Delta Opioid Receptor Expression Research Articles

Down-regulation of delta-opioid receptors in Na +/H + exchanger 1 null mutant mouse brain with epilepsy

Mice lacking Na +/H + exchanger 1 (NHE1) show a unique epilepsy phenotype although the underlying mechanisms remain unclear. Since expression of delta-opioid receptor (DOR) may be involved in control of epileptic activity, we conducted immunohistochemistry and autoradiography to investigate whether DOR expression is dys-regulated in the brain of NHE1 null mouse. Immunohistochemistry showed a decline in DOR expression in hippocampus and cortex. Autoradiographic results confirmed that the density of DOR was decreased in most cortical and hippocampal regions such as striate and temporal cortex, hippocampal CA1 and CA3 regions (reduced by 27.7 ± 6.4%, 29.4 ± 5.1%, 40.7 ± 4.4% and 20.6 ± 5.7%, respectively, P < 0.05). These data demonstrate that NHE1 null mutation leads to a reduction of DOR expression in the cortical and hippocampal regions, which provides a new clue for the genesis of epilepsy.

δ Opioid Receptors Stimulate Akt-Dependent Phosphorylation of c-jun in T Cells

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

Kappa and delta opioid receptors are expressed but down‐regulated in fibroblast‐like synoviocytes of patients with rheumatoid arthritis and osteoarthritis

To investigate the expression and regulation of the kappa-opioid receptor (KOR) and the delta-opioid receptor (DOR) in fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and to explore the potential antiarthritic mechanisms of peripheral KORs. FLS isolated from synovial tissues of 6 OA patients, 8 RA patients, and 2 healthy individuals were exposed to the selective KOR agonist U69593, the selective DOR agonist SNC 80, and kappa-opioid dynorphin A in the presence or absence of the KOR antagonist nor-binaltorphimine, the DOR antagonist naltrindole, and the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). The expression of KOR and DOR in OA and RA FLS was evaluated on the messenger RNA (mRNA) and protein levels with TaqMan real-time reverse transcriptase-polymerase chain reaction and immunofluorescence staining, respectively. KOR/DOR-mediated activation of ERK-1 and ERK-2 was investigated by Western blotting. We detected functional KOR and DOR in normal FLS and observed a reduction of both receptors in OA and RA FLS, which was more distinct in RA FLS. U69593 enhanced KOR mRNA expression in both OA and RA FLS in a KOR antagonist-reversible manner. However, the dose required for maximal enhancement in RA FLS was 10 times higher than that required in OA FLS. TNFalpha and IL-1beta both suppressed the expression of DOR and KOR mRNA in both OA and RA FLS. DOR and KOR are constitutively present in normal FLS and are suppressed under inflammatory conditions, such as RA and OA. Most interestingly, the KOR agonist U69593 may exert an antiarthritic effect via up-regulation of KOR in OA and RA FLS.

Oxygen-sensitive δ-Opioid Receptor-regulated Survival and Death Signals: NOVEL INSIGHTS INTO NEURONAL PRECONDITIONING AND PROTECTION

The detrimental effect of severe hypoxia (SH) on neurons can be mitigated by hypoxic preconditioning (HPC), but the molecular mechanisms involved remain unclear, and an understanding of these may provide novel solutions for hypoxic/ischemic disorders (e.g. stroke). Here, we show that the delta-opioid receptor (DOR), an oxygen-sensitive membrane protein, mediates the HPC protection through specific signaling pathways. Although SH caused a decrease in DOR expression and neuronal injury, HPC induced an increase in DOR mRNA and protein levels and reversed the reduction in levels of the endogenous DOR peptide, leucine enkephalin, normally seen during SH, thus protecting the neurons from SH insult. The HPC-induced protection could be blocked by DOR antagonists. The DOR-mediated HPC protection depended on an increase in ERK and Bcl 2 activity, which counteracted the SH-induced increase in p38 MAPK activities and cytochrome c release. The cross-talk between ERK and p38 MAPKs displays a "yinyang" antagonism under the control of the DOR-G protein-protein kinase C pathway. Our findings demonstrate a novel mechanism of HPC neuroprotection (i.e. the intracellular up-regulation of DOR-regulated survival signals).

Heterodimerization of μ- and δ-Opioid Receptors Occurs at the Cell Surface Only and Requires Receptor-G Protein Interactions

Homo- and heterodimerization of the opioid receptors with functional consequences were reported previously. However, the exact nature of these putative dimers has not been identified. In current studies, the nature of the heterodimers was investigated by producing the phenotypes of the 1:1 heterodimers formed between the constitutively expressed mu-opioid receptor (MOR) and the ponasterone A-induced expression of delta-opioid receptor (DOR) in EcR293 cells. By examining the trafficking of the cell surface-located MOR and DOR, we determined that these two receptors endocytosed independently. Using cell surface expression-deficient mutants of MOR and DOR, we observed that the corresponding wild types of these receptors could not rescue the cell surface expression of the mutants, whereas the antagonist naloxone could. Furthermore, studies with constitutive or agonist-induced receptor internalization also indicated that MOR and DOR endocytosed independently and could not "drag in" the corresponding wild types or endocytosis-deficient mutants. Additionally, the heterodimer phenotypes could be eliminated by the pretreatment of the EcR293 cells with pertussis toxin and could be modulated by the deletion of the RRITR sequence in the third intracellular loop that is involved in the receptor-G protein interaction and activation. These data suggest that MOR and DOR heterodimerize only at the cell surface and that the oligomers of opioid receptors and heterotrimeric G protein are the bases for the observed MOR-DOR heterodimer phenotypes.

An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect™ (vehicle), and delivered as repeated daily bolus doses (0.5 μg to 4 μg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect™ complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

Effects of peripheral nerve injury on delta opioid receptor (DOR) immunoreactivity in the rat spinal cord*1

Morphine and other opioids have direct analgesic actions in the spinal cord and chronic spinal administration of opioid agonists is used clinically in patients suffering from severe, chronic pain. Neuropathic pain resulting from peripheral nerve injury is often less sensitive to opioid therapy than other forms of chronic pain in both humans and animal models. Changes in spinal mu-opioid receptor (MOR) expression have been demonstrated in animal models of neuropathic pain. However, these changes alone fail to account for the attenuation of opioid activity. Reduced expression of delta-opioid receptors (DOR) following peripheral nerve injury has been reported but most of these reports are limited to subjective observation. The magnitude and consistency of these changes is therefore unclear. In addition, previous studies did not evaluate the effects of nerve injury on behavioral measures to confirm induction of aberrant pain symptoms. We therefore performed quantitative image analysis to evaluate the effect of peripheral nerve injury on DOR-immunoreactivity in spinal cord sections from rats previously characterized for sensory responsiveness. We observed statistically significant decreases ipsilateral to nerve injury in all three models tested: sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. These results suggest that decreases in the expression of DOR are a common feature of peripheral nerve injury.

Effects of peripheral nerve injury on delta opioid receptor (DOR) immunoreactivity in the rat spinal cord

Morphine and other opioids have direct analgesic actions in the spinal cord and chronic spinal administration of opioid agonists is used clinically in patients suffering from severe, chronic pain. Neuropathic pain resulting from peripheral nerve injury is often less sensitive to opioid therapy than other forms of chronic pain in both humans and animal models. Changes in spinal mu-opioid receptor (MOR) expression have been demonstrated in animal models of neuropathic pain. However, these changes alone fail to account for the attenuation of opioid activity. Reduced expression of delta-opioid receptors (DOR) following peripheral nerve injury has been reported but most of these reports are limited to subjective observation. The magnitude and consistency of these changes is therefore unclear. In addition, previous studies did not evaluate the effects of nerve injury on behavioral measures to confirm induction of aberrant pain symptoms. We therefore performed quantitative image analysis to evaluate the effect of peripheral nerve injury on DOR-immunoreactivity in spinal cord sections from rats previously characterized for sensory responsiveness. We observed statistically significant decreases ipsilateral to nerve injury in all three models tested: sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. These results suggest that decreases in the expression of DOR are a common feature of peripheral nerve injury.

Phosphorylation of activating transcription factor in murine splenocytes through delta opioid receptors

Delta opioid receptors (DORs) modulate TCR signaling through the mitogen-activated protein kinases (MAPKs), ERKs 1 and 2. These studies determined whether a DOR agonist alone ([ d-Ala 2- d-Leu 5]enkephalin; DADLE) affects phosphorylation of the activating transcription factor (ATF-2) and its interaction with the MAPK, c-Jun NH 2-terminal kinase (JNK). DOR expression was induced on murine splenocytes by anti-CD3 and then quiescent cells were treated with DADLE. DADLE, itself, dose-dependently induced maximal phosphorylation of ATF-2 within 5–10 min; naltrindole, a specific antagonist, abolished this. Anti-ATF-2 immunoprecipitates from control and DADLE-treated splenocytes showed a dominant 59 kDa phosphorylated band and a 71 kDa band. DADLE stimulated phosphorylation of both bands, although the 71 kDa band was selectively immunoprecipitated by anti-JNK. Thus, DADLE stimulated phosphorylation of 71kDa ATF-2 and its association with JNK, suggesting that JNK is activated through DORs. Along with previous observations, these studies suggest that lymphocyte DORs can affect the activation of MAPKs by TCR-independent stimulation (e.g., JNK) or indirectly by modulating TCR-dependent stimulation (e.g., ERK).

Expression of delta opioid receptor mRNA and protein in the rat cerebral cortex and cerebellum is decreased by growth hormone.

Hormones released from the pituitary have been shown to regulate the expression of different proteins in the central nervous system. We wanted to examine whether peripheral administration of bovine growth hormone (bGH) regulates the expression of delta-opioid receptor (DOR) in the cerebral cortex and cerebellum. Expression of the DOR protein was quantified using Western blot densitometry. DOR mRNA was quantified with a solution hybridization RNase protection assay. Hypophysectomized (Hx) and untreated normal female rats were included in the study. All Hx rats were hormonally treated with cortisol (400 microg/kg/day) and L-thyroxine (10 microg/kg/day) for 19 days. Hypophysectomy resulted in a threefold increase in cerebral cortex and a twofold increase in cerebellum of the DOR protein compared with normal rats. One subgroup of Hx rats received bGH (1 mg/kg body weight) as a daily subcutaneous injection for 19 days. This treatment normalized the levels of DOR protein in the cerebral cortex and cerebellum. Immunohistochemical experiments showed that GH decreased DOR expression especially in layers II-VI in cerebral cortex and in stratum moleculare in cerebellum. Quantification of DOR mRNA by solution hybridization RNase protection assay corresponded to the DOR protein measurements. We conclude that the expression of DORs in cerebral cortex and cerebellum is regulated by GH.

Transcriptional Regulation of Mouse δ-Opioid Receptor Gene: ROLE OF IKAROS IN THE STIMULATED TRANSCRIPTION OF MOUSE δ-OPIOID RECEPTOR GENE IN ACTIVATED T CELLS

Delta-opioid receptors (DOR) present on T cells have been shown to mediate the immunomodulatory effects of endogenous and synthetic DOR agonists on T cells. Considerable evidence indicates that there is stimulated transcription of DOR gene in activated T cells, which is correlated with augmented expression of DOR and enhanced capacity of DOR agonists to affect the T-cell's functions. However, the molecular mechanism underlying the stimulated transcription of the DOR gene in activated T cells is still unclear. In the present study, we analyzed a 1.3-kb DNA fragment immediately upstream of the translation start site (-1300 to +1 bp, with the translation start site designated as +1) of the mouse DOR gene in EL-4 cells, a mouse lymphoma T cell line that exhibits enhanced expression of DOR transcripts when activated by phytohemagglutinin. Through both in vivo and in vitro experiments, we have demonstrated that increased binding activity of Ikaros at the Ikaros-binding site (-378 to -374) in the DOR promoter is required for the stimulated transcription of DOR gene in phytohemagglutinin-activated T cells.

Expression of Delta Opioid Receptors by Splenocytes from SEB-Treated Mice and Effects on Phosphorylation of MAP Kinase

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 μg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 ± 0.62 units (mean ± SEM), increasing to 16.1 ± 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 ± 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10−8 to 10−6 M, [d-Ala2-d-Leu5]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-ϵ-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.

Differential expression of delta opioid receptors and mRNA in proliferating astrocytes during the cell cycle

Differential expression of delta opioid receptors and mRNA in proliferating astrocytes during the cell cycle

Characterization of opioid receptor types modulating acetylcholine release in septal regions of the rat brain.

Presynaptic opioid receptors of the delta- and mu-types have been shown to inhibit the release of acetylcholine (ACh) in the rat striatum and hippocampus, respectively, but it is unknown whether opioid receptors modulate the release of ACh also in the region of origin of the hippocampal cholinergic innervation, the septum. To answer this question, slices (350 microm) of the medial septal area and of the diagonal band of Broca, as well as (for comparison) of the hippocampus, were prepared from adult male Wistar rats. The slices were incubated with [3H]choline, superfused in the presence of hemicholinium-3 (10 microM) and stimulated twice (S1, S2) by electrical fields (360 pulses, 3 Hz, 2 ms, 60 mA); opioid receptor agonists were present during S2. The preferential mu-agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) inhibited the evoked ACh release by maximally about 40% in hippocampal slices and acted even more strongly in the medial septal area, or the diagonal band of Broca (about 60% or 75% maximal inhibition, respectively). These effects were reduced or abolished by the preferential mu-antagonist naloxone, which showed no effects when given alone. Using naloxone in the presence of a cocktail of peptidase inhibitors, no evidence for an endogenous tone of opioid peptides was found in the medial septal area, diagonal band of Broca or the hippocampus. Using the preferential delta-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and the delta-antagonist naltrindole, a delta-opioid receptor inhibiting evoked ACh release was clearly detectable both in the medial septal area and the diagonal band of Broca, but not in the hippocampus, whereas the preferential kappa-agonist trans-3,4-dichloro-N-methyl-N-[2(1-pyrrolidinyl)cyclo-hexyl] benzeneacetamide (U50,488H) had only weak or no effects. In addition to the functional experiments, double in-situ hybridization studies were performed, in which cells containing mRNA for choline acetyltransferase (ChAT) were labeled by an antibody-linked enzymatic staining procedure, whereas mRNAs for mu- or delta-opioid receptors were detected with radioactive probes. These experiments revealed that in the septal region mainly mu-opioid receptors were expressed by neurons positive for ChAT mRNA, whereas in the rat striatum the expression of delta-opioid receptors prevailed in those neurons. We conclude that in the septal area of the rat brain, in contrast to the rat striatum and hippocampus, both presynaptic mu- and delta-opioid receptors modulate the evoked release of ACh. Whether presynaptic mu- and delta-opioid receptors occur on the same or on different septal cells or axon terminals remains to be clarified.

Expression of delta opioid receptors and transcripts by splenic T cells.

Delta opioid receptors (DORs) and preproenkephalin-A-derived opiate peptides are expressed by mononuclear cells in various lymphoid organs. DOR ligands modulate a variety of immune functions, such as T-cell proliferation, calcium mobilization, and cytokine production. Recently, quiescent T cells were found to express low levels of DOR transcripts, which increased due to the following: cell culture of unstimulated murine splenocytes (depending on cell density); cross-linking the T-cell receptor (TCR) with anti-CD3-epsilon; and a single in vivo exposure to staphylococcal enterotoxin B (SEB). Enhanced expression of DOR mRNA was mediated transcriptionally. Moreover, PMA + ionomycin, which mimic the proliferative signal of anti-CD3, inhibited the expression of DOR mRNA. Using semiquantitative immunofluorescence to detect DORs, SEB was found to increase the fraction of T cells that expressed DOR and to enhance the relative level of DOR expression per T cell. Previous studies have shown that DOR agonists inhibited the anti-CD3-stimulated production of interleukin-2 and T-cell proliferation. Therefore, the enhanced expression of DORs by activated T cells may be capable of downregulating the T-cell activation program.

Differential expression of delta opioid receptors and mRNA in proliferating astrocytes during the cell cycle.

Previous immunohistochemical and radioligand binding studies have shown a cell cycle-dependent regulation of the delta opioid receptor (DOR). The relationship between DOR expression and mitosis in primary astroglial cultures of rat cerebral cortex was investigated in this study. The cultures were arrested during the G(1)/S transition or during mitosis. The DOR protein level increased twofold (P = 0.009) during mitosis and DOR mRNA level increased threefold (P = 0.002) during the G(1)/S transition compared to nonsynchronized cultures. DOR mRNA was also elevated (1.6-fold, P = 0.008) during the G(1)/S transition compared with mitotic cells. A premitotic increase in DOR mRNA suggests that elevated DOR protein levels during mitosis might be regulated during transcription.

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT-PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes froin CD 1 female mice as well. The results of studies using quantitative competitive RT-PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti-CD3-epsilon for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti-CD3-epsilon did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti-CD3-epsilon. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti-CD3-epsilon-stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti-CD3-epsilon regulates DOR expression differently than PMA and ionomycin.

Ca2+/calmodulin-dependent transcriptional activation of delta-opioid receptor gene expression induced by membrane depolarization in NG108-15 cells.

Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of delta-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 mM KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca2+ entry through L-type voltage-dependent Ca2+ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca2+ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca2+ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.

Quantitative analysis of mu and delta opioid receptor gene expression in rat brain and peripheral ganglia using competitive polymerase chain reaction.

Competitive polymerase chain reaction assays following reverse transcription have been developed for quantitative analysis of delta and mu opioid receptor gene expression. The assay was used to obtain quantitative measurements of mu and delta opioid receptor expression levels in different brain regions and sensory and sympathetic ganglia in the rat. The assays provide accurate estimates of the relative levels of receptor messenger RNAs by the inclusion in the assays of known amounts of internal standards with the same sequence, except for a small deletion, as the target complementary DNA. The amplification products of target and competitor can be distinguished by size, and their amounts measured by densitometry. Expression of mu and delta opioid receptor messenger RNAs in different regions of the rat brain, somatic and visceral sensory and sympathetic ganglia was investigated using this method. In the brain the highest density of delta receptor messenger RNA was detected in the olfactory bulb, followed by the striatum. The mu receptor was expressed at highest levels in the midbrain-hypothalamic region. All the sensory ganglia studied expressed both mu and delta opioid receptor messenger RNAs. In the nodose ganglion we observed the highest level of mu receptor messenger RNA of any structure studied; in the trigeminal ganglion the level was about 10 times lower than that in the nodose ganglion. Among the dorsal root ganglia, mu receptor messenger RNA density was highest in the lumbar region, followed by the thoracic and cervical regions. The sympathetic superior cervical ganglion expressed a very low level of mu message. Delta receptor messenger RNA was detected only in the sensory ganglia, at levels that were considerably lower than in the striatum. The reverse transcription polymerase chain reaction assay is quantitatively reliable for comparison of messenger RNA levels between different RNA extracts, and sensitive enough to permit the detection and assay of mu and delta opioid receptor gene expression in a single pair of sensory or autonomic ganglia from the rat.

Delta opioid receptor expression is induced by concanavalin A in CD4+ T cells.

Expression of delta opioid receptor mRNA in CD4+ T cells purified from Con A-stimulated murine splenocytes was demonstrated by reverse transcription-PCR and sequencing of the PCR products. mu and kappa opioid receptor mRNAs were not detected. Similar results were obtained in a single isolation of CD8+ T cells from Con A-stimulated splenocytes. mRNA for the delta opioid receptor was not detected in CD4+ T cells purified from either freshly isolated splenocytes or splenocytes cultured without Con A, and incubation of purified CD4+ T cells with Con A did not induce delta opioid receptor mRNA. It appears that quiescent CD4+ T cells do not express classic opioid receptors, but the delta-type receptor can be induced upon activation.