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Director's Report to the National Advisory Council on Drug Abuse - February, 2005

Research Findings - Basic Neurosciences Research

Acetaldehyde Enhances Acquisition of Nicotine Self-Administration in Adolescent Rats

Tobacco use has one of the highest rates of addiction and relapse of any abused drug. Paradoxically, however, in animal models of reinforcement nicotine appears weak compared to other abused drugs. The reinforcing effects of tobacco smoke may be greater than nicotine alone due to complex interactions between the many constituents of tobacco smoke. One such constituent, acetaldehyde, has been thought to be reinforcing when inhaled. This compound may, therefore, augment nicotine's reinforcing properties. Juvenile and adult male rats were implanted with intravenous catheters and tested for nicotine self-administration 4 days later, at postnatal day 27 or 90, respectively. Animals were offered one of the following solutions: nicotine (30 mg/kg/injection), acetaldehyde (16 mg/kg/inj), nicotine (30 mg/kg/inj) plus acetaldehyde (16 mg/kg/inj), or saline. The youngest animals responded significantly more for nicotine plus acetaldehyde than for saline or for either drug alone. Tests with receptor antagonists indicated that these drug effects are mediated by central, but not peripheral, nicotinic receptors. There was an age-related decline in self-administration of nicotine plus acetaldehyde. Taken together, these results indicate that acetaldehyde, at the low concentrations found in tobacco smoke, interacts with nicotine to increase responding in a stringent self-administration acquisition test where nicotine alone is only weakly reinforcing, and that adolescent animals are more sensitive to these actions than adults. The authors suggest that animal models of tobacco addiction could be improved by combining acetaldehyde, and possibly other smoke components, with nicotine to more accurately reflect the pharmacological profile of tobacco smoke. Belluzzi, J.D., Wang, R. and Leslie, F.M. Acetaldehyde Enhances Acquisition of Nicotine Self-Administration in Adolescent Rats. Psychopharmacology (epub), 2004.

The CB2 Receptor Shows Promise as Target for Novel Pain Therapies

NIDA funded research has been examining the potential use of CB2-selective cannabinoid agonists in the treatment of pain. CB2-selective cannabinoid agonists are of special interest because CB2 receptors are located peripherally in the nervous system; thus agents that activate these receptors are unlikely to have the abuse potential of centrally acting cannabiniods. In a recently published study, Dr. Andrea Hohmann (University of Georgia) found that a CB2-selective cannabinoid agonist (AM1241) suppressed the development of capsaicin-evoked thermal and mechanical hyperalgesia and allodynia. AM1241 also produced a dose-dependent suppression of capsaicin-evoked nocifensive behavior. This AM1241-induced suppression capsaicin-evoked pain behavior was completely blocked by a CB2 antagonist. These data provide evidence that the activation of CB2 receptors is sufficient to produce analgesia in a persistent pain state, thus bolstering the case that the CB2 receptor may be an important therapeutic target for novel pain therapies. Hohmann, A.G., Farthing, J.N., Zvonok, A.M. and Makriyannis, A. Selective Activation of Cannabinoid CB2 Receptors Suppresses Hyperalgesia Evoked by Intradermal Capsaicin, Journal of Pharmacology and Experimental Therapeutics, 308(2), pp. 446-453, 2004.

Intrathecal CART (55-102) Enhances the Spinal Analgesic Actions of Morphine in Mice

Cocaine- and amphetamine-regulated transcript (CART) peptides are peptide neurotransmitters that have been implicated in feeding, reward and reinforcement. In this study, the researchers had made the observation that CART also appears to be present in regions of the nervous system associated with pain. They hypothesized that CART peptides might be involved in regulating pain transmission. There are at least two naturally occurring, active peptides that are generated by enzymatic processing of CART. Damaj and colleagues tested the peptide referred to as CART (55-102). They found that injecting CART (55-102) alone into the cerebrospinal fluid of mice did not affect pain levels in their model. However, injecting CART (55-102) in addition to morphine enhanced the pain-reducing effects of morphine. Understanding how this enhancement occurs may provide for the development of therapies that will result in improved effectiveness of morphine, which means patients would need less morphine to get equivalent pain relief. Damaj, M.I., Hunter, R.G., Martin, B.R. and Kuhar, M.J. Intrathecal CART (55-102) Enhances the Spinal Analgesic Actions of Morphine in Mice. Brain Research, 1024(1-2), pp. 146-149, 2004.

Excitation-Neurogenesis Coupling in Adult Neural Stem/Progenitor Cells

Although many in vivo manipulations can influence neurogenesis in the adult hippocampus, it is not known whether adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Additionally, it is not known how the coupling between activity and neurogenesis plays a role in modulating hippocampal-type memory processing networks. Dr. Karl Deisseroth and his colleagues at Stanford University School of Medicine showed that excitatory stimuli can act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca(v)1.2/1.3 (L-type) Ca(2+) channels and NMDA receptors on the proliferating precursors. Excitation through this pathway inhibits the expression of the genes responsible for the expression of glia (genes Hes1 and Id2) and increases the expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an "excitation-neurogenesis coupling rule" within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories. Deisseroth, K., Singla, S., Toda, H., Monje, M., Palmer, T.D. and Malenka, R.C. Excitation-neurogenesis Coupling in Adult Neural Stem/Progenitor Cells. Neuron, 42(4), pp. 535-552, 2004.

Dopamine D1 and D2 Receptor Co-Activation Generates a Novel Phospholipase C-Mediated Calcium Signal

Although dopamine D1 and D2 receptors each belong to distinct subfamilies of dopamine receptors, it is clear that they are functionally linked. However, it is not well understood how this linkage occurs. In this study, Dr. Susan George and her research team at the University of Toronto showed that agonist stimulation of co-expressed D1 and D2 receptors increased intracellular calcium levels in a signaling pathway that was not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished after treatment with a phospholipase C inhibitor (but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C). This indicates that they couple to the G(q) pathway. George et al. also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex. Finally, they used immunohistochemistry to show that these receptors are co-expressed and co-localized in individual neurons of human and rat brains. Lee, S.P., So, C.H., Rashid, A.J., Varghese, G., Cheng, R., Lanca, A.J., O'Dowd, B.F. and George, S.R. Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal. Journal of Biological Chemistry, 279(34), pp. 35671-35678, 2004.

Rats Reared in Enriched Environments Have More Robust Glutamatergic Neurotransmission in the Prefrontal Cortex and Improved Performance in a Spatial Memory Task Than Animals Reared in Impoverished or Normal Environments

Rearing rats in impoverished (IC) and enriched (EC) environmental conditions alters synaptic plasticity and cognitive processes. Metabotropic glutamate receptors (mGluRs) are known to play a key role in synaptic and behavioral plasticity. In the present study, the effect of rearing conditions on the expression of mGluR proteins in the prefrontal cortex (PFC) was assessed by immunoblotting. A significant difference in the content of prefrontal mGluR1 and mGluR5 (ie group I) and mGluR2/3 (ie group II) was observed between IC and EC rats. To functionally characterize this difference, in vivo microdialysis was used to verify differences in mGluR regulation of extracellular glutamate in the PFC. The results indicate that the capacity of group I and II mGluRs to elevate extracellular glutamate levels was significantly blunted in the PFC of IC rats compared to either EC subjects, or rats reared in normal environmental conditions (ie NIH standards). Group II mGluR receptors regulate performance in a forced T-maze spatial memory task that involves the PFC, and IC rats demonstrated deficits in this task relative to EC rats. These data suggest that reduced mGluR transmission in the PFC produced by impoverished, relative to enriched, rearing environments may contribute to cognitive deficits. Melendez, R.I., Gregory, M.L., Bardo, M.T. and Kalivas, P.W. Impoverished Rearing Environment Alters Metabotropic Glutamate Receptor Expression and Function in the Prefrontal Cortex. Neuropsychopharmacology, 29(11), pp. 1980-1987, 2004.

Differences in Maternal Styles of Rearing May Predispose to or Protect Individuals From the Development of Addiction

While many people experiment with drugs, relatively few individuals develop a true addiction. Dr. Michael Meaney and his colleagues hypothesized that, in rats, such individual differences in the actions of addictive drugs might be determined by postnatal rearing conditions. To test this idea, they investigated whether stimulant- and stress-induced activation of nucleus accumbens dopamine transmission and dopamine-dependent behaviors might differ among adults rats that had been either repeatedly subjected to prolonged maternal separation or a brief handling procedure or left undisturbed (non-handled) during the first 14 days of life. They found that, in comparison with their handled counterparts, maternally separated and non-handled animals are hyperactive when placed in a novel setting, display a dose-dependent higher sensitivity to cocaine-induced locomotor activity and respond to a mild stressor (tail-pinch) with significantly greater increases in nucleus accumbens dopamine levels. In addition, maternally separated animals were found to sensitize to the locomotor stimulant action of amphetamine when repeatedly stressed under conditions that failed to sensitize handled and non-handled animals. Finally, quantitative receptor autoradiography revealed a lower density of nucleus accumbens-core and striatal dopamine transporter sites in maternally separated animals. They also found greatly reduced D3 dopamine receptor binding and mRNA levels in the nucleus accumbens-shell of handled animals. Together, these findings provide compelling evidence that disruptions in early postnatal rearing conditions can lead to profound and lasting changes in the responsiveness of mesocorticolimbic dopamine neurons to stress and psychostimulants, and suggest a neurobiological basis for individual differences in vulnerability to compulsive drug taking. Brake, W.G., Zhang, T.Y., Diorio, J., Meaney, M.J. and Gratton, A. Influence of Early Postnatal Rearing Conditions on Mesocorticolimbic Dopamine and Behavioural Responses to Psychostimulants and Stressors in Adult Rats. European Journal of Neuroscience, 19, pp. 1863-1874, 2004.

Classifying New Drugs of Abuse - "Flatliners" and "Blue Mystic"

Within the past few years, two sulfur-containing agents have appeared on the clandestine market, and their increasing popularity has resulted in actions to schedule them under the Controlled Substances Act. One is 1-(4-methylthio-phenyl)-2-aminopropane or 4-MTA (known as "Flatliners" and "Golden Eagles"), and the other is 2-(2,5-dimethoxy-4-n-propylthiophenyl)-1-aminoethane or 2C-T-7 (known as "T7," "Blue Mystic," and "Tripstasy"). Both of these substances are substituted phenylalkylamines, as are the amphetamines and mescaline. However, sulfur-containing drugs of abuse are rare, and there is little information on the behavioral actions of 4-MTA and 2C-T-7. A reasonable approach to the scientific understanding of new drugs of abuse is to test them in rats trained to distinguish (i.e., to discriminate) known, well-characterized drugs of abuse from vehicle. Behaviorally active drugs of various pharmacologic categories provide relatively distinct internal stimuli to the rats, which are trained to press a "drug-appropriate" lever for a food reward when they detect that they have been administered a drug like the training drug. They are trained to press a different lever when they are administered the vehicle. Tests of stimulus generalization (substitution) were performed with 4-MTA and 2C-T-7 using a two-lever drug discrimination task with groups of rats trained to discriminate either the hallucinogen DOM [1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane], or the stimulant cocaine, or the "empathogen" MDMA ("Ecstasy") from vehicle. 4-MTA (ED50 = 0.8 mg/kg) substituted only for the MDMA stimulus, whereas 2C-T-7 (ED50 = 0.8 mg/kg) substituted only for the DOM stimulus. Thus, 4-MTA appears to be MDMA-like, and 2C-T-7 seems best classified as an hallucinogen. These results are consistent with what little is known about the action of 4-MTA and 2C-T-7 in humans. Khorana, N., Pullagurla, M.R., Dukat, M., Young, R. and Glennon, R.A. Stimulus Effects of Three Sulfur-containing Psychoactive Agents. Pharmacology Biochemistry and Behavior, 78, pp. 821-826, 2004.

VTA Dopamine Neurons are Less Sensitive to NE Inhibition During the Early Phases of Cocaine-seeking Behavior - A Cellular Phenotype Correlated with Cocaine-seeking Behavior, but not Cocaine

The reinforcing properties of psychostimulants depend critically on their effects on dopamine (DA) neurons in the ventral tegmental area (VTA). Dr. John Williams's group characterized cellular responses associated with the rewarding component of drug seeking behavior by evaluating the DA neuronal activities measured in triple-yoked animals that self-administered cocaine or that received cocaine or saline passively. His group evaluated the adaptive changes of DA neurons in slice preparations of the VTA. They found that the natural dis-inhibitory regulation of norepinephrine (NE) on the metabotrophic glutamate receptor (mGluR)-mediated hyperpolarization of VTA DA neurons, always found in normal animals, was also unchanged in animals passively given cocaine for 3 days. In contrast, animals that self-administered the cocaine for 3 days showed a transient, reduced dis-inhibitory effect of NE. The adaptation of VTA neuron sensitivity to NE drive at early state of the development of drug-seeking behavior represented a cellular phenotype of self-motivation behavior that was not evident in animals receiving cocaine via passive administration. That is, the adaptation correlated with efforts of the animal to attain cocaine. The diminished dis-inhibitory effect of NE on VTA neurons was still detectable 14 days later in self-administering rats. A similar response developed by 14 days in animals receiving cocaine. It is not yet known whether the same molecules are responsible for the adaptation at both early and later stages of cocaine administration. Paladini, C.A., Mitchell, J.M., Williams, J.T. and Mark, G.P. Cocaine Self-administration Selectively Decreases Noradrenergic Regulation of Metabotropic Glutamate Receptor-mediated Inhibition in Dopamine Neurons. Journal of Neuroscience, 24(22), pp. 5209-5215, 2004.

Different Calcium Signaling Mechanisms Underlie the Interaction of Neuroactive Molecules at VTA Dopamine Neurons

VTA dopamine neurons receive glutamatergic, adrenergic and muscarinic inputs. Receptors that couple to phosphoinositide hydrolysis, which include metabotropic glutamate receptors (mGluRs), adrenoceptors and muscarinic receptors, can either activate or inhibit VTA dopamine cells, depending on the pattern of receptor stimulation. Transient activation of alpha1 adrenoceptors with norepinephrine produces an outward current recorded in brain slices that contain midbrain dopamine neurons. The norepinephrine-mediated outward (hyperpolarizing) current was induced by activation of a potassium conductance through release of calcium from intracellular stores. In contrast to the mGluR-mediated outward current, the outward current induced by alpha1 adrenoceptors often consisted of multiple peaks. Activation of alpha1 adrenoceptors also induces a wave of calcium release that spread through the soma and proximal dendrites without a decline in amplitude or rate of propagation and, therefore, differs qualitatively from that induced by mGluRs. Finally, the alpha1 adrenoceptor-activated outward current was more sensitive to the calcium store-depleting agents ryanodine and caffeine. Thus, although both alpha1 adrenoceptors and mGluRs mobilize calcium from intracellular stores, they do it differently and mobilize different pools of calcium. The results suggest that noradrenergic innervation of dopamine cells can directly inhibit the activity of dopamine cells. Psychostimulants, such as amphetamine, will therefore have a direct effect on the firing pattern of dopamine neurons through a combination of actions on dopamine and alpha1 adrenoceptor activation. Paladini, C.A. and Williams, J.T. Noradrenergic Inhibition of Midbrain Dopamine Neurons. Journal of Neuroscience, 24(19), pp. 4568-4575, 2004.

Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane Inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. Dr. Szeto and colleagues have developed peptide antioxidants that target the IMM, and used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. Zhao, K., Zhao, G-M., Wu, D., Soong, Y., Birk, A.V., Schiller, P.W. and Szeto, H.H. Journal of Biological Chemistry, 279(33), pp. 34682-34690, 2004.

Cannabinoids and Pregnancy

Ectopic pregnancy is a major reproductive health issue. Although other underlying causes remain largely unknown, one cause of ectopic pregnancy is embryo retention in the fallopian tube. In a recent study, Dr. Haibin Wang and his associates, Vanderbilt University of Medical Center, Nashville, TN, show that genetic or pharmacologic silencing of cannabinoid receptor CB1 causes retention of a large number of embryos in the mouse oviduct, eventually leading to pregnancy failure. This was reversed by isoproterenol, a beta-adrenergic receptor agonist. Impaired oviductal embryo transport is also observed in wild-type mice treated with methanandamide. Collectively, their results suggest that aberrant cannabinoid signaling impedes coordinated oviductal smooth muscle contraction and relaxation crucial to normal oviductal embryo transport. Colocalization of CB1 and beta2-adrenergic receptors in the oviduct muscularis implies that a basal endocannabinoid tone in collaboration with adrenergic receptor coordinates oviductal motility for normal journey of embryos into the uterus. Besides uncovering a new regulatory mechanism, this study could be clinically relevant to ectopic pregnancy. Wang, H., Guo, Y., Wang, D., Kingsley, P.J., Marnett, L.J., Das, S.K., DuBois, R.N. and Dey, S.K. Nature Medicine, 10, pp. 1074-1080, 2004.

Plasmon Resonance Spectroscopy

Binding of an agonist to a GPCR such as the delta receptor (DOR) is believed to involve an overall change in the receptor "inactive" or ground state conformation, including movement of helices III, VI, and VII, to generate one or more "active" receptor states which can then couple to various G-proteins which interact with the intracellular loops at the cytoplasmic side or face of the receptor. Receptor ground state disruption of various hydrophobic, ionic, and hydrogen bond amino acid residue contacts between the N-terminus and extracellular loops 1-3 is brought about by ligand binding, which takes place largely at the opposite face of the receptor, in relation to the G-protein binding sites. Agonist delta ligands having differing structures (such as peptide or non-peptide) may activate the receptor by stabilizing different delta receptor conformations, and these can interact with different G-proteins (such as Go or Gi), leading to activation of different second messenger pathways. The binding of the agonist to the receptor promotes nucleotide exchange of GTP for GDP, and dissociation of the G-protein GABG heteromer into GA and BG subunits. Dr. Victor Hruby and his associates have recently reported on the use of an optical technique known as surface plasmon resonance, based on the theory of thin films, in order to measure the affinity of a delta receptor-ligand complex, and that of a receptor-ligand-GTPGS complex. A thin metal film (silver or gold) is coated upon a silicon dioxide dielectric layer, the latter being in contact with an aqueous buffer in which ligand or GTPGS can be introduced. A solubilized receptor can be pre-incorporated into a lipid bilayer which is then spread on the silica surface. Incident laser light, at a particular incidence angle, on the metal film will excite electronic oscillations known as plasmons, and the resonance is detected as a decrease in the reflected light intensity. Molecules such as receptors and their complexes immobilized at the silica surface can change the resonance angle as a function of their orientation, and their contribution to layer thickness and refractive index. The method does not require radiolabeling, fluorescent labels, separate GTPGS or cAMP assays, or the use of antibodies against particular G-protein subtypes. Since it depends upon relative changes in angle (millidegree), it does not require knowing the actual concentration of G-protein and receptor in the bulk aqueous solution in order to determine binding constants. Generally, the ligand may be pre-bound to the receptor before immobilization in the lipid, and the G-protein or GTPGS then introduced into the aqueous solution. Some of the results of this work include: (a) dissociation constants could be determined for full and partial agonists, and for antagonists binding to the human DOR in the absence of added G protein, which were comparable to in-vitro radiolabeled binding assay values. (b) considerable selectivity was shown in the binding of various ligands to different G protein subtypes; the presence of different G-protein subtypes also produced different GTPGS binding results. (c) agonist affinity (but not antagonist affinity) was increased by the presence of the G-protein, and in general, the presence of BG subunits improved the ligand binding compared to the presence of GA subunits alone. (d) significant differences in resonance angles were found depending on whether the incident light was parallel or perpendicularly polarized, i.e., the technique depends on the orientation of the immobilized receptor in the lipid bilayer. Since both ligand binding and G-protein binding can be separately measured, it is assumed that some receptors can be immobilized with the extracellular face pointing toward the aqueous phase, and some with the intracellular face pointing toward the aqueous phase. (e) adding GTPGS to a ligand-receptor complex produced a decrease in resonance angle associated with the disassembly of the G-protein heteromer, and exchange of GTP for GDP. (f) the technique can be used to follow the kinetics of binding an agonist such as DPDPE to the DOR, which was found to be biphasic (suggesting the formation and dissociation of an intermediate in the absence of G-proteins, and monophasic (and comparatively faster) in the presence of G-proteins. One prospect for the further use of this technique is to develop ligands selective for various G-protein subtypes, and thereby selective for a particular signaling pathway (such as analgesia) without activating side effect pathways. Alves, I.D., Ciano, K.A., Boguslavski, V., Varga, E., Salamon, Z., Yamamura, H.I., Hruby, V.J., and Tollin, G., Journal of Biological Chemistry, 279(43), pp. 44673-44682, 2004.

Further Structurally Constrained Analogues of cis-(6-Benzhydrylpiperidin-3-yl) benzyamine with Elucidation of Bioactive Conformation: Discovery of 1,4- Diazabicyclo[3,3,1] nonane Derivatives and Evaluation of Their Biological Properties for the Monoamine Transporters

The purpose of this study was to develop a series of 3,6-disubstituted piperidine derivatives, structurally constrained versions of flexible piperidine analogues, with preferential affinity for dopamine tranporter (DAT). In an attempt to further rigidify this structure to study influence of rigidity on binding and in vivo activity, the PI and his coworkers have developed a series of 4,8-disubstituted 1,4-diazabicyclononane derivatives and tested them for their affinity at the DAT, serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [3H]WIN 35,428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also tested for their ability to inhibit uptake of [3H]DA. The SAR study led to the discovery of potent lead compound which exhibited high affinity and selectivity for DAT (IC50 = 22.5 nM; SERT/DAT = 384 and NET/DAT > 444). The PI and coworkers further stated that overall current SAR results corresponded well with the results from less constrained 3,6-disubstituted versions of these compounds albeit the former class exhibited more stringent requirements in molecular structure for activity. However the potent compounds in the current series exhibited greater selectivity for the DAT compared to their corresponding lesser constrained 3,6-disubstituted versions indicating an effect of rigidity in selective interaction with transporter protein. In an attempt to elucidate the bioactive conformational structure of the lead molecules in the current and the 3,6-disubsituted series, a preliminary molecular modeling study was carried out where the most rigid derivative was used as a template structure. Two compounds (-)-2 and (-)-10c (see in the publication) exhibited stimulant activity in locomotor tests in mice in which (-)-2 exhibited a slower onset and longer duration of action compared to (-)-10c. Both compounds occasioned complete cocaine-like responding in mice trained to discriminate 10 mg/kg ip cocaine from vehicle. Kolhatkar, R., Cook, C.E., Ghoral, S.K., Deschamps, J., Beardsley, P.M., Reith, M.E.A. and Dutta, A.K. Journal of Medicinal Chemistry, 47, pp. 5101-5113, 2004.

Cannbinoid Systems Related to Disease Progression

There have been clinical studies indicating marijuana (THC) alleviates progression and some symptoms of multiple sclerosis (M.S.). As found in other studies focusing on AIDS-related diseases, THC inhibits some immune functions. This report provides some understanding of the basic neural-immune factors that may be key to this process. Lymphocyte/endothelial interactions are not only an area related to M.S., they are also important in HIV invasive processes. Multiple sclerosis (MS) is the most common of the immune demyelinating disorders of the central nervous system (CNS). Leukocyte/ endothelial interactions are important steps in the progression of the disease and substances that interfere with these activities have been evaluated as potential therapeutic agents. Cannabinoid receptor agonists have been shown to down-regulate immune responses and there is preliminary evidence that they may slow the progress of MS. The purpose of this investigation was to determine how cannabinoid receptor agonists interfere with leukocyte rolling and adhesion. This was investigated in an experimental auto-immune encephalomyelitis (EAE) model using six to eight week old C57BL/6 mice. Mouse myelin oligodendrocyte protein and pertussis toxin were used to induce EAE. WIN 55212-2, CB1 and CB2 antagonists were given. By use of in vivo intravital microscopy, leukocyte/endothelial interactions were evaluated via a cranial window implanted two days before. The results demonstrated that EAE increases leukocyte rolling and firm adhesion in the brain, and that this increased leukocyte/endothelial interaction can be attenuated by administration of WIN 55212-2. Furthermore, use of the selective antagonists for the CB1 receptor (SR141716A) and the CB2 receptor (SR144528) in this study demonstrated that the cannabinoid's inhibitory effects on leukocyte/endothelial interactions can be mediated by activating CB2 receptor. Ni, X., Geller, E.B., Eppihimer, M.J., Eisenstein, T.K., Adler, M.W. and Tuma, R.F. Win 55212-2, A Cannabinoid Receptor Agonist, Attenuates Leukocyte/endothelial Interactions in an Experimental Autoimmune Encephalomyelitis Model. Multiple Sclerosis 10, pp. 158-164, 2004.

Cannabinoids and Cell Death

An important issue in cellular science focuses on attrition of certain cells and systems: apoptosis. This mitochondrial directed process is very important in the turnover of immune cells that are constantly being replaced. This study focuses on dendritic cell systems and observed stimulation of apoptosis by the cannabinoid system. The precise role of cannabinoid receptors CB1 and CB2, as well as endogenous ligands for these receptors, on immune cells remains unclear. In the current study, the NIDA supported investigators examined the effect of endogenous and exogenous cannabinoids on murine bone marrow-derived dendritic cells (DCs). Addition of Delta(9)-tetrahydrocannabinol (THC), a major psychoactive component found in marijuana or anandamide, an endogenous cannabinoid, to DC cultures induced apoptosis in DC. DCs expressed CB1 and CB2 receptors and the engagement of both receptors was necessary to trigger apoptosis. Treatment with THC induced caspase-2, -8, and -9 activation, cleavage of Bid, decreased mitochondrial membrane potential, and cytochrome c release, suggesting involvement of death-receptor and mitochondrial pathways. DCs from Bid-knockout mice were sensitive to THC-induced apoptosis thereby suggesting that Bid was dispensable. THC treatment induced phosphorylation and enhanced the transcription of several apoptotic genes regulated by NF-kappaB. Moreover, inhibition of NF-kappaB was able to block THC-induced apoptosis in DCs. Lastly, in vivo treatment of mice with THC caused depletion of splenic DCs. Together, this study demonstrates for the first time that endogenous and exogenous cannabinoids may suppress the immune response through their ability to induce apoptosis in DCs. Do, Y., McKallip, R.J., Nagarkatti, M. and Nagarkatti, P.S. Activation Through Cannabinoid Receptors CB1 and CB2 on Dendritic Cells Triggers NF-kappa B-dependent Apoptosis: Novel Role for Endogenous and Exogenous Cannabinoids in Immunoregulation. Journal of Immunology, 173, pp. 2373-2382, 2004.

Opioid and Chemokine Systems Interaction in Neuron Activity

Chemokine receptors were early identified with movement of immune cells to target infectious agents. Subsequently, studies demonstrated the importance of these systems (plus the CD4 receptor) for the entry of HIV into immune cells. More recently, this group has found chemokines on neurons. This study provides some insight into what happens with these receptors. Both the chemokine SDF-1-alpha and the human immunodeficiency virus-1 (HIV-1) coat protein gp120 can bind to CXCR4 chemokine receptors but with different signaling consequences. To understand the molecular basis for these differences, the NIDA supported investigators tagged the rat CXCR4 receptor with enhanced cyan (ECFP) and yellow (EYFP) derivatives of the green fluorescent protein and investigated CXCR4 receptor dimerization in human embryonic kidney cells using fluorescence resonance energy transfer (FRET). Elevated FRET was detected under basal conditions from chemokines receptor-cotransfected cells indicating a high level of CXCR4 receptor dimerization. In comparison, chemokine and mu-opioid receptor-cotransfected cells displayed a much lower FRET signal. The FRET signal resulting from EYFP-CXCR4- and ECFP-CXCR4-expressing cells could be attenuated by coexpressing nontagged CXCR4 receptors suggesting competition with fluorophore-tagged receptors in the membrane. Nontagged mu-opioid, kappa-opioid, and muscarinic receptors also decreased the FRET between the tagged CXCR4 receptor pairs but to a lesser extent. Application of the CXCR4 receptor agonist SDF-1alpha further increased the FRET signal from tagged CXCR4 receptors, an effect that was inhibited by the CXCR4 antagonist. FRET analysis of tagged hCD4 constructs demonstrated that there was significant association of human CD4 and CXCR4, as well as dimerization. These data suggest that CXCR4 dimerization is involved in SDF-1alpha- and gp120-induced signaling events. Toth, P.T., Ren, D.J., Miller, R.J. Regulation of CXCR4 Receptor Dimerization by the Chemokine SDF-1 Alpha and the HIV-1 Coat Protein gp120: A Fluorescence Resonance Energy Transfer (FRET) Study. Journal of Pharmacology and Experimental Therapeutics, 310, pp. 8-17, 2004.

Delta Opioids on Immune Cell Maturation

The different types of opiate receptors differentially affect immune processes. The delta opioid receptor appears important in the maturation process of lymphocytes, especially T-cells. The following study focuses on the early stage of T-cell delineation process. The delta-opioid receptor-1 (DOR-1) as well as delta-opioid enkephalin peptides are expressed during maturation of T cells, although the functional significance of their expression remains unclear. Based on results which show that the administration of the highly selective delta-opioid agonist D-Pen(2), D-Pen(5)]enkephalin (DPDPE) induces an altered pattern of T-cell differentiation in fetal thymic organ culture (FTOC), these NIDA supported researchers hypothesized that DOR-1 is involved in the negative selection process. Our results show that superantigen-induced clonal deletion is promoted by DPDPE and significantly impaired in DOR-1-deficient mice. These results suggest that delta-opioids may play a homeostatic role in the negative selection process during T-cell development. McCarthy, L.E., Nitsche, J.F., Pintar, J.E. and Rogers, T.J. The Delta-opioid Receptor Participates in T-cell Development by Promoting Negative Selection. Journal of Neuroimmunology, 153, pp. 91-98, 2004.

Nicotine Activation of 4* Receptors: Sufficient for Reward, Tolerance, and Sensitization

Addiction to nicotine has been shown by twin studies to have a significant genetic component. Previous studies have also shown that beta2 subunit and the alpha4 subunits of the nicotinic acetylcholine receptor is required for the rewarding properties of nicotine. These previous studies do not show that either subunit of the receptor is sufficient (acting by itself) to mediate the actions of nicotine on reward, tolerance, and sensitization. To demonstrate that gene variants of the alpha 4 subunit of the nicotinic acetylcholine receptor is sufficient, Tapper and his colleagues created mutant mice in which the ninth amino acid, a leucine, is changed to an alanine in the pore-forming M2 domain. The alpha4beta2 nicotinic acetylcholine receptors were made hypersensitive by the amino acid substitution. Mutant mice when challenged with low doses of nicotinic agonists were more sensitive to the rewarding properties of nicotine and showed enhanced rates of tolerance and sensitization. These results suggest that gene variants in the alpha4 subunit of the nicotinic acetylcholine receptor can alter the sensitivity of the receptor to nicotinic receptors and may alter vulnerability to addiction to nicotine. Consistent with the idea that variants or polymorphisms in the alpha4 nicotine receptor alter vulnerability to nicotine addiction is the finding by Feng et al. Feng and his colleagues report that two single nucleotide polymorphisms in the coding region of exon 5 are associated with a protective effect against addiction. Tapper A.R., McKinney, S.L., Nashmi, R., Schwarz, J., Deshpande, P., Labarca, C., Whiteaker, P., Marks, M.J., Collins, A.C. and Lester, H.A. Science, 306 (5698), pp. 1029-1032, 2004; Feng,.Y., Niu, T., Xing, H., Xu, X., Chen, C., Peng, S., Wang, L., Laird, N. and Xu, X. American Journal of Human Genetics, 75(1), pp. 112-121, 2004.

Reduced Cellular Expression and Activity of the P129T Mutant of Human Fatty Acid Amide Hydrolase: Evidence for a Link Between Defects in the Endocannabinoid System and Problem Drug Use

Fatty acid amide hydrolase (FAAH) inactivates the activity of a large class of signaling lipids, including anandamide, N-palmitoyl ethanolamine, and oleomide. Animal studies indicate that FAAH serves as the primary catabolic regulator of fatty acid amide signaling in the nervous system and thus plays an important role in many neurobehavioral processes. Drs. Sipe and Cravatt and colleagues have identified a single nucleotide polymorphism in the FAAH gene that converts a conserved proline residue to a threonine at amino acid position 129 (P129T). This polymorphism was previously shown by these researchers to be associated with problem drug and alcohol use. To determine the functional effect of this polymorphism, Dr. Cravatt's group compared the expression and activity of the protein containing the proline allele to the protein containing the variant (threonine) allele in both peripheral T lymphocytes and transfected cells. The purified proteins of both forms were found to exhibit similar catalytic properties and structural stability; however the P129T variant protein was expressed at significantly lower levels than the more common proline containing protein. They examined whether post-translational mechanisms, differences in rates of cellular turnover, or proteosome mediated degradation could be responsible for the decreased levels of the P129T variant protein, but no significant differences were found. Altogether, these studies suggest that the reduced cellular expression levels of the P129T-FAAH protein may be caused by a post-translational mechanism that precedes the productive folding of this enzyme. In summary, this is the first evidence that the natural polymorphism in the human FAAH gene associated with problem drug use produces a mutant enzyme (P129T) with defective biochemical and cellular properties. This study highlights the idea that significant functional variability exists in the endocannabinoid system in the human population, and can be explained in part by genetic variation. Chiang, K.P., Gerber, A.L., Sipe, J.C., and Cravatt, B.F. Reduced Cellular Expression and Activity of the P129T Mutant of Human Fatty Acid Amide Hydrolase: Evidence for a Link Between Defects in the Endocannabinoid System and Problem Drug Use. Human Molecular Genetics, 13, pp. 2113-2119, 2004.

System-based Proteomic Analysis of the Interferon Response in Human Liver Cells

NIDA supports a P30 Center Grant to Dr. Michael Katz at the University of Washington in which proteomics and gene expression profiling are used to study the host response to hepatitis C (HCV), a sequealae and major public health problem in intravenous drug users. One of the major host factors used to fight hepatitis C and other viral infection are interferons. In response to viral infection, infected cells synthesize interferon. Gamma interferon is used to treat HCVy infection. However, many HCV infected individuals do not respond to interferon therapy. To determine the mechanism by which interferon acts to clear HCV infection of liver Dr. Katze and his colleagues conducted a global quantitative proteomic analysis in a human hepatoma cell line (Huh7) in the presence and absence of IFN treatment. Of the 1364 proteins observed, 54 were upregulated more than 2 fold while another 24 were down regulated. Using this approach Dr. Katze discovered two new proteins, LAMB1 and ADRM, induced by interferon that are cell adhesion molecules. These cell adhesion molecules may be important interferon mediated cell migration, attachment, and cell cell interaction. Other classes of molecules upregulated by interferon were G coupled and Jak-State signaling pathways, proteins involved in ubiquination (required for protein degredation), and four liver specific interferon response proteins. These finding validate the proteomics approach because some of these proteins like the Jak-Stat pathway were known to be involved in the mediating the actions of interferon on HCV infection. Proteins suppressed by interferon included proteins in the wnts and keratins involved regulating cytoskeleton function. Using a bioinformatics software package, Cytoscape, Dr. Katze was able to identify proteins that interact with one another. Such bioinformatics tools permit detailed visualization of biological network that has not been possible previously. The identification of new molecules induced by interferon and the protein network that mediates interferon response will enable scientists to identify new drug targets and treatments for HCV infection. Yan, W., Lee, H., Yi, E.C., Reiss, D., Shannon, P., Kwieciszewski, B.K., Coito, C., Li, X.J., Keller, A., Eng, J., Galitski, T., Goodlett, D.R., Aebersold, R. and Katze, M.G. Genome Biology, 5(8), p. R54, 2004.


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