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H H Loh

Publications and source records attributed to H H Loh.

At least 73 records · Page 4Linked to original sources
PubMed1997

Dual promoters of mouse mu-opioid receptor gene.

Two transcriptional initiation sites, distal and proximal, located at approximately 500 bp apart in mouse mu-opioid receptor gene were identified recently. Using deletional and transfection analyses, the distal or proximal promoter alone displayed similar activity in neuronal cells constitutively expressing mu-opioid receptors (SH-SY-5Y). The presence of both promoters did not result in an increase in activity. However, when distal promoter linked with 3'-downstream sequences without the proximal promoter region, the distal promoter activity was abolished. Transfection analysis using various cell lines further suggested that only the proximal promoter preferentially directed the neuronal subtype expression. To verify the physiological importance of each promoter, the ratio of mu-receptor transcripts initiated by either promoter was examined by quantitative RT-PCR using mouse adult brain mRNA. We found that receptor mRNA was predominantly initiated by the proximal promoter. These studies suggested that the proximal promoter was the major functional promoter.

Animals↗
PubMed1997

Agonist activation of delta-opioid receptor but not mu-opioid receptor potentiates fetal calf serum or tyrosine kinase receptor-mediated cell proliferation in a cell-line-specific manner.

Activation by opioid receptors of cell proliferation was examined with fibroblast cell lines stably expressing either delta-opioid or mu-opioid receptors. Addition of [D-Ala2, D-Leu5]-enkephalin or [D-Pen2,D-Pen5]-enkephalin to Chinese hamster ovary (CHO) cells transfected with delta-opioid receptor cDNA resulted in an agonist concentration-dependent potentiation of fetal calf serum (FCS)-stimulated cell proliferation. This potentiation by delta-opioid agonists was antagonized by naloxone and was not observed with the kappa-opioid receptor selective agonist U50,488 or the mu-opioid receptor selective agonist [D-Ala2,N-MePhe4, Gly-ol5]-enkephalin. This delta-opioid agonist effect was not observed at FCS concentrations > 0.1% and could be blocked by pretreating cells with pertussis toxin, indicating that Gi/Go were involved in this action. In addition, delta-opioid agonists could potentiate CHO cell proliferation stimulated by those growth factors that are mediated by tyrosine kinase receptors (i.e., insulin, insulin-like growth factor 1, and fibroblast-derived growth factor b). This delta-opioid agonist potentiation of growth apparently was dependent on the level of delta-opioid receptors that were expressed and had cell-line selectivity. Activation of delta-opioid receptors expressed in Rat-1 or NIH3T3 fibroblast did not result in a modulation of the cell growth induced by FCS or by growth factors. Interestingly, in CHO cells transfected with mu-opioid receptor cDNA, activation with agonists did not produce a potentiation of FCS-stimulated proliferation. This lack of mu-opioid receptor effect was not due to the differences among CHO clones. In a CHO cell line transfected with both delta-opioid receptor cDNA and mu-opioid receptor cDNA, activation of delta-but not mu-opioid receptors resulted in a potentiation of growth. These data suggest that delta- and mu-opioid receptors in CHO cells activate similar but divergent second messenger pathways, resulting in the differential regulation of cell growth.

3T3 Cells↗
PubMed1997

Studies of dual promoters of mouse kappa-opioid receptor gene.

Dual promoters were identified in the mouse kappa-opioid receptor (KOR) gene. The distal promoter was located in the 5'-upstream region of exon 1 and the proximal promoter was located in the first intron of this gene. The transcription initiation site of the proximal promoter was mapped to the -93rd nucleotide position from the ATG codon in a primer extension experiment. The expression of KOR mRNAs transcribed from these two promoters in mouse central nervous system and an embryonal carcinoma cell line P19 was confirmed in a ribonuclease protection assay. In non-neuronal tissues, only the transcripts initiated from the distal promoter were detected. The biological activities of these two promoters were determined in transient transfection of P19 cells with a series of reporters, each truncated at various 5'-upstream regions. It was concluded that the distal promoter was located between nucleotide positions -990 and -570, and the proximal promoter was located between nucleotide positions -330 and -93, relative to the translation initiation codon. The presence of dual promoters in the KOR gene suggested potential regulation of KOR expression by using different promoters.

Animals↗
PubMed1997

The mu-opioid receptor down-regulates differently from the delta-opioid receptor: requirement of a high affinity receptor/G protein complex formation.

Chronic opioid treatment of Neuro2A cells stably expressing either delta-opioid receptor (DOR) or mu-opioid receptor (MOR) resulted in agonist-dependent receptor down-regulation. Although there is high homology in the DOR and MOR amino acid sequences, there is an apparent difference in the regulation of the cellular levels of these two receptors. The ability of 24-hr [D-Pen2,D-Pen5]enkephalin (DPDPE) treatment to internalize and down-regulate DORs expressed in Neuro2A remained intact after pertussis toxin (PTX) pretreatment, which uncouples the receptor from G proteins. In contrast, the ability of [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) to internalize and down-regulate MORs in Neuro2A cells was completely abolished by PTX pretreatment. The requirement of functional MOR but not DOR in agonist-induced receptor down-regulation was further demonstrated by site-directed mutagenesis of the receptors. When Asp114 in transmembrane 2 of MOR was converted to alanine, the ability was abolished of DAMGO or morphine to inhibit forskolin-stimulated [3H]cAMP production in Neuro2A cells stably expressing this mutant receptor. There was a parallel decrease in agonist affinity and elimination of the agonist-induced receptor down-regulation. On the other hand, although the equivalent mutation of Asp95 to alanine in DOR likewise resulted in the inability of DPDPE to inhibit [3H]cAMP production, the ability of DPDPE to down-regulate this mutant receptor after 24-hr treatment was unaffected. This difference in MOR and DOR down-regulation could be caused by the differences in the ability of these two receptors to form high affinity complexes with G proteins. DOR retained the ability to form high affinity complexes even after PTX pretreatment or after mutation of Asp95 in transmembrane 2. In contrast, MOR existed only in the low affinity, uncoupled state after PTX pretreatment or after conversion of Asp114 to alanine. Therefore, in Neuro2A cells, agonist-induced opioid receptor down-regulation seems to depend directly on the formation of the high affinity receptor complexes and not on the activation of the receptors and subsequent transduction of the signals.

Animals↗
PubMed1996

Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.

Animals↗
PubMed1996

Orphanin FQ/nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord.

Antisera were developed that specifically recognize orphanin FQ/nociceptin, the 17 amino acid peptide reported to be the endogenous ligand for the orphan opioid receptor. Immunocytochemical localizations in rat spinal cord demonstrated that orphanin FQ /nociceptin-immunoreactivity (-ir) was abundant in superficial dorsal horn, lateral spinal nucleus and the region dorsal to the central canal, areas that also exhibit prominent enkephalin-and dynorphin-ir. Orphanin FQ/nociceptin-ir was not affected by dorsal rhizotomy, indicating that in spinal cord the peptide is produced by central rather than primary afferent neurons. thus, the distribution of orphanin FQ/nociceptin-ir appeared in neuronal circuits that parallel those containing enkephalin- and dynorphin-ir, with only modest co-existence of these peptides.

Amino Acid Sequence↗
PubMed1996

Differential effects of opioids on the proliferation of a macrophage cell line, Bac 1.2F5.

Chronic treatment of mice with morphine selectively abrogates the terminal differentiation of committed bone marrow progenitor cells to form macrophage colony forming units. To understand the molecular mechanisms involved in morphine-mediated suppression of myeloid cell differentiation, we investigated the use of a macrophage cell line, Bac 1.2 F5. In vitro proliferation of this cell line is dependent on the exogenous supply of macrophage colony stimulating factor. Treatment of Bac 1.2F5 cells in vitro with morphine showed a dose-dependent inhibition of proliferation which was associated with morphological changes. Characterization of the binding site revealed that the binding site for morphine on these cells is different from the classical opioid receptors described in the brain. In addition to the putative novel class of morphine receptors, Bac 1.2F5 cells also expressed the delta opioid receptors as determined by RT-PCR analyses. These studies show that Bac 1.2F5 cells are suitable for the molecular characterization of opioid effects on the proliferation and differentiation of myeloid progenitor cells.

Animals↗
PubMed1996

Effects of opioids on the immune system.

Both therapeutic and chronic uses of opioids compromise the optimal functioning of the immune system. Overwhelming evidence suggests that opioid use affects both innate immunity and adaptive immunity. Chronic administration of opioids decreases the proliferative capacity of macrophage progenitor cells and lymphocytes. Additionally, the differentiated function of immune cells is significantly affected by opioids. These effects are mediated by either a direct action of opioids on the target cells or by indirect centrally mediated pathways. Molecular biological and biochemical characterization suggest that immune cells differentially express classical opioid receptors. Interestingly, these studies also reveal the presence of a novel class of opioid receptors in immune cells. We believe that this low affinity morphine binding site mediates the antiproliferative effects of morphine.

Animals↗
PubMed1996

Differential expression of opioid receptor genes in human lymphoid cell lines and peripheral blood lymphocytes.

The existence of receptors for opioid compounds on cells of the immune system has long been hypothesized, but has been very difficult to demonstrate unequivocally. We have used reverse-transcription polymerase chain reaction to obtain cDNA clones from the human MOLT-4 and CEM-3 T-leukemic cell lines which are nearly identical to portions of the delta and kappa opioid receptor cDNAs recently isolated from human brain and placenta, respectively. Northern analyses with riboprobes derived from the delta and kappa opioid receptor clones indicate these sequences are expressed at low levels in human peripheral blood lymphocytes and in several human lymphoid cell lines. Sequences corresponding to the mu opioid receptor cDNA were not detected in this study. The results suggest that delta and kappa opioid receptors may be responsible for mediating some direct effects of opioids in immune cells.

Base Sequence↗
PubMed1996

Expression cloning of a full-length cDNA encoding delta opioid receptor from mouse thymocytes.

A delta opioid receptor complementary DNA (cDNA) was cloned by expression of cDNA library from activated thymocytes in Cos 7 cells. The deduced amino acid sequence of this receptor was similar to that described in the brain. As analyzed by southern blot hybridization, the delta opioid receptor transcripts are constitutively expressed in unactivated thymocytes. In addition, neither kappa nor mu opioid receptor transcripts were detected in thymocytes, suggesting tissue-specific opioid receptor gene expression in the immune system. The studies represent the first report of a full-length opioid receptor in the immune system.

Amino Acid Sequence↗
PubMed1996

Expression of OBCAM-related cDNA clones in Cos 1 cells: evidence for a phosphatidylinositol linkage to the cell membrane.

Previously, our laboratory purified and isolated the cDNA for OBCAM (opioid binding cell adhesion molecule) from bovine brain, as well as highly homologous rat brain cDNA clones, SG13 and DUZ-1. Structural similarities with members of the immunoglobulin superfamily suggest a possible role for OBCAM in cell adhesion and recognition, while studies in our own laboratory suggest that OBCAM is important in the regulation of opioid binding and signal transduction. However, OBCAM lacks a putative transmembrane domain, and its possible mode of linkage to the cellular membrane has not been studied. Upon transfection of Cos 1 cells with SG13 and DUZ-1 cDNAs, the OBCAM-homologous proteins were expressed on the surface of the Cos 1 cells. These proteins were released from the membrane of the Cos 1 cells upon digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), demonstrating that they are linked to the membrane via a phosphatidylinositol (PI) linkage. These results are consistent with a role for OBCAM in cell recognition and adhesion, as well as in cellular signaling.

Animals↗
PubMed1996

Activation of rat thymocytes selectively upregulates the expression of somatostatin receptor subtype-1.

Somatostatin and other neuropeptides are known to modulate the proliferative capacity of immune cells. In the present study, we investigated the expression of Somatostatin receptor (SSTR) subtypes on rat thymocytes. RT-PCR analysis of fresh thymocytes showed significant levels of transcripts for the SSTR2 whereas transcripts for the SSTR1 and SSTR3 were not detectable. Interestingly, when the thymocytes were activated with low concentration of Phytohemagglutinin and interleukin 1, the transcript for SSTR1 was markedly increased. Lymphokine induced activation of thymocytes selectively upregulated the SSTR1 since, transcripts for SSTR2 remained the same after activation and SSTR3 was not detectable. PCR amplified fragment of SSTR1 from the activated thymocytes showed identical sequence to the rat brain receptor. The physiological significance of the increase of SSTR1 mRNA in thymocytes after activation remains to be elucidated but it may be possible that these two different subsets of receptors (SSTR1 and SSTR2) are involved in the modulation of thymocyte proliferation and differentiation.

Amino Acid Sequence↗
PubMed1995

The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids.

Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin.

Amino Acid Sequence↗
PubMed1995

Distribution of neuropeptide receptors. New views of peptidergic neurotransmission made possible by antibodies to opioid receptors.

The cloning of receptors for neuropeptides made possible studies that identified the neurons that utilize these receptors. In situ hybridization can detect transcripts that encode receptors and thereby identify the cells responsible for their expression, whereas immunocytochemistry enables one to determine the region of the plasma membrane where the receptor is located. We produced antibodies to portions of the predicted amino acid sequences of delta, mu, and kappa opioid receptors and used them in combination with antibodies to a variety of neurotransmitters in multicolor immunofluorescence studies visualized by confocal microscopy. Several findings are notable: First, the cloned delta opioid receptor appears to be distributed primarily in axons, and therefore most likely functions in a presynaptic manner. Second, the cloned mu and kappa opioid receptors are found associated with neuronal plasma membranes of dendrites and cell bodies and therefore most likely function in a postsynaptic manner. However, in certain, discrete populations of neurons, mu and kappa opioid receptors appear to be distributed in axons. Third, enkephalin-containing terminals are often found in close proximity (although not necessarily synaptically linked) to membranes containing either the delta or mu opioid receptors, whereas dynorphin-containing terminals are often found in proximity to kappa opioid receptors. Finally, a substantial mismatch between opioid receptors and their endogenous ligands was observed in some brain regions. However, this mismatch was characterized by complementary zones of receptor and ligand, suggesting underlying principles of organization that underlie long-distance, nonsynaptic neurotransmission.

Afferent Pathways↗
PubMed1995

Complementary DNA cloning of a mu-opioid receptor from rat peritoneal macrophages.

Treatment with opioid agonists in vitro and in vivo has been shown to affect the function of the immune system. Several investigators have suggested that immune cells may express opioid receptors, but it had been very difficult to demonstrate their presence on these cells by direct binding assays. Our earlier studies have shown that macrophage progenitor cells are highly sensitive to morphine treatment in vitro and in vivo. In the current investigation, we determined, unequivocally, the expression of mu-opioid receptor related transcripts in rat peritoneal macrophages by reverse transcriptase-polymerase chain reaction (RT-PCR) studies. In order to further characterize the transcript, the RT-PCR product was cloned and sequenced. The sequence analyses indicate that the transcripts from rat peritoneal macrophages are identical to those for the mu-opioid receptor described in the rat brain. To further confirm the presence of mu-opioid receptors, immunoreactivity to an antiserum raised against the carboxyl terminal fifteen amino acid residues of the mu-opioid receptor was determined. These studies show for the first time that rat peritoneal macrophages express a mu-opioid receptor.

Amino Acid Sequence↗
PubMed1995

Cloning and promoter mapping of mouse kappa opioid receptor gene.

We have isolated mouse kappa opioid receptor (KOR) genomic clones containing the complete 5'-untranslated region, a portion of the 3'-untranslated region, and its coding region. The mouse KOR gene spans at least 16 kb and contains at least 4 exons. Exon I encodes the major portion of 5'-untranslated sequence and transcription initiation is determined, by primer extension experiments, to reside at 3 major sites, located at 334, 340, and 716 nucleotides upstream from the translation initiation codon. No typical TATA or CAAT boxes can be identified within an approximately 1-kb upstream region. Exon II contains 271 nucleotides, including 14 nucleotides of 5'-untranslated sequence. Exon III contains 353 nucleotides and exon IV begins at amino acid position #204 and encodes the rest of the 3'-end sequence of the cloned cDNA. Based upon Northern blot analysis, KOR transcript is estimated to be approximately 6 kb in length.

Amino Acid Sequence↗
PubMed1995

Genomic structure of the mouse delta opioid receptor gene.

Using mouse delta opioid receptor (DOR) cDNA sequence to probe genomic libraries in bacteriophage lambda and P1 vectors, clones traversing the entire DOR coding sequence and 5' and 3' flanking regions were isolate. Genomic sequence encoding mature DOR message, including 5' and 3' untranslated sequence, is divided by two introns of 26 kb and 3 kb, resulting in the gene occupying 32 kb of chromosomal DNA. Multiple putative transcription initiation sites were located, by RNase protection assay, in TATA-less G+C rich sequence between 390 and 140 nucleotides upstream from the ATG translation start codon. A polyadenylation site was located 1.24 kb downstream from the TGA translation stop codon. Examination of 1.3 kb of 5'flanking sequence revealed potential binding sites for several known transcription factors including: Sp1, Ap-2, NF-kappa B, NF-IL6, and NGFI-B.

Animals↗