Director's Report to the National Advisory Council on Drug Abuse
The human dopamine transporter (hDAT) serves to regulate the ratio of extracellular to intracellular levels of the neurotransmitter dopamine. Inward transport or influx involves binding of synaptic dopamine at the surface of a neuron, transport across the cell membrane, and release to the intracellular plasma. The process is saturable, and is coupled with the inward transport of sodium and chloride ions. In the reverse or efflux direction, dopamine or related monoamines can be released to the outside of the cell. For kinetic analysis of influx and efflux, useful models of the transporter have proposed an "outward-facing" DAT conformation(s), and an "inward-facing" conformation(s) which would allow external or internal binding of ligands, respectively. One feature of these models is that they might operate in a gated fashion, alternately providing binding sites at the extracellular side of the membrane while blocking the intracellular side, and vice versa. It is presently not known whether the same set of amino acid residues control such transporter conformations of the DAT in both directions, and mutational studies are being extensively carried out to identify the critical residues of dopamine and drugs of abuse. Transport and the DAT are of considerable significance in drug abuse research because monoamines such as amphetamine can be transported, the neurotoxic cation MPP+ can be internalized by transport, and cocaine is known to block or inhibit the uptake of dopamine, by binding to the DAT at one or more sites. Dr. Ulrik Gether and collaborators have previously identified an endogenous high affinity zinc binding site on the DAT which involves the close association in space of histidine at position 193 (extracellular loop 2), histidine 375 (transmembrane helix 7), and glutamate 396 (extracellular loop 4, top of transmembrane helix 8). This zinc binding site, which is largely extracellular, has been shown to act as a noncompetitive inhibitor of dopamine uptake at micromolar zinc concentrations. As an extension of this work, Dr. Gether's research group has now reported that when the intracellular loop 3 residue tyrosine 355 was mutated to alanine, dopamine uptake velocity (Vmax) was reduced to less than 1% of the DAT wild type velocity, and produced a twenty fold increase in the inhibition constant Ki for dopamine binding, all in the absence of zinc. In the presence of ten micromolar zinc, the velocity of uptake was 75% restored. This work was done with COS-1 cells transiently expressing the hDAT. Double mutations, such as mutating both histidine 335 and positions 193, 375, or 396, were not effective in promoting transport of dopamine. The authors have separately shown that the DAT mutated at position 355 appeared to be functional, in that it could be tagged with green fluorescent protein, expressed in HEK-293 cells, and visualized at the cell surface, and then internalized by activating a protein kinase. It has also been possible to show that the binding of cocaine (which is not transported) was reduced over one hundred fold by the single mutation at position 355, in the absence of zinc, and this reduction was partially reversed in the presence of zinc. The authors have suggested a model in which histidine 355 is needed to stabilize conformations permitting inward transport, with zinc influencing the equilibrium distribution of these conformations. Loland, C.J., Norregaard, L., Litman, T., and Gether, U. Proceedings of the National Academy of Sciences, 99(3), pp. 1683-1688, 2002.
Crystal Structure of Biphalin - Multireceptor Opioid Peptide
The opioid system plays the most important role in pain signal modulation. This system combines transmembrane G-protein coupled receptors, and their endogenous ligands and opioid peptides released by neurocells. It is widely accepted that there are at least three opioid receptor types, μ, λ, and κ. All opioid receptors have binding sites for benzomorphan alkaloids. Extensive structure-activity studies have shown that recognition of benzomorphan tyramine moiety is a common feature of all opioid receptors. The same tyramine moiety is a part of N-terminal tyrosine, the active site of endogenous opioid peptides. Large numbers of endogenous opioid peptides, which have been identified, could be divided into groups represented by the endomorphins (large peptide with preference to μ), enkephlins (with affinity with both μ and γ), dynorphins (with selectivity to K), and endomorphins (with selectivity to μ). Opioid analgesic drugs, like morphine, activate opioid receptors that initiate a cascade of events, which results in blocking the pain signal. However, the opioid system is involved in a number of homeostatic neurological and immunological functions. As a result, activation of the opioid system results in a number of side effects, including respiratory depression and dependency. Therefore, one of the most common ways of searching for new opioid analgesic drugs is developing the compounds that have the highest possible selectivity to the receptor and the least unwanted side effect(s). Nevertheless, all opioid receptors are involved in pain transmission modulation. Therefore, the contradictory approach of opioid analgesic development is to search for drugs with affinities to the very broad spectrum of receptors modulating the pain signals. The discovery of biphalin is the best example of the success of using this type of approach. This peptide expresses high affinity to all three opioid receptor types, with some preference for μ receptors. When administered directly into the brain, it has been shown to be more potent than morphine and etorphine at eliciting antinociception (pain relief). Currently biphalin is under intensive preclinical studies. Benzomorphan alkaloid analogues that express different receptor activity, as agonist or antagonist, all possess tyramine moiety in common freeze conformation. This may suggest that opioid peptides, during interaction with receptors, also adopt respective conformation of the tyramine part of N-terminal tyrosine. The presence of other functional groups in opioid peptide analogue and possibility of adopting specific topographical conformation(s) by these groups, determine receptor selectivity and potency. Topographical requirements of different opioid receptors are different even for the same group. Biphalin expresses high affinity to all opioid receptor types. This means that biphalin (i) possesses groups which guarantee high affinity to all types of opioid receptors, (ii) does not possess groups which could negatively interfere with particular receptor subtypes, and (iii) has a peptide chain that is flexible enough to adopt topographical requirements of all opioid receptor types. Flippen-Anderson, J.L., Deschamps, J.R., George, C., Hruby, V.J., Misicka, A., and Lipkowski, A.W. Journal of Peptide Research, 59, pp. 123-133, 2002.
Epileptiform Events in CA3 Hippocampus Depressed by Activation of the Opioid-Receptor-like-1 Receptor
The CA3 hippocampal region is important in the generation of hippocampal seizures. The opioid-receptor-like-1 (ORL-1) shares a high sequence homology with opioid receptors and is highly expressed in rat hippocampus. Activation of the receptor robustly depressed spontaneous epileptiform bursting without desensitization, and this was reversed by application of the receptor antagonist. This depressive action is consistent with the general inhibitory nature that occurs as a result of the activation of the ORL-1 receptor as it reduces the spontaneous miniature excitatory post synaptic currents (EPSCs) as well as electro-stimulation induced EPSCs. These observations also indicate that both pre- and post-synaptic mechanisms are involved in the depressive effect of epileptiform activity in CA3. This is interesting as it was thought that in CA1, CA3 and dentate, the ORL-1 receptor had inhibitory postsynaptic actions and seemed to lack the disinhibitory actions. Two membrane currents (channels) activated by the ORL-1 receptor agonist nociceptin were found to mediate the depressive effect of this drug on the epileptiform activity. The relation of the cell membrane outward potassium current (the M-current activated by nociceptin) to the CA3 epileptiform activity is confirmed by two observations. Similar to the effect of nociceptin, activation of the M-channel (by the channel activator retigabine) reduces epileptiform bursting. Blockade of this outward current (with channel blocker Linopirdine) increased the duration of CA3 epileptiform bursting. A cellular membrane inward rectifier potassium current (and therefore the channel, activated by nociceptin) was also identified. Blockade of the inward current (by adding Ba++ to the superfusion solution), while leaving the M-current intact, diminished the depressive effect of nociceptin on the spontaneous epileptiform bursting. Madamba, S.G., Schweitzer, P., and Siggins, G.R. J Neurophysiol, 82(4), pp. 1776-1785, 2001.
Ultrastructural Immunocytochemical Localization of the Dopamine D2 Receptor and Tyrosine Hydroxylase in the Rat Ventral Pallidum
The mesopallidal dopamine system plays a role in locomotor activity and reward. To understand the potential contribution of the dopamine D2 receptor (D2R) to the action of dopamine in the ventral pallidum (VP), investigators used electron microscopic immunocytochemistry to examine the cellular and subcellular localization of an antipeptide antiserum against the D2R in both ventromedial and dorsolateral VP compartments. In each region the majority of the total D2R-labeled profiles (n = 1,132) were axon terminals (55%) and small unmyelinated axons (27%). These terminals were often apposed to other axon terminals or dendrites and formed almost exclusively symmetric, inhibitory-type axodendritic synapses. Immunogold D2R labeling in axon terminals was seen on the plasmalemma and membranes of nearby synaptic vesicles. In ventral pallidal sections processed for dual detection of D2R peptide and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH), D2R labeling was detected in a few axons and axon terminals containing TH immunoreactivity as well as in axons contacted by TH-labeled terminals. In most cases, however, the D2R-labeled profiles were located at a distance from small axons and terminals containing TH. The results provide the first ultrastructural evidence that D2Rs in the two VP subterritories are strategically located for primary involvement in modulation of the presynaptic release of nondopaminergic inhibitory transmitters. They also suggest that in this region the presynaptic D2 receptors are 1) minimally involved in autoregulation of dopaminergic transmission, and 2) differentially activated by dopamine, depending in part on levels and distance from release sites. Mengual, E., and Pickel, V.M., Synapse, 43, pp. 151-162, 2002.
Impaired Prohormone Convertases in Cpe(fat)/Cpe(fat) Mice
A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene results in a loss of CPE activity that correlates with the development of late onset obesity. Examination of the level of neuropeptides in these mice showed a decrease in mature bioactive peptides as a result of a decrease in both carboxypeptidase and prohormone convertase activities. A defect in CPE is not expected to affect endoproteolytic processing. Drs. Berman and Devi and their research team at New York University have addressed the mechanism of this unexpected finding by directly examining the expression of the major precursor processing endoproteases, prohormone convertases PC1 and PC2 in Cpe(fat) mice. They found that the levels of PC1 and PC2 are differentially altered in a number of brain regions and in the pituitary. Since these enzymes have been implicated in the generation of neuroendocrine peptides (dynorphin A-17, beta-endorphin, and alpha-melanocyte-stimulating hormone) involved in the control of feeding behavior and body weight, they compared the levels of these peptides in Cpe(fat) mutant and wild type mice. They found a marked increase in the level of dynorphin A-17, a decrease in the level of alpha-melanocyte-stimulating hormone, and an alteration in the level of C-terminally processed beta-endorphin in Cpe(fat) mice. These results suggest that the impairment in the level of these and other peptides involved in body weight regulation is mainly due to an alteration in carboxypeptidase and prohormone convertase activities and that this may lead to the development of obesity in mice. Berman, Y., Mzhavia, N., Polonskaia, A., and Devi, L.A.. Impaired Prohormone Convertases in Cpe(fat)/Cpe(fat) Mice. J Biol Chem, 276(2), pp. 1466-1473, Jan 12, 2001.
Possible Role of Basal μ Opioid Receptor Signaling in Narcotic Dependence
The µ opioid receptor (MOR) displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPγgS binding to cell membranes and cAMP levels in intact cells. β-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6α- and 6β-naltrexol and -naloxol, and 6β-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6β-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction. Wang, D., Raehal, K.M., Bilsky, E.J. and Sadée, W. Inverse Agonists and Neutral Antagonists at µ Opioid Receptor (MOR): Possible Role of Basal Receptor Signaling in Narcotic Dependence J. of Neurochem., 77(6), pp. 1590-1600, 2001.
Neural Systems involving Cannabinoids: Focusing on the CB1 Receptors
Current dogma focuses on CB1 as the main cannabinoid receptor in the brain and CB2 as the main cannabinoid receptor in the periphery. Although microglia are brain cells, their systems are much different from those of nerve cells and considered to have actions commensurate with those systems of the macrophages of the peripheral immune cells. Herein, the authors have described a cannabinoid system in microglia more similar to that of nerve cells involving the CB1 receptor system. However, in these microglia, they utilized stimulation by lipopolysaccharide (LPS) to study the actions, an activator often used to modulate peripheral lymph cell systems. Activated brain microglial cells release inflammatory mediators such as nitric oxide (NO) that may play important roles in central nervous system antibacterial, antiviral, and antitumor activities. However, excessive release of these factors has been postulated to elicit immune-mediated neurodegenerative inflammatory processes and to cause brain injury. Recent studies using the rat animal model indicate that select cannabinoids may modulate production of these inflammatory factors. Treatment of neonatal rat brain cortical microglial cells with the cannabinoid paired enantiomers CP55940 and CP56667 resulted in a stereoselective differential effect on inducible NO production. The analog CP55940 exerted a dose-dependent inhibition of interferon gamma (IFN gamma)/bacterial lipopolysaccharide (LPS)-inducible NO production which was significantly greater than that exerted by CP56667. Pretreatment of microglial cells with the CB1 cannabinoid receptor-selective antagonist SR141716A reversed this CP55940-mediated inhibition. MRT-PCR demonstrated the presence of CB1 receptor mRNA within microglial cells consistent with the presence of CB1 receptors. Collectively, these results indicate that the cannabinoid analog CP55940 selectively inhibits inducible NO production by microglial cells and that this inhibition is effected, at least in part, through the CB1 receptor. Cabral, G.A., Harmon, K.N., and Carlisle, S.J. Cannabinoid-Mediated Inhibition of Inducible Nitric Oxide Production by Rat Microglial Cells: Evidence for CB1 Receptor Participation. Adv. Exper. Med. Biol., 493, pp. 207-214, 2001.
Peripheral Systems Involving Cannabinoids: Focusing on the CB1/CB2 Receptors in Lymphocytes
Although studies have shown that CB2 cannabinoid receptors are more abundant in immune cells than are CB1 receptors, both are increased/decreased by different classes of activators when immune cells are stimulated to proliferate and differentiate into different types of mature lymphocytes. Thus, it appears that cannabinoid receptors are playing unique, but different roles in the presence of different antigens. The following two studies report how each type of cannabinoid receptor is down-regulated under different conditions by particular antigens. In the first study, cannabinoid receptor 2 (CB2) was identified as the most abundant cannabinoid receptor subtype in the immune system. Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells, inducing proliferation and differentiation into antibody secreting cells. It has been reported that CB2 receptor expression is upregulated during human, tonsillar B cell activation through CD40. It was of interest to investigate the expression of CB2 mRNA using another B cell activator, LPS. Using northern blot analysis, they measured CB2 mRNA levels in murine splenocytes and enriched B cells. Results indicated that the 4.0 kb CB2 transcript was 2 fold higher in abundance in murine B cells than in whole splenocyte preparations. This observation confirmed data from others and from their previous RT-PCR studies that the expression of CB2 mRNA is more abundant in B cells. Upon LPS stimulation, CB2 transcripts were decreased 46% and 42% at 4 hours and 24 hours, respectively, when compared to unstimulated populations. An examination by flow cytometry of the CD69, early activation marker, on splenocytes, showed that the majority of the B cells were activated at 24 hrs. Thus, these results suggested that LPS stimulation of murine B cells caused a decrease in CB2 mRNA expression in contrast to the increase observed following human B cell stimulation through CD40. Lee, S.F., Newton, C., Widen, R., Friedman, H., and Klein, T.W. Down-regulation of Cannabinoid Receptor 2 (CB2) Messenger RNA Expression during in vitro Stimulation of Murine Splenocytes with Polysaccharide. Adv. Exper. Med. Biol., 493, pp. 223-228, 2001.
The second study showed significant evidence that cannabinoids have the ability to exert immunomodulatory effects. The identification of cannabinoid receptors in immune tissues has therefore led to questions about whether these immunomodulatory effects occur via these cannabinoid receptors. The cannabinoid receptor 1 (CB1), although expressed primarily in the brain, is also expressed in lower amounts in peripheral tissues. Of interest is the fact that CB1 is expressed in immune tissues such as spleen, albeit at lower levels than the peripheral cannabinoid receptor, CB2. To examine the function of CB1 in immune cells, activation experiments were performed using different stimuli e.g., anti-CD3, phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io), and PMA/Io + IL-2. Whole spleen cells were cultured in the presence of different stimuli for 0, 2, 4, and 24 hours, harvested at each time point, RNA isolated, and RT-PCR performed. FACS analysis was also performed using CD69 (an early activation marker) to determine whether cells were actually being activated. Results from anti-CD3 stimulation indicated a decrease in CB1 mRNA expression following activation. CB1 mRNA expression in murine splenocytes that were stimulated with PMA/Io in the presence or absence of IL-2 was also modulated. Expression of the message was enhanced upon stimulation with PMA/Io and PMA/Io + IL-2, however, stimulation with PMA/lo + IL-2 led to a stronger increase within 2 to 4 hours with CB1 returning to at or below baseline levels by 24 hours. Expression of CD69 was detected in all stimulated samples thereby indicating that the splenocytes were becoming activated. In summary, anti-CD3 stimulation appeared to decrease CB1 mRNA expression while PMA/Io + IL-2 stimulation significantly increased CB1 mRNA expression. These results demonstrate that the expression of CB1 mRNA is modulated upon cellular activation and that this modulation is dependent on the stimulus that is used. Noe, S.N., Newton, C., Widen, R., Friedman, H., and Klein, T.W. Modulation of CB1 mRNA Upon Activation of Murine Splenocytes Adv. Exper. Med. Biol. 493, pp. 215-221, 2001.
Functional Phenotypic Switch of RVM Neurons Provides a Novel Mechanism of Inflammatory Pain
NIDA-grantee Dr. Ronald Dubner of the University of Maryland and his colleagues examined N-methyl-D-aspartate (NMDA) receptor gene expression and neuronal activity in the rostral ventromedial medulla (RVM), an important area in the pain modulatory circuitry, after hindpaw inflammation in rats. Reverse transcription polymerase chain reaction analysis showed that there was an upregulation of mRNA encoding NMDA receptor subunits in the RVM after inflammation that lasted for up to 7 days. Electrophysiological studies demonstrated that this increased NMDA-gene expression correlated with the activation of RVM of cells that are normally not active during pain states. This functional phenotypic switch of RVM neurons appears to provide a novel mechanism underlying inflammatory pain states. Miki, K., Zhou, Q.Q., Guo, W., Guan, Y., Terayama, R., Dubner, R. and , Ren, K. Changes in Gene Expression and Neuronal Phenotype in Brain Stem Pain Modulatory Circuitry after Inflammation. J Neurophysiol., 87(2), pp. 750-760, Feb 2002.
Regulation of Opioid Receptor Trafficking and Morphine Tolerance by Receptor Oligomerization
Opiates such as morphine, can be and are used as analgesics. However, treatment of chronic pain means long-term use and this can lead to tolerance and even dependence. While morphine acts by binding to the mu opioid receptor, it does not induce desensitization and endocytosis. These investigators looked at the effect of a mu opioid receptor agonist, DAMGO, on endocytosis of the mu-opioid receptor itself and consequently the effect of DAMGO on tolerance. What they observed was that HEK 293 cells treated with a saturating concentration of DAMGO show robust endocytosis of the mu opioid receptors. HEK 293 cells treated with morphine at the same concentration show that the receptors generally have not been internalized. Those cells that are treated simultaneously with morphine and a sub-saturating dose of DAMGO show endocytosis of the mu opioid receptor like that seen with a saturating dose of DAMGO alone. DAMGO and morphine have a similar affinity for the mu-opioid receptor. If the mu opioid receptors in a cell are independent of one another (monomers), in the last scenario one would expect that the saturating dose of morphine would cause a morphine-like response. Instead, a DAMGO-like response was observed. That is, many of the mu opioid receptors were internalized. Therefore, the investigators suspect that the activated receptors are not monomeric, but instead form oligomeric complexes. In order to test the hypothesis that endocytosis of the mu opioid receptor delays tolerance, these investigators examined the effect of DAMGO, morphine and co-administration of both drugs on the development of tolerance in an animal model. In their in vivo model, they found that some of the mu opioid receptors of lamina II of the spinal cord had moved to intracellular compartments in response to sub-antinociceptive levels of DAMGO administered twice daily to animals receiving nociceptive levels of morphine. The presence of these receptors in intracellular compartments suggests endocytosis of those receptors. Furthermore, these animals did not develop tolerance to morphine during the seven days of the experiment. This is in contrast to tolerance developed within four days in the animals receiving morphine alone. The precise mechanism by which this response is elicited is unclear. Nonetheless, these results have important implications for the treatment of chronic pain and suggest that the development of tolerance to morphine can be delayed by co-administration of drugs that promote endocytosis. He, L., Fong, J., von Zastrow, M. and Whistler, J.L. Cell,108, pp. 271-282, January 25, 2002.
Mutation of Drosophila Homer Disrupts Control of Locomotor Activity and Behavioral Plasticity
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. One class of receptors activated by glutamate is the metabotropic Class I glutamate receptors. This class consists of mGluR1 and mGluR5 glutamate receptors. These receptors are coupled to phospholipase C. Activation of these receptors causes the hydrolysis of phosphoinositides to produce diacylglycerol, an activator of protein kinase c and inositol-3-phosphate (IP3), a molecule that releases intracellular calcium stores. Research has shown that knockout of mGluR5 or pharmacological blockade of mGluR5 blocks both the reinforcing and locomotor stimulant effects of cocaine without affecting food reward. In related work NIDA grantee Dr. Paul Worley has shown that homer proteins play an important role in regulating the function of mGluR1 and mGluR5. Homer proteins act as scaffolding proteins to couple the mGluR5 receptor to inositol-3-phosphate receptor. By regulating the coupling of the mGluR5 receptor to the IP3 receptor, homer may modulate the amount of intracellular calcium released. In addition, homer proteins bind to shank, a post synaptic protein associated with the NMDA glutamate receptors. Shank has been shown to regulate dendritic morphology. The changes in dendritic morphology are dependent on shank forming a complex with homer. This result suggests that homer may play an important role in regulating synaptic plasticity at glutamatergic synapses. To test the role of homer in synaptic and behavioral plasticity, Dr. Worley and colleagues isolated and characterized the gene for homer in drosophila. As in mammals, the drosophila homer protein is localized to dendrites and the endoplasmic reticulum. The drosophila mutation, lacking the homer gene, are viable and show coordinated locomotor activity. This suggests that homer is not essential for normal synaptic transmission. However, the mutants displayed deficits in courtship conditioning, an associative learning paradigm in drosophila. In addition, the homer mutant flies showed increased spontaneous locomotor activity. These results suggest that homer may play a role in modulating locomotor activity and behavioral plasticity. Behavioral and synaptic plasticity have been implicated in the cellular processes mediating addiction. Future studies on homer in mammals may reveal the role of homer in regulating synaptic plasticity and the role that homer plays in cocaine addiction. Diagana, T.T., Ulrich Thomas, U., Sergei, N. Prokopenko, S.N., Xiao, B., Worley, P.F., and Thomas, J.B. J. Neuroscience 22, pp. 428-436, 2002.
Mu Knockout Mice and Immune Function
To understand the role of the mu opiate receptor in the modulation of immune function, several studies have used mu knockout mice to study this system. There are still several actions of morphine that modulate immune functions in these mu knockout mice. This study identifies the kappa opioid system as a site where morphine may elicit action. Opioids such as morphine are potent analgesic and addictive compounds. Chronic morphine use also induces immunomodulatory and immunosuppressive effects, as especially evident in HIV-infected patients. Morphine acts on the immune cells primarily through its binding to mu-opioid receptors on the plasma membrane. However, morphine modulation of immune functions still exists in mu-opioid receptor knockout mice, suggesting that in addition to the mu opioid receptors, morphine may also act by mechanisms mediated by either delta or kappa opioid receptors. To determine whether morphine activates kappa opioid receptors (KOR), a quantitative competitive RT-PCR procedure was utilized to quantify the KOR gene expression of morphine-treated cells. A segment of KOR transcript spanning the second extracellular loop, which has the reported dynorphin specificity, and the seventh transmembrane domain of the receptor was amplified from the total RNA of morphine-treated CEM x174 lymphocytes, along with a competitor molecule. The competitor was constructed by deleting a 33-nucleotide fragment from KOR. The results of the competitive RT/PCR indicated that CEMx174 cells expressed KOR mRNA constitutively, in the order of femtograms. Treatment of 10 muM of morphine resulted in the up-regulation of KOR gene expression 24 hr post-treatment. The observed morphine effect could be reversed by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-Binaltorphimine (a KOR-selective antagonist). Suzuki, S., Chuang, T.K., Chuang, L.F., Doi, R.H. and Chuang, R.Y. Morphine Upregulates Kappa-Opioid Receptors of Human Lymphocytes. Adv Exper Med Biol, 493, pp. 81-87, 2001.
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