Eva V. Varga

Plasmon Resonance Studies of Agonist/Antagonist Binding to theHuman δ-Opioid Receptor: New Structural Insights into Receptor-Ligand Interactions

Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.

Cannabinoid CB1 receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB(1) receptors in the trabecular meshwork and increase aqueous outflow.

Endomorphin-1 and endomorphin-2 are partial agonists at the human μ-opioid receptor

Recently two tetrapeptide ligands that bind preferentially to the mu-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human mu-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPgammaS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the mu-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the mu-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.

Involvement of Raf-1 in chronic δ-opioid receptor agonist-mediated adenylyl cyclase superactivation

Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.

Identification of adenylyl cyclase isoenzymes in CHO and B82 cells

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.

Phosphorylation of adenylyl cyclase VI upon chronic δ-opioid receptor stimulation

Abstract An immunoprecipitation method was used to measure [ 32 P ]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human δ-opioid receptor. Chronic SNC 80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide) (1 μM, 24 h) treatment increased the incorporation of [ 32 P ] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 μM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.

Cloning of the rat m3, m4 and m5 muscarinic acetylcholine receptor genes by the Polymerase Chain Reaction (PCR) and the pharmacological characterization of the expressed genes

The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.

Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner.

Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.

Properties of TAN-67, a nonpeptide δ-opioid receptor agonist, at cloned human δ-and μ-opioid receptors

Abstract 2-methyl-4a α(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a α-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67) is a nonpeptidic δ-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse δ and μ-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, [D-Pen2, 4′-Cl-Phe4, D -Pen5]enkephalin (pCl-DPDPE) were measured by the radioligand binding inhibition studies at mouse and human variants of the δ and μ-opioid receptor using [3H]Naltrindole and [3H] D -Phe- Cys  Tyr  D  Trp  Orn  Thr  Pen -Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human δ-opioid receptor and high δ-opioid receptor binding selectivity (> 1000-fold) relative to the human μ-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human δ-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human μ-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human δ and μ-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a δ-opioid receptor agonist.

Agonist-specific down-regulation of the human δ-opioid receptor

Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.