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H H Loh

Publications and source records attributed to H H Loh.

At least 37 records · Page 2Linked to original sources
PubMed2001

Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide.

The major objective of this paper is to characterize the mechanism by which morphine modulates lymphocyte function and if these effects are mediated through the mu-opioid receptor. We evaluated the in vitro effects of morphine on lymphocytes that were freshly isolated from lymph nodes from wild type (WT) and mu-opioid receptor knock-out (MORKO) mice. Results show that morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-gamma synthesis in a dose-dependent manner. This effect was abolished in lymph node cells isolated from MORKO mice. The inhibition of T-cell function with low-dose morphine was associated with an increase in caspase-3- and caspase-8-mediated apoptosis. The inhibition of T-cell function with high-dose morphine was associated with an increase in the inducible NO synthase mRNA expression. N(G)-nitro-L-arginine methyl ester (L-NAME) antagonized the apoptosis induced by high-dose morphine. Our results suggest that low-dose morphine, through the mu-opioid receptor, can induce lymph node lymphocyte apoptosis through the cleavage activity of caspase-3 and caspase-8. Morphine at high doses induces NO release. This effect of morphine is also mediated through the mu-opioid receptor present on the surface of macrophages.

Animals↗
PubMed2000

Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization.

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.

Amino Acid Sequence↗
PubMed2000

An intronic Ikaros-binding element mediates retinoic acid suppression of the kappa opioid receptor gene, accompanied by histone deacetylation on the promoters.

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in mouse embryonal carcinoma P19 stem cells and suppressed by retinoic acid (RA) in cells undergoing neuronal differentiation. A negative regulatory element is located within intron 1 of the KOR gene, which contains an Ikaros (Ik)-binding site (GGGAAgGGGAT). This sequence is an Ik-1 respondive, functionally negative element as demonstrated in the context of both natural KOR and heterologous promoters. The two underlined G residues of the second half-site are critical for Ik-1 binding and Ik-mediated repression of the KOR gene. RA induces Ik-1 expression within 1 day of treatment and suppresses KOR expression between 2 and 3 days. Overexpression of Ik-1 in P19 suppresses endogenous KOR gene expression, accompanied by increased binding of Ik-1 to the Ik-binding site and chromatin histone deacetylation on KOR promoters. It is proposed that in an RA-induced P19 differentiation model, RA elevates Ik-1 expression, which recruits histone deacetylase to intron 1 of the KOR gene and silences KOR gene promoters.

Acetylation↗
PubMed2000

Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor.

Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations </=90 fmol/mg of protein were rapid desensitizations (t(12) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser(363) in the carboxyl tail of the delta-opioid receptor to Ala completely abolished the deltorphin II-induced receptor phosphorylation but not the desensitization response. Although the magnitude of desensitization was attenuated, the rate of deltorphin II-induced receptor desensitization remained the same in the S363A mutant as compared with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediated receptor internalization was blocked by 0.4 m sucrose that the deltorphin II-induced receptor desensitization was abolished in the S363A mutant. Similarly, 0.4 m sucrose could partially block the agonist-induced rapid desensitization in HEK293 cells expressing the wild type delta-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitization of the delta-opioid receptor involves both the phosphorylation and the internalization of the receptor.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Association of kappa opioid receptor mRNA upregulation in dorsal root ganglia with mechanical allodynia in mice following nerve injury.

This study examined the contribution of nerve injury alone or nerve injury with signs of neuropathic pain to alteration of kappa opioid receptor (KOR) mRNA expression. Two groups of mice, both of them were subjected to unilateral transection of the inferior and superior caudal trunks at the S1spinal nerve, were compared with respect to KOR mRNA expression by reverse transcriptase-polymerase chain reaction. One group showed exclusive pain behavior (PB+) as mechanical allodynia, and the other group exhibited no enhanced sensitivity to innocuous mechanical stimulation to the tail (PB-). Expression of total KOR and variants B and C mRNA increased ipsilaterally in dorsal root ganglia (DRG) of PB+ mice, whereas KOR variant A mRNA was not detected in DRG. These results show that KOR mRNA expression differs between PB+ and PB- groups of mice after nerve injury, and suggest an association of KOR expression with mechanical allodynia.

Animals↗
PubMed2000

Comparison of G-protein activation in the brain by mu-, delta-, and kappa-opioid receptor agonists in mu-opioid receptor knockout mice.

Mice lacking the mu-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for mu, delta-, and kappa-opioid receptors in mu-opioid receptor knockout mice. Selective agonists of mu- (DAMGO), delta- (DPDPE), and kappa- (U-69,593) opioid receptors stimulated [(35)S]GTPgammaS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [(35)S]GTPgammaS binding in these regions of mu-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a mu-opioid receptor agonist in mu-opioid receptor knockout mice, and demonstrated that coupling of the kappa-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the delta-opioid receptor agonist, DPDPE, was reduced in the mu-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.

Alleles↗
PubMed2000

Involvement of mu-opioid receptors in potentiation of apomorphine-induced climbing behavior by morphine: studies using mu-opioid receptor gene knockout mice.

The present study examined the hypothesis that mu-opioid receptors contribute to a behavioral stimulation produced by stimulation of dopamine receptors by comparing responses in mu-opioid receptor knockout and wild type mice. Apomorphine-induced climbing behavior was augmented by 65%, in wild type mice, but not in mu-knockout, following subcutaneous administration of morphine (15 mg/kg). Moreover, pretreatment with either naloxone (an opioid receptor antagonist) or haloperidol (a mixed D(1)/D(2) receptor antagonist) eliminated the enhancement by morphine of climbing behavior in wild type mice. These results indicate that expression of mu-opioid receptors plays an important role in the enhancement of climbing behavior induced by the dopamine receptor agonist, apomorphine. Furthermore, this augmentation is mediated by interaction between dopamine and mu-opioid receptors.

Analgesics, Opioid↗
PubMed2000

An autoradiographic study in mu-opioid receptor knockout mice.

An autoradiographic study was carried out to study the opioid binding sites in a mu-opioid receptor knock-out mouse line whose exon 2 and 3 were deleted. Mu-opioid binding sites were undetectable in this knock-out mouse line while the binding of the other two types of receptors were unaltered. Our results suggest destroying functional mu-opioid receptor does not affect the expression of the other two opioid receptors.

Animals↗
PubMed2000

Morphine self-administration in mu-opioid receptor-deficient mice.

Morphine-induced place preference was demonstrated recently in wild-type mice, whereas this conditioned behaviour was not observed in mu-opioid receptor-deficient mice. In the present study, we investigated locomotor effects of subcutaneously (s.c.) injected morphine as well as intracerebroventricular (i.c.v.) morphine self-administration in mu-opioid receptor-knockout mice. After s.c. morphine injection, locomotor activity significantly increased in wild-type animals. As expected, in the self-administration test the rate of self-administration constantly increased in wild-type mice reflecting reward effects of morphine. This increase was independent of locomotor/motor activity. In contrast, self-administration rates and locomotor/motor activity significantly decreased in the receptor-deficient animals. It was shown that this aversive effect might partly be due to kappa-opioid receptor interaction.

Analysis of Variance↗
PubMed2000

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies.

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.

Animals↗
PubMed2000

Receptor density and recycling affect the rate of agonist-induced desensitization of mu-opioid receptor.

Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.

Brefeldin A↗
PubMed2000

Molecular mechanisms and regulation of opioid receptor signaling.

Cloning of multiple opioid receptors has presented opportunities to investigate the mechanisms of multiple opioid receptor signaling and the regulation of these signals. The subsequent identification of receptor gene structures has also provided opportunities to study the regulation of receptor gene expression and to manipulate the concentration of the gene products in vivo. Thus, in the current review, we examine recent advances in the delineation basis for the multiple opioid receptor signaling, and their regulation at multiple levels. We discuss the use of receptor knockout animals to investigate the function and the pharmacology of these multiple opioid receptors. The reasons and basis for the multiple opioid receptor are addressed.

Adenylyl Cyclases↗
PubMed1999

Transcriptional regulation of mouse delta-opioid receptor gene.

Three major types of opioid receptors, mu (MOR), delta (DOR), and kappa (KOR), have been cloned and characterized. Each opioid receptor exhibits a distinct pharmacological profile as well as a distinct pattern of temporal and spatial expression in the brain, suggesting the critical role of transcription regulatory elements and their associated factors. Here, we report the identification of a minimum core promoter, in the 5'-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line. In vitro protein-DNA binding assays and in vivo transient transfection assays indicated that members of both the upstream stimulatory factor and Sp families of transcription factors bound to and trans-activated the DOR promoter via the E box and GC box, respectively. Furthermore, functional and physical interactions between these factors were critical for the basal as well as maximum promoter activity of the DOR gene. Thus, the distinct developmental emergence and brain regional distribution of the delta opioid receptor appear to be controlled, at least in part, by these two regulatory elements and their associated factors.

Animals↗
PubMed1999

Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure.

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Antibody Specificity↗
PubMed1999

Opioid receptor gene: cytokine response element and the effect of cytokines.

Previous studies demonstrated that cytokines regulate opioid and opioid receptor gene expression in neuronal and immune cells. The gene sequence analysis of opioid receptors revealed that mu-, delta- and kappa-opioid receptor promoter regions contain potential cytokine response elements (NF-IL6 binding sites). It was postulated that the response elements present in opioid receptor promoter regions may have a role in the cytokine effects on opioid receptor gene expression through cis-trans interaction. The present study investigated whether cytokines have an effect on opioid receptor gene expression by cytokine-induced transcription factor, NF-IL6, using a number of immune cell lines which respond to cytokines. Further investigation was made to determine whether the potential cytokine response element DNA sequences on opioid receptor promoter region have functional significance which may be affected by the DNA context of the opioid receptor promoter in immune cell lines. Tandem repeats of conserved cytokine response elements of IL-6 gene fused to a heteropromoter SV40 were utilized as a positive control and expressed 2-fold increased promoter activity after cytokine stimulation. Transient transfection studies in time courses (24-72 h) and different dose treatments (100-500 U/ml for IL-6 and 50-200 U/ml for IL-1 alpha+beta) were also carried out to investigate the possibility that the upregulated gene expression may be transient or cytokine-dose-dependent. Our data demonstrated that there was no significant cytokine-stimulated opioid receptor gene expression in immune cells tested. In addition, the cytokine response elements (NF-IL6 binding sites) in opioid receptor genes are not functional. These results contradict the previous reports in which cytokines modulated the expression of opioid and opioid receptors in neuronal and immune cells. The possible reasons regarding the different results were discussed.

Binding Sites↗
PubMed1999

Promoter activity of mouse kappa opioid receptor gene in transgenic mouse.

The biological activity of mouse kappa opioid receptor (KOR) gene promoter was examined in transgenic mice using a beta-galactosidase (lacZ) reporter strategy for the first time. A lacZ cDNA was inserted at the 5th amino acid in the coding region of a mouse KOR genomic segment containing 3 kb of the 5' regulatory region, to generate a Kor-lacZ fusion gene which was then used to generate transgenic mice. The expression of transgene was demonstrated at the RNA level by reverse transcription-polymerase chain reaction (RT-PCR), and at the protein level by in situ lacZ enzyme assay. From studying three independent transgenic mouse lines that express this transgene, it is concluded that Kor-lacZ expression begins at embryonic day 9.5 (E9.5) and increases in several brain areas and neural tube as embryos develop. At E12.5 and E13.5, Kor-lacZ expression is found primarily in the mantle layer of midbrain, hindbrain and medulla oblongata, cranial ganglion and vagus nerve. At E15.5 and E17.5, the transgene is expressed in eye, ear, neopallial cortex, caudate putamen, lateral ventricle, thalamus, hypothalamus and pons. Therefore, the 3 kb 5' regulatory sequence of the mouse KOR gene is functional in transgenic animals and directs a specific expression pattern recapitulating that of the endogenous KOR gene expression during developmental stages. However, in adult animals, this transgene is only expressed in the brain, indicating that the regulatory information for peripheral expression in the adult is not encoded within this 3 kb upstream sequence.

Age Factors↗
PubMed1999

The absence of a direct correlation between the loss of [D-Ala2, MePhe4,Gly5-ol]Enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation.

Chronic activation of the mu-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) receptors were expressed in the same system, overexpression of beta-adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.

Adenylyl Cyclase Inhibitors↗