Biomedical subjects

H H Loh

Publications and source records attributed to H H Loh.

At least 19 recordsLinked to original sources

PubMed1992

Regulation of an opioid-binding protein in NG108-15 cells parallels regulation of delta-opioid receptors.

An opioid-binding protein has recently been purified from bovine brain and cloned, and its cDNA sequence has been obtained. Indirect evidence suggests that this protein has a role in opioid-receptor function. However, because direct testing of its function by expression of its cDNA has not yet been possible and because its structure bears no resemblance to G protein-coupled receptors, the role of this protein in opioid-receptor activity is still in question. An antibody raised to a portion of the predicted amino acid sequence of opioid-binding cell-adhesion molecule (OBCAM) specifically labeled the surface of NG108-15 cells, as visualized by immunofluorescence with confocal microscopy. Furthermore, chronic treatment of these cells with opioid agonist, which down-regulates opioid receptors, reduced OBCAM immunoreactivity (ir). Down-regulation of both opioid receptors and OBCAM-ir was greatest after chronic treatment of NG108-15 cells with delta-opioid agonists, as well as with nonselective agonists such as etorphine, whereas other agonists including [D-Ala2-N-MePhe4-Gly-ol]enkephalin, morphine, levorphanol, dynorphin A-(1-13), and U-50,488H were less effective or ineffective. Chronic treatment of NG108-15 cells with muscarinic agonists had no effect on OBCAM-ir. Furthermore, NG108-15 cells transfected with an antisense construct to OBCAM have a reduced density of opioid-binding sites as well as reduced OBCAM-ir. Taken together, these results strongly suggest that OBCAM has a role in opioid-receptor function in NG108-15 cells.

Amino Acid Sequence↗

PubMed1992

Suppression by dynorphin A-(1-13) of the expression of opiate withdrawal and tolerance in mice.

Dynorphin A-(1-13) has been shown to suppress the expression of opiate withdrawal and tolerance dose dependently in morphine-dependent mice when administered i.v. The ED50 of naloxone to precipitate withdrawal jumping was increased by 1.5- and 7-fold when morphine-dependent mice were pretreated with 2.5 and 5.0 mumol/kg of dynorphin A-(1-13), respectively. When dynorphin A-(1-13) (5.0 mumol/kg, i.v.) was administered after the precipitation of withdrawal with naloxone, the ED50 of naloxone was still increased by over 2-fold. Also, the expression of tolerance which was estimated by noting the antinociceptive ED50 of morphine, was inhibited by over 70% with a dynorphin A-(1-13) dose of 2.5 mumol/kg and completely suppressed by pretreatment with 5.0 mumol/kg of dynorphin A-(1-13) i.v. The mechanism by which dynorphin A-(1-13) produces these effects when given i.v. remains to be elucidated.

Animals↗

PubMed1992

Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library.

A rat brain cDNA library was screened with probes constructed from portions of the cDNA (OBCAM) encoding the opioid-binding cell adhesion molecule (OBCAM). Three clones of interest were isolated and sequenced. The largest clone, DUZ1, had a putative open reading frame (ORF) essentially identical to OBCAM in its C-terminal 318 amino acids (aa), but differing in its N-terminal aa (20 vs. 27 in OBCAM), and in all of its 5'-noncoding regions. The other clones, SG8 and SG13, had a putative ORF essentially identical to that of OBCAM, but differed from each other in a portion of their 5'-noncoding region. This study suggests that there is a family of OBCAM-like genes.

Amino Acid Sequence↗

PubMed1992

Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.

Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.

3T3 Cells↗

PubMed1992

Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.

Amino Acid Sequence↗

PubMed1992

Antisense proenkephalin cDNA transfection decreases opioid binding in NG 108-15 cells.

The proenkephalin A (PENK) gene codes for several opioid peptides, including Met-enkephalin, an endogenous ligand of opioid receptors. These peptides are thought to play an important role in a variety of neural processes. To study the role of the PENK gene in opioid related processes, an antisense sequence of PENK gene was subcloned into pSVL SV 40 late promoter expression vector and stably transfected into NG 108-15 cells, which contain opioid receptors. The sense orientation of the same fragment was also cloned and transfected, serving as a control. The presence and expression of transfected recombinant plasmids in NG 108 cells were confirmed by DNA and RNA PCR and by subsequent sequencing. Surprisingly, the endogeneous PENK message level was found to be 3 times higher in antisense cells than in sense or NG 108 cells. This high steady-state mRNA level seemed to be due to the increased stability of PENK mRNA rather than to an elevated transcription rate. Nevertheless, the level of total Met-enkephalin was found to be reduced in antisense-transfected cells, though free Met-enkephalin content did not differ from sense-transfected or non-transfected cells. We suggest that both the increased PENK message and the unchanged levels of free Met-enkephalin may be the result of compensatory mechanisms induced by translational inhibition by antisense, although the underlying processes remain to be determined. Binding of the opioid ligand [3H]diprenorphine was significantly reduced by 50-80% in the antisense-transfected cell lines, but not in the sense cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells.

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.

Adenosine Diphosphate Ribose↗

PubMed1992

Characterization of [3H]morphine binding to interleukin-1-activated thymocytes.

We have previously reported that interleukin-1-induced proliferation of thymocytes is accompanied by the appearance of [3H]morphine binding sites on these cells. In the present study, we have characterized these binding sites. They differ from classical opioid receptors in the brain in several ways, including: 1) lack of stereoselectivity; 2) relatively low affinity (Kd = 50 nM) and high capacity (Bmax = 3 pmol/mg of protein); 3) binding is strongly inhibited by Ca++, Mg++, Mn++ and Cl- ions and 4) binding is inhibited by proteinase K or E and by phospholipase A2 but not trypsin treatment of thymocyte membranes. The binding sites, which were found largely on the CD4+ subset of T-cells, also showed a preference for opioid alkaloids over peptides. These [3H]morphine binding sites may mediate a negative feedback effect on interleukin-1-induced proliferation of thymocytes in vivo.

Animals↗

PubMed1992

Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1991

Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins.

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.

Adenosine Diphosphate Ribose↗

PubMed1991

Dynorphin blocks opioid inhibition of macrophage-colony stimulating factor-induced proliferation of bone marrow cells.

We previously reported that morphine inhibits macrophage-colony stimulating factor (M-CSF)-induced proliferation of mouse bone marrow cells, both in vivo and in vitro, in a dose-dependent manner. We now report that either dynorphin A-(1-13) or dynorphin A-(1-10) amide, though having no effect on proliferation by themselves at concentrations less than 0.1 mM, can block the inhibitory effect of morphine both in vivo and in vitro, in a dose-dependent manner. The opioid antagonist naloxone can also block morphine's inhibitory effect on bone marrow cell proliferation in vivo, but has no effect in vitro. Dynorphin A-(1-13) was also able to block the dramatic reduction of spleen weight observed in animals chronically treated with morphine. Thus dynorphin, which has previously been shown to antagonize morphine analgesia, is also able to antagonize some of the immunosuppressive effects of morphine.

Analgesics↗

PubMed1991

Chronic effect of [D-Pen2,D-Pen5]enkephalin on rat brain opioid receptors.

In previous studies, we have demonstrated that chronic etorphine or [D-Ala2,D-Leu5]enkephalin (DADLE) treatment of rats results in the reduction of mu- and delta-opioid receptor binding activities as tolerance develops. As both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu- and delta-receptors, these studies could not determine whether down-regulation of a specific receptor type occurs. Therefore, in the present studies, animals were rendered tolerant to the delta-opioid receptor-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and receptor binding activities were measured. Treating Sprague-Dawley rats with increasing doses of DPDPE (80-160-240-320 micrograms/kg) i.c.v. for 1 to 4 days resulted in a time-dependent increase in the AD50 of DPDPE to elicit an antinociceptive response. When delta-receptor binding was determined by using [3H]DPDPE, a 40-50% decrease in binding in the midbrain and cortex, and 25-35% decrease in binding in the striatum were observed after 3 or 4 days of DPDPE treatment. Scatchard analysis of the [3H]DPDPE saturation binding data revealed a decrease in Bmax values and no significant change in Kd values. To our surprise, when mu-receptor binding was determined by using [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), a 10-15% decrease in binding was also observed in the midbrain and cortex after 4 days of DPDPE treatment. Our conclusion is that chronic DPDPE treatment preferentially reduces delta-opioid receptor binding activity. Its minor effect on the mu-opioid receptor maybe due to an interaction between delta cx and mu cx binding sites.

Analgesics↗

PubMed1991

[3H]morphine binding is enhanced by IL-1-stimulated thymocyte proliferation.

Mouse thymocytes incubated in vitro with increasing concentrations of interleukin-1 (IL-1) in the presence of phytohemagglutinin (PHA) exhibited a dose-dependent increase in cell proliferation, as measured by [3H]thymidine incorporation. Under these conditions, there was a parallel dose-dependent increase in specific [3H]morphine binding, with a maximum increase of approximately 5-fold over basal levels. The binding sites differ from classical opioid receptors in that they are not stereo-selective. Interleukin-2 was ineffective in promoting either cell proliferation or enhanced opioid binding, but the effects of IL-1 could be mimicked by phorbol myristate acetate (PMA), suggesting the involvement of tyrosine phosphorylation. These results indicate that morphine-binding sites on immune cells can be regulated by cytokine activation.

Animals↗

PubMed1991

Chronic morphine treatment selectively suppresses macrophage colony formation in bone marrow.

Opioids have been shown to have diverse effects on the immune system, both in vivo and in vitro, but their interactions on immature progenitor cells have been little studied. We have examined the effects of chronic morphine treatment of mice on colony formation by bone marrow cells in vitro. Bone marrow cells from mice implanted with morphine pellets for 72 h showed a 65% decrease in their response to macrophage colony stimulating factor (M-CSF). In contrast, chronic morphine treatment had no effect on the response of bone marrow cells to granulocyte/macrophage colony stimulating factor (GM-CSF). Removal of the morphine pellets from the mice resulted in a time-dependent reversal of the inhibition of macrophage colony formation, and the inhibition was completely blocked by simultaneous administration of naloxone and morphine pellets to the mice. No inhibition of colony formation was observed in bone marrow cells from mice treated with a single acute dose of morphine. Incubation of bone marrow cells from untreated mice for 7 days with in vitro morphine concentrations as low as 25 microM also reduced macrophage colony formation, and the opioid peptide beta-endorphin was even more potent, significantly reducing macrophage colony formation at concentrations as low as 0.25 microM. In agreement with the in vivo effects, neither opioid in vitro had a significant effect on granulocyte/macrophage colony formation. These results suggest that opioids may significantly alter the maturation of immune cells, which could result in potent effects on overall immune competence.

Animals↗

PubMed1991

Problems and approaches in studying membrane opioid receptors.

Considerable progress has been made in recent years in understanding opioid receptors at the molecular level. Several laboratories have reported purification of opioid receptors by standard procedures involving solubilization of brain membranes in nondenaturing detergent, followed by affinity chromatography and other fractionation methods. In addition, several alternative approaches have been used with some degree of success. Several investigators have labeled receptors with covalent ligands, then characterized them under denaturing conditions such as SDS gel electrophoresis. Antibodies have been prepared to several pure or partially pure opioid receptor preparations that can be used to (1) map the brain regional distribution of opioid receptors, (2) determine the role of specific regions of the receptor molecules in ligand binding and in interaction with other functional molecules, (3) compare the structural features of different opioid receptor types, and (4) purify receptors further. One group also has reported cloning of the cDNA for an opioid-binding protein using antibodies as affinity ligands to purify receptors from solubilized material and the application of gene cloning techniques. The successful use of these varied approaches ensures that future progress in this field will be rapid. The purification, reconstitution, and detailed structural characterization of different opioid receptor types appears feasible within a few years. The next challenge in the molecular understanding of these receptors will be to identify and characterize the second messenger systems in the cell membranes that mediate their pharmacological functions.

Animals↗

PubMed1991

Effect of chronic D-Ala,2 D-Leu5-enkephalin or pertussis toxin treatment on the high-affinity state of delta opioid receptor in neuroblastoma x glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗

PubMed1990

Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL017 treatment.

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2, D-Leu5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, mu-opioid receptor binding capacity was attenuated together with the delta-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu and delta receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the mu-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20 micrograms/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED50 of the peptide to elicit the catatonic effect. When mu- and delta-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the mu-opioid receptor binding but not in delta-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence, mu-opioid receptors can be specifically regulated by ligand in the brain as delta-receptors are in neuroblastoma x glioma NG 108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Opioid-binding protein (OBCAM) is rich in beta-sheets.

Based on circular dichroism (CD) and the sequence-predictive method, the opioid-binding cell adhesion molecule (OBCAM) consisted of one half beta-sheets and one fourth alpha-helices. This is consistent with significant sequence homology of the protein to several members of the immunoglobulin (Ig) superfamily, particularly cell adhesion molecules, which are rich in beta-sheets. Hydropathy analysis suggests that hydrophobic and hydrophilic regions were evenly distributed along the sequence, but the NH2- and COOH-termini were hydrophobic. Hydrophobic moments and Fourier-transform amphipathic analyses further suggest that residues 23-30 and 83-93 were amphipathic beta-sheets. The overall conformation of OBCAM was unaltered by adding linoleic acid, which is required for opioid ligand binding.

Animals↗