National Institute on Drug Abuse
Director's Report to the National Advisory Council on Drug Abuse
September, 1998
Research Findings
Basic Research
Mouse Model for Cannabinoid Withdrawal Described
In addition to published reports by various research groups demonstrating cannabinoid dependence in monkeys and rats, Dr. Billy Martin and colleagues (Medical College of Virginia) have provided evidence of dependence in a mouse model. The work was based on behavioral observations made following administration of the cannabinoid antagonist SR141716A, used to precipitate withdrawal in mice previously given delta-9-THC. The manifestations of withdrawal in mice (e.g., tremors, disorganized movement) paralleled those found in the rat, and were found to be dependent both on the dose of chronically administered delta-9-THC (two, three, seven, and 14 days), and on the dose of SR 141716A. Cook, S.A., Lowe, J.A., Martin, B.R. J. Pharm. Exp. Therapeutics 285, pp. 1150-1156, 1998.
Structure-Activity Relationships in Hallucinogen Analogs
Dr. David Nichols and coworkers at Purdue University have prepared a series of tetrahydronaphtho-furans designed to incorporate chemical features of hallucinogenic phenethylamines. The compounds were expected to bind to the serotonin receptor subtype 5-HT2A based on hydrogen binding possibilities with the receptor, and on other stereochemical information. They also contain an alkylamine side chain coplanar with the phenyl ring of the phenylethylamine. The results of the study indicate that, although one of the compounds binds to the human cloned 5-HT2A and 5-HT2C receptors, they all lack LSD-like behavioral effects in drug discrimination studies, and instead, several of the compounds are potent ligands for binding to the muscarinic receptor. It was concluded that the binding site(s) of the serotonin receptor fit LSD and phenethylamines differently, possibly involving different receptor amino acid residues. Monte, A.P., Marona-Lewicka, D., Lewis, M.M., Mailman, R.B., Wainscott, D.B., Nelson, D.L., Nichols, D.E. J. Med. Chem. 41, pp. 2134-2145, 1998.
MDMA Administration Induces Expression of HSP70 in the Rat Brain
Heat stress or heat shock proteins were induced after a variety of neuronal injuries and pathological conditions. The inducible 70-kDA heat shock protein (HSP70) has been shown to protect against glutamate neurotoxicity and may play a similar role in ischemic tolerance. Previous studies have shown that HSP70 expression is also induced in the rat by administration of amphetamine or methamphetamine at doses of 10 mg/kg. Dr. Seiden at the University of Chicago and his research team investigated the effect of a neurotoxic dose of MDMA (40 mg/kg), a substituted amphetamine, on HSP70 synthesis in the rat at 30°C ambient temperature, using 30°C and 42°C ambient temperatures as control temperatures for the saline-treated rats. These results show that rats treated with MDMA (average core temperature 39.3°C; maximum core temperature 40.3°C) at an ambient temperature of 30°C had core temperatures comparable to those observed in saline-treated rats exposed to an ambient temperature of 42°C (average 39.6°C; maximum 40.6°C) and higher than saline-treated rats exposed to 30°C environment (average 37.7°C). Moreover, the MDMA-treated rats also expressed HSP70 levels similar to those observed in saline-treated rats exposed to a 42°C environment. The HSP70 levels expressed by MDMA-treated rats were higher than those observed in saline-treated rats exposed to an ambient temperature of 30°C. Since HSP70's are neuroprotective in other types of toxicity, this finding suggests that HSP70's may play a role in reducing damage induced by amphetamine derivatives such as MDMA. Malberg, J.E. and Seiden, L.S., Poster, 1997. Society for Neuroscience Annual Meeting, New Orleans, LA, October 25-30, 1997.
Nitrous Oxide, the Inhalational General Anesthetic and Drug of Abuse, Is an NMDA Antagonist
Nitrous oxide (N2O) is a general inhalational anesthetic, used widely in medicine and dentistry, but also widely abused. Its mechanism of action was unknown. Unlike other general anesthetics such as barbiturates and halothane, it does not increase inhibitory neurotransmission through GABAA receptors. An important recent discovery by John W. Olney, MD and Vesna Jevtovic-Todorovic, MD, Ph.D., of the departments of Psychiatry and Anesthesiology, Washington University, St. Louis, is that N2O is a N-methyl-D-aspartate (NMDA) receptor antagonist; i.e. it decreases glutamate excitatory transmission by blocking NMDA glutamate receptors at relevant concentrations. Thus, N2O resembles the intravenous dissociative anesthetics and drugs of abuse, ketamine and phencyclidine (PCP). Like these and other NMDA antagonists, N2O inhibits excitotoxic neurodegeneration mediated through NMDA receptors, but unlike them produces a distinctive vacuolar injury to specific pyramidal neurons of the posterior cingulate and retrosplenial cortices (the "Olney lesion"). The Olney lesion can be prevented by drugs that enhance GABAergic inhibition. The neurotoxic properties of N2O have the same age dependency that Olney's group had previously shown for other NMDA antagonists. One reason N2O's mechanism was unknown was the inability of researchers to achieve appropriate concentrations in lab animals while maintaining an adequate oxygen supply. Olney's group overcame this problem with a hyperbaric chamber. Jevtovic- Todorovic, Todorovic, Mennerick, Powell, Dikranian, Benshoff, Zorumski and Olney, Nature Medicine 4(4), pp. 460-463, April 1998.
Receptor Mechanisms of Opioid Tolerance and Dependence by Agonists of Varying Efficacies
Dr. Fedor Medzihradsky of the University of Michigan Medical School and his team have been investigating the molecular mechanisms involved in the development of tolerance by agonists of varying efficacies. Prolonged exposure to agonists induced desensitization of the receptor as estimated by a reduction in the maximal stimulation of [35S]GTPyS binding by DAMGO. In C6 glioma cells stably expressing the mu opioid receptor, treatment with maximally efficacious concentrations of partial and full agonists reduced DAMGO-stimulated [35S]GTPyS binding. The reduction correlated with agonist efficacy where the more efficacious agonists reduced DAMGO stimulated [35S]GTPyS binding to a greater extent. Guanine nucleotide regulation of agonist binding was lower in membranes from tolerant cells. The potency of DAMGO to inhibit cAMP accumulation was lower in morphine and DAMGO tolerant cells. Pertussis toxin treatment of the cells prior to agonist treatment did not prevent the down-regulation by full agonists such as DAMGO while the ability of partial agonists was greatly impaired showing that down-regulation by full agonists proceeds almost completely in the absence of a functional G protein. These results indicate important differences in the inactivation pathways of receptors triggered by full and partial agonists. Yabaluri, N. and Medzihradsky, F. Molecular Pharmacology, 52, pp. 896-902, 1997.
Sigma Receptors & Abused Drugs
In papers published recently, Dr. Vadivel Ganapathy and his colleagues report isolating and cloning sigma receptors from mouse cDNA and from rat brain. They are the first group to observe that an identical sigma receptor is present in the liver, placenta, and brain of the rat. The structural information generated on this gene, especially in the mouse, has potential utility in the development of transgenic and knock-out models. Such models would be useful for studying the physiological significance of sigma receptor function and for understanding the pathologial consequences of sigma receptor dysfunction. In a parallel study, Dr. Vadivel Ganapathy and his colleagues report the structure and organization of human gene coding for the type 1 sigma receptor. Kekuda, R., et al., Biochem. Biophysical Res. Commun. 229, pp. 553-55, 1996. Seth, P., Leibach, F.H. and Ganapathy, V. Biochem. Biophysical Res. Commun. 241, pp. 535-540, 1997.
Lung Cancer Risk and Habitual Smokers of Marijuana, Cocaine and/or Tobacco
More frequent alterations in molecular markers and histopathologic parameters were found in the bronchial epithelium of habitual marijuana and/or cocaine smokers, who may or may not also smoke tobacco, than in non-smokers. These findings suggest that smoking marijuana and/or cocaine, like tobacco smoking, exerts field cancerization effects on bronchial epithelium which may place smokers of these substances at increased risk for the subsequent development of lung cancer. An accompanying editorial appeared with this paper. Barsky, S.H., Roth, M.D., Kleerup, E.C., Simmons, M. and Tashkin, D.P., Journal of the National Cancer Institute, 90(16), pp. 1198-1205, August 19, 1998.
Presynaptic Recording of Quanta from Midbrain Dopamine Neurons and Modulation of the Quantal Size
Neurons communicate with each other by releasing chemical substances from nerve terminals at specialized junctions called synapses. More than thirty years ago it was shown by Sir Bernard Katz that neurotransmitters are secreted from synaptic vesicles. The amount released from a synaptic vesicle is known as quanta because the amount of excitation produced by the release of transmitter released from a single vesicle is the same. As more vesicles are released, the amount of excitation increases in step wise or quantal fashion. The strength of the signal between two neurons can be increased by either increasing the amount of neurotransmitter stored in a single vesicle, increasing the number of vesicles that release their contents, or by increasing the sensitivity of the neighboring neurons to the neurotransmitter being released. Thus, analysis of quantal events and how synaptic transmission is regulated is key to understanding how neurons communicate with each other. The mechanisms of quantal transmission are well studied at synapses where the neurotransmitter has a rapid onset and offset. However, for a transmitter such as dopamine, a neurotransmitter implicated in mediating addiction, the mechanisms of quantal release are not well characterized because the action of dopamine has a slow onset. To overcome this problem, Dr. Sulzer and his colleagues used a technique called amperometry. Released neurotransmitter is rapidly detected as a change in current by an electrode connected to an amperometer. Dr. Sulzer and his colleagues report that dopamine is released in quantal fashion that is likely to originate from small vesicles instead of large dense core vesicles because the amount released is smaller than the amount seen in neuroendocrine cells. In addition, they observed that L-Dopa, the precursor for dopamine and glia derived growth neurotrophic factor (GDNF), a survival factor for dopamine neurons, increases the amount of neurotransmitter packaged in a single vesicle. The work by Dr. Sulzer and his colleagues lays the foundation for elucidating the modulatory actions of growth factors and neurotransmitters on dopamine transmission in the brain. By understanding how dopamine release is modulated, new therapies can be devised for the treatment of addiction, Parkinson's Disease, and schizophrenia because the neurotransmitter dopamine has been implicated in the etiologies of these diseases. Pothos, E.N., Davila, V., and Sulzer, D. The Journal of Neuroscience, 18(11), pp. 4106-4118, June 1, 1998.
Increased Vulnerability to Cocaine in Mice Lacking the Serotonin 1B Receptor
NIDA grantee Dr. Rene Hen and his coworkers at Columbia University, demonstrated that knockout mice lacking one serotonin receptor subtype (serotonin 1B), are more sensitive to both the motor and rewarding effects of cocaine. As a result, these knockout mice lacking the serotonin 1B subtype are more motivated to self-administer cocaine than wild-type mice. Dr. Hen and his coworkers have characterized the biochemical changes which occurred in these knockout mice which they believe relate to their preference for cocaine. These changes include an increase in the immediate early protein FosB that has been shown to be upregulated following chronic cocaine exposure. Dr. Hen and his colleagues used a large panel of techniques, such as intra-venous self-administration of cocaine and in vivo microdialysis in freely moving mice, that until recently had not been applied to transgenic mice. They demonstrated that changes in the dopaminergic system took place in these knockout mice and that these changes are likely to explain their increased responsiveness to cocaine and psychostimulants. Dr. Hen and his coworkers are in the process of generating and characterizing several lines of tissue-specific and inducible knockout mice of the 5-HT1B receptor. In conclusion, this is the first example of a gene that might be related to the large individual differences in vulnerability to cocaine that are found in rodents and humans. The identification of a candidate gene that, when mutated, is responsible for increased vulnerability to psychostimulants, has considerable implications for the understanding of the genetics of drug abuse in humans. Rene Hen, et.al. Nature, 393, pp. 175, June 18, 1998.
Dopamine Neurons Make Glutamatergic Synapses In Vitro
Dopamine is a neurotransmitter synthesized by neurons located in the substantia nigra and ventral tegmental areas of the midbrain. Dopamine plays a key role in the initiation of movement. Degeneration of dopamine neurons in the substantia nigra lead to the development of Parkinson's disease. Furthermore, all drugs of abuse and pleasurable stimuli appear to cause increased release of dopamine. Thus, most of the work on how dopamine neurons communicate with neighboring neurons, known as synaptic transmission, has focused on the synthesis and release of dopamine as well as the effect of dopamine on neighboring neurons. The onset of the actions of dopamine on neighboring neurons is slow and does not explain the reason that stimulating dopamine neurons also produces rapid effects on neighboring neurons. One possibility is that dopamine neurons release, in addition to dopamine, another neurotransmitter such as glutamate that has a rapid action on a neighboring neuron. To test the possibility that dopamine and glutamate are co-localized in the same neurons, Dr. David Sulzer and his colleagues at Columbia University used anatomical and electrophysiological techniques. They report that the enzyme glutaminase needed for the synthesis of glutamate, and the enzyme tyrosine hydroxylase needed for the synthesis of dopamine, are colocalized in neurons of the substantia nigra and ventral tegmental region in both rat and monkey. Excitatory actions were blocked by a glutamate antagonist suggesting that glutamate is released onto the dopamine neuron being stimulated. Application of dopamine onto the neuron prior to stimulation blocked or reduced the amount of excitation, suggesting that dopamine acts to inhibit the release of glutamate. These results suggest that neurons of the substantia nigra and ventral tegmental area exert their rapid actions via glutamate and their slower and modulatory actions via dopamine. Sulzer, D., Joyce, M.P., Lin, L., Geldwert, D., Haber, S.N., Hattori, T., and Rayport, S. Dopamine Neurons Make Glutamatergic Synapses In Vitro. The Journal of Neuroscience, 18(12), pp. 4588-4602, June 15, 1998.
Morphine Induces Sepsis in Mice
A recent publication has identified an action whereby morphine causes dissemination of bacteria from the gut into areas causing an immune response, inflammation and hyperemia. The end result can be toxic shock which can result in the death of the animal. Although these studies were conducted in mice, a similar result may occur in some humans as gram-negative sepsis and subsequent endotoxic shock remain major health problems in the United States. The present study examined the role of morphine in inducing sepsis. Mice who were administered morphine by the subcutaneous implantation of a slow-release pellet developed colonization of the liver, spleen, and peritoneal cavity with gram-negative and other enteric bacteria. In addition, the mice became hypersusceptible to sublethal endotoxin challenge. The effects were blocked by the simultaneous implantation of a pellet containing the opioid antagonist naltrexone. These findings show that morphine pellet implantation in mice results in the escape of gram-negative organisms from the gastrointestinal tract, leading to the hypothesis that morphine used postoperatively or chronically for analgesia may serve as a cofactor in the precipitation of sepsis and shock. In addition, morphine-induced sepsis may provide a physiologically relevant model of gram-negative sepsis and endotoxic shock. Hilburger, M.E., Adler, M.W., Truant, A.L., Meissler, J.J., Jr., Satishchandran, V., Rogers, T.J., and Eisenstein, T.K. Morphine Induces Sepsis in Mice, Journal of Infectious Diseases. 176, pp. 183-188, 1997. (Text taken largely from article abstract).
Reduced Delayed Hypersensitivity: Skin Reactions in Opiate Addicted Patients (OAP)
Despite the in vitro and animal evidence that opiates impair cell-mediated immunity (CMI), it is unknown whether human opiate dependence is associated with significant suppression of CMI manifested by delayed hypersensitivity skin reaction (DHR) response. Seventy-four OAP enrolled in a methadone clinic and 86 healthy volunteers (HV), were skin tested intradermally using five standardized antigens: tetanus (neat); mumps(neat); candida albicans (1:100); trichophyton mentagrophytes (1:100); and tuberculin (5 T.U.). Subjects were excluded if they had a significant illness, were on immunosuppressive medications, were HIV (+), or had known tuberculin DHR (+). Mean induration diameter (forearm long axis + perpendicular/2) were measured by "ball point pen" method using an electronic digital caliper. Demographics showed no significant age differences between OAP and HV, but significantly more males (p=<.001) and non-Caucasians (p<.001) were in the addict group. Analysis of DHR prevalence showed no significant differences between OAP & HV for tetanus (p=.55), candida (p=.77), tuberculin (p=.85), trichophyton (p=.24); borderline significance for mumps (p=.05); and no significant differences for individuals showing any of five reactions (p=.53). However, highly significant decreases in size (mm) of DHR were seen in the OAP for tetanus 32.2 percent smaller. (P=<.001) and mumps 30.4 percent smaller (p=<.001), and the other groups were too small to analyze. In the OAP, no significant differences were seen either in the prevalence or degree of DHR reactivity for any antigen between male or female or Caucasian and non-Caucasian subgroups. One group of OAP demonstrated highly significant hypoergy rather than anergy to DHR antigens. Since tuberculin skin testing is the standard public health method for identifying infected individuals, this study suggests that criteria for positivity of DHR in OAP needs to be adjusted downward. Steinberg, P., McFadden, R.,Carlson, G.A., Bullock, M.L., Pheley, A.M., O'Hara, C.M. and Peterson, P.K. Reduced Delayed Hypersensitivity: Skin Reactions in Opiate Addicted Patients (OAP). Journal of Allergy and Clinical Immunology, 1(1, Part 2), S55, 1998. (Text taken largely from article abstract).
Opioid Receptors in Immune Function This publication describes the expression and relation of opioid receptors in immune cells and their possible role in combating disease, especially in relation to humans. Delta opioid receptor (DOR) transcripts and binding sites are expressed by lymphocytes and lymphoid cell lines from several species. Direct modulation of lymphocyte function through DORs affects T cell proliferation, interleukin-2 production, chemotaxis, and intracellular signaling. Moreover, in human DOR-transfected T cells (DOR-Ju.1), delta opioids have been shown previously to mobilize intracellular calcium rapidly, to inhibit forskolin- stimulated cyclic AMP production, and to activate the mitogen-activated protein kinases ERKs 1 and 2. These observations led us to consider whether delta agonists modify T cell functions, thus affecting the expression of human immunodeficiency virus-1 (HIV-1) by CD4+ T cells. To test this hypothesis, DOR-Ju.1 cells, derived from Jurkat cells (a modified human cell line) stably transfected with a cDNA encoding the neuronal DOR, were stimulated with deltorphin or benzamide,4-[[2,5-dimethyl-4- (2-propenyl)-1-piperazinyl] (3-methoxyphenyl)methyl]-N,-, [2S-[1(S*),2a,5b]]-(9Cl) (SNC-80) prior to the addition of HIV-1. Both deltorphin and SNC-80 concentration dependently inhibited the production of p24 antigen, an index of HIV-1 expression. Inhibition was maximal with 10-13 to 10-9 M SNC- 80 (>60% reduction) or 10-15 to 10-11 M deltorphin (> 50% reduction). At higher concentrations, less inhibition of p24 antigen production was found. Naltrindole (NTI, 10-11 M), a selective DOR antagonist, abolished the inhibitory effects of 10-9 M SNC-80, whereas 10-13 M NTI partially reversed the effect of SNC-80. Thus activation of DORS expressed by CD4+ T cells significantly reduced the expression of HIV-1 by these cells. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard anti-viral approaches to HIV-1 infection. Sharp, B., Gekker, G., Li, M.D., Chao, C.C., Peterson, P. Delta Opioid Suppression of Human Immunodeficiency Virus-1 Expression in T Cells, Biochemical Pharmacology, August 1998. (Text taken largely from article abstract).
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