base-line level, was required for anchorage-independent growth. Primary fibroblasts that expressed high levels of receptors displayed a ligand-dependent change in morphology and an increase in saturation density but did not acquire a fully transformed phenotype. The results demonstrate that when amplified, this ubiquitous growth factor receptor behaves like an oncogenic protein and is capable of promoting neoplastic growth in vivo.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

A G protein-coupled receptor encoded by rhesus rhadinovirus is similar to ORF74 of Kaposi's sarcoma-associated herpesvirus

Rhesus rhadinovirus (RRV) is a gamma-2 herpesvirus and is the rhesus macaque homologue of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. DNA sequence analysis of RRV indicates that it shares numerous open reading frames (ORFs) with HHV-8, including one (ORF74) encoding a seven-transmembrane-spanning G protein-coupled receptor (GPCR) with similarity to cellular chemokine receptors. Examination of the predicted amino acid sequence of RRV ORF74 reveals that it encodes a seven-transmembrane-spanning GPCR sharing 40.8% amino acid sequence identity with HHV-8 ORF74 and 24.1% amino acid sequence identity with rhesus macaque CXCR2. In addition, immunofluorescence studies indicate that an epitope-tagged version of RRV ORF74 is expressed on the surfaces of transfected cells, suggesting that this protein is in fact a membrane receptor. In in vitro cell culture assays, RRV ORF74 possesses transforming potential, as NIH 3T3 clones stably expressing the receptor demonstrate an increased ability to grow in soft agarose and to induce tumor formation in nude mice. Further analysis of RRV ORF74 indicates that expression of the receptor in NIH 3T3 cells causes an increased secretion of vascular endothelial growth factor and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway. The results of these studies suggest that RRV ORF74 encodes a GPCR with properties similar to those of its homologue in HHV-8 and that this gene may play a role in RRV-associated pathogenesis.     

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.