Lee Yuan Liu-Chen

Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization.

The opiates are well‐established immunomodulatory factors, and recent evidence suggests that μ‐ and δ‐opioid receptor ligands alter chemokine‐driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of μ‐ and δ‐opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell‐derived factor‐1α/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the μ‐opioid receptor to determine whether cross‐desensitization of CCR5 was a result of receptor internalization. Using radiolabeled‐binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross‐desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV‐1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV‐1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor‐dependent HIV‐1 infection. The interference with HIV‐1 uptake by heterologous desensitization of CCR5 suggests that HIV‐1 interaction with this receptor is not passive but involves a signal transduction process.

Dual ultrastructural localization of μ-opiate receptors and substance P in the dorsal horn

Opiates active at the μ‐opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence‐specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP‐immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR‐labeled dendrites that received any afferent input were contacted by at least one SP‐containing axon or terminal. Although SP‐immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP‐containing spinal interneurons may be regulated by MOR ligands. A few SP‐labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second‐order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post‐ and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics. Synapse 36:12–20, 2000.

Isoquinoline Alkaloids Isolated from Corydalis yanhusuo and Their Binding Affinities at the Dopamine D1 Receptor

Bioactivity-guided fractionation of Corydalis yanhusuo has resulted in the isolation of eight known isoquinoline alkaloids - tetrahydropalmatine, isocorypalmine, stylopine, corydaline, columbamine, coptisin, 13-methylpalmatine, and dehydro-corybulbine. The tertiary alkaloids were further analyzed by chiral HPLC to determine the ratios of d-and l-isomers. The isolated compounds were screened for their binding affinities at the dopamine D1 receptor. Isocorypalmine had the highest affinity (Ki = 83 nM). The structure-affinity relationships of these alkaloids are discussed.

N-Methylacetamide Analog of Salvinorin A: A Highly Potent and Selective κ-Opioid Receptor Agonist with Oral Efficacy

Several preclinical studies indicate that selective κ-opioid receptor (KOR) antagonists have antidepressant-like effects, whereas KOR agonists have opposite effects, suggesting that each might be useful in the treatment of mood abnormalities. Salvinorin A (salvA) is a valuable KOR agonist for further study due to its high potency and receptor selectivity. However, it has short lasting effects in vivo and limited oral bioavailability, probably due to acetate metabolism. We compared the in vitro receptor binding selectivity of salvA and four analogs containing an ethyl ether (EE), isopropylamine (IPA), N-methylacetamide (NMA), or N-methylpropionamide (NMP) at C-2. All compounds showed high binding affinity for the KOR (Ki = 0.11–6.3 nM), although only salvA, EE, and NMA exhibited KOR selectivity. In a liver microsomal assay, salvA was least stable, whereas NMA and IPA displayed slower metabolic transformations. Intraperitoneal (i.p.) administration of salvA, NMA, and NMP dose-dependently elevated brain reward thresholds in the intracranial self-administration (ICSS) test, consistent with prodepressive-like KOR agonist effects. NMA and NMP were equipotent to salvA but displayed longer lasting effects (6- and 10-fold, respectively). A dose of salvA with prominent effects in the ICSS test after i.p. administration (2.0 mg/kg) was inactive after oral administration, whereas the same oral dose of NMA elevated ICSS thresholds. These studies suggest that, although salvA and NMA are similar in potency and selectivity as KOR agonists in vitro, NMA has improved stability and longer lasting actions that might make it more useful for studies of KOR agonist effects in animals and humans.

Ablation of Kappa-Opioid Receptors from Brain Dopamine Neurons has Anxiolytic-Like Effects and Enhances Cocaine-Induced Plasticity

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR−/−) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KORlox/lox). Autoradiography demonstrated complete ablation of KOR binding in the KOR−/− mutants, and reduced binding in the DAT-KORlox/lox mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR−/− mice appeared normal in the open field and light/dark box tests, DAT-KORlox/lox mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR−/− mutants, but was exaggerated in DAT-KORlox/lox mutants. Increased sensitivity to cocaine in the DAT-KORlox/lox mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR−/− mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

Ligand directed signaling differences between rodent and human κ-opioid receptors.

Background: Dysphoric effects of κ-opioid receptor (KOR) agonists require p38 MAPK activation in mice, but sequence differences in human KOR may affect this mechanism. Results: Differences in p38 activation were observed between human and rodent KOR for pentazocine and butorphanol. Conclusion: Species differences affect signaling. Significance: Rodent models may not predict adverse effects of KOR agonists in humans. KOR activation of Gβγ dependent signaling results in analgesia, whereas the dysphoric effects of KOR agonists are mediated by a different pathway involving G protein receptor kinase and non-visual arrestin. Based on this distinction, a partial KOR agonist that does not efficiently activate arrestin-dependent biased signaling may produce analgesia without dysphoria. No KOR-selective partial agonists are currently available, and preclinical assessment is complicated by sequence differences between rodent (r) and human (h) KOR. In this study, we compared the signaling initiated by the available partial agonists. Pentazocine was significantly more potent at activating p38 MAPK in hKOR than rKOR expressed in HEK293 cells but equally potent at arrestin-independent activation of ERK1/2 in hKOR and rKOR. Similarly, butorphanol increased phospho-p38-ir in hKOR-expressing cells but did not activate p38 in rKOR-HEK293. Like pentazocine, butorphanol was equally efficacious at activating ERK1/2 in rKOR and hKOR. In contrast, levorphanol, nalorphine, and U50,488 did not distinguish between hKOR and rKOR in p38 MAPK activation. Consistent with its low potency at p38 activation, pentazocine did not produce conditioned place aversion in mice. hKOR lacks the Ser-369 phosphorylation site in rKOR required for G protein receptor kinase/arrestin-dependent p38 activation, but mutation of the Ser-358 to asparagine in hKOR blocked p38 activation without affecting the acute arrestin-independent activation of ERK1/2. This study shows that hKOR activates p38 MAPK through a phosphorylation and arrestin-dependent mechanism; however, activation differs between hKOR and rKOR for some ligands. These functional selectivity differences have important implications for preclinical screening of partial KOR agonists.

Detection of К-Opioid Receptor mRNA in Immature T Cells

Opioid receptors were first detected on the cells of the nervous system (1,2). Using radiolabeled ligands, cell lines developed from cells of the immune system have been shown to express the kappa opiate receptor (3,4). One example is the R1.1 T-thymoma developed from a C58/J mouse. This cell line has been shown to express the surface markers Thy 1.2 and H-2 k(5). This cell line has also been shown to express the kappa receptor by receptor binding assays (4). Little is known about the differentiation stage or functional capacity of this T cell line.

The region in the μ opioid receptor conferring selectivity for sufentanil over the δ receptor is different from that over the κ receptor

We determined the binding domains of sufentanil and lofentanil in the μ opioid receptor by comparing their binding affinities to seven μ/δ and six μ/κ chimeric receptors with those to μ, δ and κ opioid receptors. TMHs 6 and 7 and the e3 loop of the μ opioid receptor were important for selective binding of sufentanil and lofentanil to the μ over the κ receptor. TMHs 1–3 and the e1 loop of the μ opioid receptor conferred binding selectivity for sufentanil over the δ receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1–3 and TMHs 6–7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the μ receptor.

Effects of acute agonist treatment on subcellular distribution of κ opioid receptor in rat spinal cord

We investigated whether acute treatment with agonists affected the subcellular distribution of κ opioid receptor (KOPR) in the dorsal horn of the rat lumbar spinal cord by using immunoelectron microscopy. Rats were injected intrathecally (i.t.) with U50,488H (100 nmole), dynorphin A(1–17) (15 nmole), or vehicle. The doses chosen have been shown to induce antinociception. Rats were perfused transcardially 30 min later, and lumbar spinal cords were removed and processed for electron microscopic analysis. KOPR was stained with KT‐2, a specific polyclonal antibody against the rat/mouse KOPR(371–380) peptide, followed by gold‐labeled secondary antibody and silver intensification. The silver grains were present in axons, terminals, dendrites, and somata, and the association with plasma membranes was quantified in dendrites, because KOPR immunoreactivity was most frequently observed in these profiles. In vehicle‐treated rats, ∼27% of KOPR immunoreactivity was associated with plasma membranes. U50,488H, i.t., did not cause a significant change in the percentage of KOPR present on plasma membranes, whereas dynorphin A, i.t., significantly decreased cell surface KOPR to ∼19%. In summary, these data indicate that U50,488H and dynorphin A differentially regulate the subcellular distribution of endogenous KOPR.

Opioid peptide receptor studies. 7. The methylfentanyl congener RTI-4614-4 and its four enantiomers bind to different domains of the rat μ opioid receptor

Mutational analysis of opioid receptors supports the hypothesis that dissimilar receptor domains contribute to the binding affinity of different ligands. To determine whether enantiomeric ligands can serve to distinguish between different binding pockets (which focuses the analysis on asymmetric structural factors while avoiding confounding changes in physiochemical characteristics), we analyzed the binding of the 3‐methylfentanyl congeners RTI‐4614‐4 [(±)‐cis‐N‐[1‐(2‐hydroxy‐2‐phenylethyl)‐3‐methyl‐4‐piperidyl]‐N‐ phenylpropanamide HCl)], its four stereoisomers [(2S,3R,4S)‐1a, (2R,3R,4S)‐1b, (2R,3S,4R)‐1c, and (2S,3S,4R)‐1d], and other μ agonists with cloned rat μ opioid receptors stably expressed in HEK‐293 cells and μ/κ receptor chimeras. Chimera III (κaminoacids 1–141/μaminoacids 151–398), chimera IV (μaminoacids 1–150/κaminoacids 142–380), and chimera XII (κaminoacids 1–262/μaminoacids 269–398) bound [125I]IOXY (6β‐iodo‐3,14‐dihydoxy‐17‐cyclopropylmethyl‐4,5α‐epoxymorphinan) with high affinities. The Ki values of 1a, 1b, 1c, and 1d at the wild‐type μ receptor were 0.55 nM, 0.66 nM, 124 nM, and 59.2 nM, respectively. When the region from the N terminal to the start of the transmembrane helix 3 (TMH3) of the μ receptor was substituted by that of the κ receptor (chimera III), the Ki value of 1b was increased (relative to the μ receptor) 590‐fold compared to a 73‐fold increase for 1a. When this portion of the κ receptor was replaced by that of the μ receptor (chimera IV), the loss of affinity was not as great: 11.7‐fold for 1a and 58.5‐fold for 1b. Replacement of the middle of the third intracellular loop and third extracellular loop (e3) of the κ receptor with that of the μ receptor (chimera XII) lowered (relative to their Ki values at the κ receptor) the Ki values of [D‐Ala2,D‐Leu5]enkephalin and [D‐Ala2‐MePhe4,Gly‐ol5]enkephalin to a much greater extent than the Ki values of the isomers. The κ/chimera XII shift was greater for isomers 1c and 1d than for 1b and 1a. Viewed collectively, these data suggest that the region from the N terminal to the start of the TMH3 of the μ opioid receptor determines the binding affinity of RTI‐4614‐4 and its isomers and that the e3 loop also plays a major role in determining the binding affinity of μ agonist peptides. These data also show that the stereoisomers of RTI‐4614‐4 probably bind to different domains of the μ receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain‐specific ligands. Synapse 28:117–124, 1998.