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H H Loh

Publications and source records attributed to H H Loh.

At least 91 records · Page 5Linked to original sources
PubMed1995

Isolation of a cDNA encoding a novel zinc-finger protein from neuroblastoma x glioma NG108-15 cells.

A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the delta-opioid agonist D-Ala2-D-Leu5 enkephalin (DADLE) to down-regulate the delta-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein containing 14 tandemly repeated zinc fingers (Zf) with high homology to the Krüppel family of Zf proteins. NGD16-4 also contains a region homologous to the A element of the Krüppel Associated Box (KRAB) domain, a domain recently linked to transcriptional repression. Southern and Northern analyses indicate that NGD16-4 is derived from the mouse genome. Northern analysis also demonstrates that expression of NGD16-4 mRNA is much higher in several mouse neuroblastoma cell lines than in mouse brain or other tissues. Although the function of NGD16-4 is unclear, the expression pattern of NGD16-4 indicates a possible association with the processes of differentiation or transformation in the mouse.

Amino Acid Sequence↗
PubMed1995

Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual G alpha subunit and is similar to that of other opioid receptors.

The purpose of the present study was to examine the coupling pattern of a recently cloned kappa-opioid receptor stably transfected in CHO cells to individual G alpha subunits with subsequent comparison to that observed previously for delta- and mu-opioid receptors. Data presented in the current study indicate the successful stable expression of a kappa-opioid receptor in CHO cells. This is supported by experiments in which ligands with selectivity for kappa-, but not delta- or mu-opioid receptors demonstrated high affinity for the expressed receptor and were able to potently and efficaciously produce inhibition of adenylyl cyclase activity. In addition, only kappa-opioid agonists were able to induce dose-dependent increases in the incorporation of [32P]azidoanilido-GTP into four G alpha subunits, three of which were identified as Gi3 alpha, Gi2 alpha and Go2 alpha. Further, the amount of kappa-opioid agonists required to induce 50% maximal labeling of any individual G alpha subunit was similar. Although kappa-opioid agonists produced equivalent maximal labelling of Gi3 alpha, Gi2 alpha and Go2 alpha, significantly less agonist-induced labeling was observed for an unknown G-protein designated as G? alpha. Although these results are slightly different than those observed previously for both delta- and mu-opioid receptors, it appears that all opioid receptors stably transfected in CHO cells interact with multiple G-proteins and that this coupling is not selective for any individual G alpha subunit.

Adenylyl Cyclases↗
PubMed1995

Neuroblastoma Neuro2A cells stably expressing a cloned mu-opioid receptor: a specific cellular model to study acute and chronic effects of morphine.

Several cellular systems display desensitization and downregulation of opioid receptors upon chronic treatment, suggesting that they could be used as a model system to understand opioid tolerance/dependence. However, a model system containing a homogeneous population of mu-opioid receptors, the receptors at which morphine and related opioids act, has been lacking. To approach this problem, the mu-opioid receptor (MOR-1) was stably expressed in murine neuroblastoma Neuro2A cells after transfection. The expressed receptor was negatively coupled to adenylyl cyclase through Gi/Go proteins, displayed high affinity ligand binding, and was expressed in high number (2.06 pmol/mg of [3H]diprenorphine binding sites). In addition, loss of ability of mu-opioids to acutely inhibit forskolin-stimulated cAMP formation was observed after 4-24 h of chronic exposure to these agonists with concentrations as low as 300-500 nM. The effects of chronic morphine or [D-Ala2,N-MePhe4,Gly-ol]enkephalin (DAMGO) administration were found to be time- and concentration-dependent. Cross 'tolerance' was also observed. Thus the IC50 value of DAMGO to inhibit adenylyl cyclase was increased by 27-fold from 4.3 nM in control cells to 117 nM in cells pretreated with 300 nM morphine; there was no effect on the inhibition of adenylyl cyclase mediated by muscarinic receptors. Further, receptor downregulation accompanied the desensitization process. However, different time-dependence for these two processes suggests, in line with other studies, that these are entirely different cellular adaptation processes. In addition, the opioid antagonist naloxone induced an acute increase in intracellular cAMP level (2-3 times above the control level) following chronic agonist exposure. This process was also concentration-dependent. Overall, these results suggest that the cell line utilized in this study has a homogeneous population of mu-opioid receptors, providing an ideal cellular model to study the molecular mechanisms underlying chronic morphine treatment.

Analgesics↗
PubMed1995

Molecular cloning of a novel protein regulated by opioid treatment of NG108-15 cells.

A new cDNA clone, NGD5, has been identified from a subtraction cDNA library constructed with mRNA isolated from control neuroblastoma x glioma NG108-15 cells and cells treated for 48 h with the delta-opioid agonist, D-Ala2, D-Leu5 enkephalin (DADLE). NGD5 mRNA is decreased, in a naloxone-reversible manner, upon long-term treatment of NG108-15 cells with DADLE, indicating that this clone may be related to opioid receptor function. Northern analysis indicates that NGD5 mRNA is expressed in rat brain. Two similar nearly full-length NGD5 clones, NGD5A and NGD5B, were isolated from a lambda gt10 NG108-15 cDNA library and sequenced. The predicted 40-kDa peptide encoded for by the NGD5 cDNA has no significant homology to the recently cloned mu, delta or kappa opioid receptors nor to any other known proteins.

Amino Acid Sequence↗
PubMed1995

Expression of alternate forms of brain opioid 'orphan' receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines.

We screened a PHA (phytohemagglutinin)-activated human lymphocyte cDNA library for clones with homology to the recently cloned brain opioid receptors. A cDNA clone, AT7-5EU, was isolated which encodes the opioid 'orphan' receptor, a molecule with very high homology to the opioid receptor gene family, but which has not been shown to bind opioids or any other known compounds. The protein coding region of AT7-5EU has complete homology with a reported opioid 'orphan' clone isolated from human brain, but the 5' untranslated regions of AT7-5EU and the human brain clones are divergent, suggesting mechanisms for tissue-specific expression of this receptor. Northern analysis of AT7-5EU mRNA demonstrates the expression of this message in human lymphocytic cell lines of both B-and T-cell lineages. Furthermore, analysis of mRNA from human peripheral blood lymphocytes demonstrates that activation of the lymphocytes with PHA results in at least a 10-fold induction of the AT7-5EU message. These results suggest that the opioid 'orphan' receptor may have an important immunological function in addition to its function in the nervous system.

Base Sequence↗
PubMed1995

Expression of the mu-opioid receptor in CHO cells: ability of mu-opioid ligands to promote alpha-azidoanilido[32P]GTP labeling of multiple G protein alpha subunits.

The identities of heterotrimeric G proteins that can interact with the mu-opioid receptor were investigated by alpha-azidoanilido[32P]GTP labeling of alpha subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed mu-opioid receptor cDNA (MOR-1). This clone expressed 1.01 x 10(6) mu-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the mu-opioid-selective ligands [D-Ala2,N-MePhe4, Gly-ol]-enkephalin and [N-MePhe3,D-Pro4]-morphiceptin, relative to the delta-selective opioid agonist [D-Pen2,D-Pen5]-enkephalin or the kappa-selective opioid agonist U-50,488H. mu-Opioid ligands induced an increase in alpha-azidoanilido[32P]GTP photoaffinity labeling of four G alpha subunits in this clone, three of which were identified as Gi3 alpha, Gi2 alpha, and Go2 alpha. The same pattern of simultaneous interaction of the mu-opioid receptor with multiple G alpha subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of alpha-azidoanilido[32P]GTP incorporation into Gi2 alpha (160-280%) and Go2 alpha (110-220%) than for an unknown G alpha (G? alpha) (60%) or Gi3 alpha (40%) was produced by three different mu-opioid ligands tested. In addition, slight differences were also found between the ability of various mu-opioid agonists to produce half-maximal labeling (ED50) of any given G alpha subunit, with a rank order of Gi3 alpha > Go2 alpha > Gi2 alpha = G? alpha. In any case, these results suggest that the activated mu-opioid receptor couples to four distinct G protein alpha subunits simultaneously.

Affinity Labels↗
PubMed1995

delta-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin.

We have recently developed antisera which recognize epitopes of the cloned delta-opioid receptor (DOR; Dado et al., 1993). In the present report we have further characterized these antisera, and raised additional antisera in rats. We used these antisera to determine the distribution of DOR-like immunoreactivity (-Ll) in rat spinal cord and brainstem in relation to serotoninergic, noradrenergic, and enkephalinergic neurons. We found DOR-Ll in fibers and varicosities distributed throughout the spinal cord gray matter, with highest densities in the superficial dorsal horn, in autonomic regions, around the central canal as well as in the ventral horn motor nuclei. In the brainstem a dense innervation of DOR-immunoreactive (-IR) fibers was found in several nuclei such as spinal trigeminal nuclei, midline raphe nuclei, parabrachial nuclei, periaqueductal gray matter (PAG), interpeduncular nucleus, ans substantia nigra. A group of DOR-positive cells was seen in the laterodorsal tegmental nucleus. In addition, a few DOR-IR cell bodies were demonstrated in the parabrachial nuclei, interpeduncular nucleus, PAG, and superior and inferior colliculi as well as around the central canal in the spinal cord. All DOR-positive cells showed a punctuate staining pattern within the cytoplasm of the cell body and in primary dendrites. No plasma membrane staining of cells or dendrites could be demonstrated using the DOR antisera. Double-labeling experiments for DOR and 5-hydroxytryptamine (5HT, serotonin) revealed that some 5HT-IR neurons in the raphe complex were surrounded by DOR-IR fibers. In the spinal cord a high degree of coexistence was found between DOR and 5HT in nerve fibers and varicosities in the neuropil around the motoneurons and in lamina V of the dorsal horn. In autonomic regions of the spinal cord, a low degree of colocalization was seen between DOR and 5HT; in the superficial dorsal horn no coexistence was found. Tyrosine hydroxylase (TH)-positive neurons in the brainstem (in the A5 area, locus coeruleus, and A7 area) were apposed by DOR-positive fibers. However, no coexistence could be seen between DOR and TH in any part of the spinal cord. A close relation, but no coexistence, was observed between DOR- and enkephalin (ENK)-IR fibers in the spinal cord ventral horn; in the intermediolateral nucleus a low degree of colocalization was observed. Thus, a delta-opioid receptor may affect the activity of descending serotoninergic and noradrenergic neurons by means of modulating the release of neurotransmitters from afferents to these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Ca2+ channel and adenylyl cyclase modulation by cloned mu-opioid receptors in GH3 cells.

Members of the three classes of opioid receptors (mu, delta, and kappa) have been cloned and characterized in unexcitable cell lines using biochemical techniques. However, an important function of these cloned receptors, their coupling to voltage-activated Ca2+ channels, remains untested. We stably transfected cloned rat mu-opioid receptor cDNAs into clonal pituitary GH3 cells. GH3 cells expressing mu-opioid receptors (GH3MOR cells) bound the receptor-specific ligands [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and morphine with high affinity (Ki = 1.0 and 7.2 nM, respectively), and these ligands also potently inhibited adenylyl cyclase activity (IC50 = 21.9 and 55.2 nM, respectively). Functional coupling of mu-opioid receptors to voltage-activated Ca2+ channels was compared with that of endogenous somatostatin (SRIF) receptors in GH3MOR cells, using the patch-clamp technique, with Ba2+ as the charge carrier. DAMGO (1 microM) and SRIF (1 microM) inhibited Ba2+ currents by 23.8 +/- 1.0% and 22.9 +/- 2.5%, respectively. DAMGO (0.1 nM to 10 microM) dose-dependently inhibited Ba2+ currents, with an IC50 of 105 nM. The mu-opioid receptor agonist morphine (1 microM) inhibited currents by 13.5 +/- 1.1% and the delta-opioid receptor-selective ligand [D-Pen2,5]-enkephalin (1 microM) caused only 3.5 +/- 2.1% inhibition. The inhibitory actions of DAMGO, morphine, and [D-Pen2,5]-enkephalin were reversed by naloxone. Ba2+ current inhibitions by DAMGO and SRIF were attenuated by pertussis toxin pretreatment. Nimodipine reduced the amplitude of Ba2+ current inhibition by DAMGO, suggesting that mu-opioid receptors couple to L-type Ca2+ channels in GH3MOR cells.

Adenylate Cyclase Toxin↗
PubMed1995

Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord.

Opioid receptors regulate neuronal activity by both pre- and postsynaptic mechanisms. We recently reported that the cloned delta-opioid receptor (DOR1) is primarily targeted to axons, suggesting a presynaptic role. In the present study we have studied the distribution and targeting of another opioid receptor, the mu-opioid receptor (MOR1), by raising anti-peptide antisera to the C-terminal peptide of MOR1. The specificity of the antisera was determined by analysis of transfected cells, Western blots, and immunoisolation studies. Immunohistochemistry showed that MOR1 immunoreactivity was enriched in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus, and the superficial laminae of the dorsal horn. Moreover, MOR1-expressing neurons seem to target this receptor preferentially to their somatodendritic domain as determined by double-labeling experiments with MAP2. However, discrete populations of neurons target MOR1 to their axons, including some primary afferent neurons that express DOR1. In many regions enkephalin-containing axons were complementary to MOR1, suggesting by their proximity that enkephalins may be physiologically relevant ligands for this receptor. Thus, these results provide a morphological basis for understanding pre- and postsynaptic functions mediated by MOR1.

Afferent Pathways↗
PubMed1994

Genomic structure analysis of promoter sequence of a mouse mu opioid receptor gene.

We have isolated mouse mu opioid receptor genomic clones (termed MOR) containing the entire amino acid coding sequence corresponding to rat MOR-1 cDNA, including additional 5' flanking sequence. The mouse MOR gene is > 53 kb long, and the coding sequence is divided by three introns, with exon junctions in codons 95 and 213 and between codons 386 and 387. The first intron is > 26 kb, the second is 0.8 kb, and the third is > 12 kb. Multiple transcription initiation sites were observed, with four major sites confirmed by 5' rapid amplification of cDNA ends and RNase protection located between 291 and 268 bp upstream of the translation start codon. Comparison of the 5' flanking sequence with a transcription factor database revealed putative cis-acting regulatory elements for transcription factors affected by cAMP, as well as those involved in the action of gluco- and mineralocorticoids, cytokines, and immune-cell-specific factors.

Amino Acid Sequence↗
PubMed1994

Ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density.

To determine whether the previously demonstrated ability of delta-opioid receptors to interact simultaneously with multiple G-proteins was a function of high receptor levels, this interaction was investigated in Chinese hamster ovary cells stably expressing 10 different levels of cloned delta-opioid receptors, ranging from 18,000 to 1.6 x 10(6) receptors/cell. The opioid agonist D-Ala2,D-Leu5-enkephalin (DADLE) inhibited forskolin-stimulated adenylyl cyclase activity in all 10 clones with variable maximal inhibitory levels. Furthermore, opioid agonists altered incorporation of [alpha-32P]azidoanilido-GTP into at least four G-protein alpha-subunits in all 10 clones, three of which were determined to be Gi3 alpha, Gi2 alpha and Go2 alpha. This effect was concentration-dependent, naloxone-reversible, and delta-opioid agonist-specific and was blocked by pretreatment with pertussis toxin. Although DADLE induced an increase in the incorporation of [alpha-32P]azidoanilido-GTP into three of the four G alpha proteins that was independent of receptor density, the magnitude of this response was greater as receptor density increased. In addition, concentrations of DADLE required to promote 50% maximal labeling were similar for all four G alpha proteins within each clone and did not appear to be affected by receptor density. Therefore, the ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density and there is also no apparent correlation between the amount of G-protein activated and the maximal effect of an agonist.

Amino Acid Sequence↗
PubMed1994

U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells.

The aminosteroid U73122 inhibited phospholipase C (PLC)-mediated intracellular Ca2+ release in differentiated and undifferentiated NG108-15 cells, as well as rat dorsal root ganglion (DRG) neurons grown in primary culture. 1 microM U73122 blocked bradykinin (BK)-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) measured in single cells with indo-1-based dual emission microfluorimetry. A close structural analog, U73343, was without effect. The effects of U73122 were time and concentration-dependent. 1 microM drug produced half maximal inhibition in approximately 3 min. The IC50 for a 20-min exposure was approximately 200 nM. The effects of the compound were irreversible for the duration of experiments as long as 1 h. Treatment with 1 microM U73122, but not U73343 produced a small but significant increase in [Ca2+]i which resulted from Ca2+ release from an intracellular store. It is not clear whether this [Ca2+]i increase resulted from inhibition of PLC or an action on the store directly. In differentiated NG108-15 cells U73122 blocked completely depolarization-induced Ca2+ influx. In contrast, in DRG neurons U73122 inhibited only slightly voltage-sensitive Ca2+ channels. Thus, we caution that U73122 may not be selective at concentrations required for maximal block of PLC and that the selectivity of U73122 is dependent on cell type. Overall, our results are consistent with U73122 inhibiting PLC in neuronal cells and indicate that under the appropriate conditions, this compound is a useful tool for studying inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ mobilization.

Animals↗
PubMed1994

Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors.

A rat brain cDNA library was screened for clones homologous to the recently cloned mouse delta-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57-58% identical to the amino acid sequences of the cloned delta, mu and kappa opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.

Amino Acid Sequence↗
PubMed1994

Analysis of delta-opioid receptor activities stably expressed in CHO cell lines: function of receptor density?

The question of whether short- and long-term opioid agonist activities could be affected by receptor density is being addressed with Chinese hamster ovary (CHO) cell lines stably expressing delta-opioid receptors. CHO cells expressing different levels of delta-opioid binding sites were isolated and characterized. The opioid binding sites in these cell lines have stereoselective high affinity for 3H-diprenorphine (0.18 to 0.91 nM), with agonist binding sensitive to both Na+ and GTP gamma S, and are of the delta-2-opioid receptor subtype. This is conclude from observations that the delta-2-opioid receptor-selective agonist naltriben (NTB) has higher affinity than the delta-1-opioid receptor-selective agonist 7-benzylidenenaltrexone (BNTX). It also could be demonstrated that delta-opioid agonists inhibited forskolin-stimulated intracellular 3H-cAMP production and that agonist inhibition could be blocked by pretreating cells with pertussis toxin. Again, NTB is more potent than BNTX or naloxone in reversing DPDPE inhibition of intracellular 3H-cAMP production. When the ability of [D-Ala2,D-Leu5]enkephalin (DADLE) to regulate adenylyl cyclase activity was examined in three separate CHO cell lines: CHODORX1-15, CHODORX1-8 and CHODORX1-4, which express 1.42 +/- 0.08, 0.74 +/- 0.07 and 0.27 +/- 0.04 pmol/mg-protein of receptor, respectively, maximal inhibitory levels in clones X1-15 and X1-8 were similar, whereas maximal inhibitory level for clone X1-4 was 66% of the other two clones. However, when maximal inhibitory levels of other opioid receptor-selective agonists were examined, different levels of inhibition were observed among these three CHO clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1994

Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells.

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Interaction of delta-opioid receptors with multiple G proteins: a non-relationship between agonist potency to inhibit adenylyl cyclase and to activate G proteins.

The purpose of the present investigation was to determine whether the coupling of delta-opioid receptors to multiple G proteins in NG108-15 neuroblastoma x glioma cells is a characteristic limited to only this cell line (because of the high density of delta-opioid receptors) and to ascertain whether there is any correlation between delta-opioid agonist potency to inhibit adenylyl cyclase and to activate G proteins. Interactions between receptors and G proteins were investigated using agonist-stimulated incorporation of the photoreactive GTP analog azidoanilido[alpha-32P]GTP ([alpha-32P]AA-GTP) into G protein alpha subunits, with subsequent separation by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membranes, four alpha subunits (Gi2 alpha, one isoform of Gi3 alpha, and both isoforms of Go alpha) in the 39-41-kDa region were labeled with [alpha-32P]AA-GTP. The delta-opioid agonist [D-Ala2,D-Leu5]-enkephalin (DADLE) produced a dose-dependent, naloxone-reversible increase of [alpha-32P]AA-GTP incorporation into all four alpha subunit subtypes, in all cell lines tested. In addition, with the single exception of Gi3 alpha in NG108-15 cells, the maximal increases in incorporation of the photoaffinity label into all G alpha subunits induced by DADLE were similar. The Bmax values determined for delta-opioid receptors in NG108-15, NS20Y, and N1E115 cell membranes were 570, 370, and 120 fmol/mg of protein, respectively. Finally, although the IC50 values to inhibit intracellular cAMP production and affinity for DADLE were similar across the three cell lines, the EC50 values to produce labeling of the G alpha subunits between cell lines differed by > 100-fold. In fact, only in NS20Y cells were the IC50 and ED50 values comparable. Firstly, these results suggest that simultaneous coupling of the delta-opioid receptor to multiple G protein alpha subunits occurs in a variety of cell lines that express a range of receptor densities. Secondly, the magnitudes with which delta-opioid receptors interact with available G alpha subunits in response to agonist are approximately the same. Finally, there appears to be no relationship between the potency of agonists to inhibit adenylyl cyclase and that required for activation of G proteins.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Immunofluorescent identification of a delta (delta)-opioid receptor on primary afferent nerve terminals.

Antisera were produced against synthetic peptides predicted from the recent cloning of a delta opioid receptor, DOR-1. Confocal microscopic examination of immunostained spinal cord sections revealed that DOR-1 immunoreactive (-ir) nerve fibers and terminals form a moderately dense plexus within the superficial dorsal horn of rats and mice. These fibers decreased dramatically following dorsal rhizotomy and consistent with these observations a population of small diameter neurons in ganglia exhibited DOR-1-ir. DOR-1-ir ganglion neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), and their terminals in the spinal cord contained both CGRP- and DOR-1-ir, the latter presumably located as a 'presynaptic' receptor. Interestingly, terminals containing DOR-1-ir appeared to be closely apposed by fibers and terminals containing enkephalin (ENK)-ir, which suggests that ENK may be a physiologically relevant ligand for the receptor encoded by DOR-1, and that DOR-1 may act to regulate the release of transmitters from small diameter primary afferent neurons.

Animals↗