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National Institute on Drug Abuse

Director's Report to the National Advisory Council on Drug Abuse

September, 1999

Research Findings

Basic Research

Gene Therapy Provides Potential New Treatment for Chronic Pain

Dr. David Yeomans of the University of Illinois and his colleagues describe a potential new chronic pain control method that uses a novel genetic therapeutic approach. These researchers applied to the skin of mice a recombinant herpes simplex virus that contained specially programmed complementary DNA (cDNA) that held the gene that produces the precursor to the opiates that stop pain. This virus then traveled up the nerve to a part of the brain that is involved in sensing pain (i.e. the spinal dorsal horn). Models of chronic pain failed to elicit a pain response in the virus-treated mice. In addition, after the virus treatment these mice appeared normal in their response to acute pain. These findings demonstrate that it is possible to specifically block chronic pain in mice using gene therapy with few side effects. This research raises hopes that gene therapy may be useful in the treatment of chronic pain in humans. Wilson, S.P., Yeomans, D.C., Bender, M.A., Lu, Y., Goins, W.F., and Glorioso, J.C. Proceedings of the National Academy of Science, 96(6), pp. 3211-3216, 1999.

G-Protein-Coupled Receptor Heterodimerization Modulates Receptor Function

The opioid system modulates physiological processes such as analgesia, stress, the immune response, and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid-receptor types, delta, kappa and mu, that mediate these diverse effects. Structural and biochemical studies with a variety of G-protein coupled receptors (GPCRs) indicate that a number of them exist as homodimers (i.e. pairs of the same protein). Relatively few studies have examined heterodimerization (i.e. pairs of different proteins) between these receptors. NIDA grantees Bryen Jordan and Lakshmi Devi have recently published a paper in Nature in which they found biochemical and pharmacological evidence for two fully functional opioid receptors, kappa and delta, to heterodimerize and generate a new receptor. The agonist binding properties of the heterodimer are distinct from the properties of either of the receptors. The heterodimer synergistically binds highly selective agonists leading to potentiation of signal transduction. Heterodimerization of GPCRs represents a novel mechanism that could modulate receptor function. These findings on heterodimerization between two fully functional GPCRs improve our understanding of the complex regulation of GPCR function. Heterodimerization also points to additional targets for the development of drug therapies. Jordan, B.A. and Devi, L.A. Nature, 399, pp. 697-700, 1999.

Anandamide Transport

The mechanism of action of the endogenous cannabinoid ligand anandamide continues to be actively studied. Drs. Daniele Piomelli and Alexandros Makriyannis reported their evidence for the existence of an anandamide transporter capable of carrying labeled anandamide into human astrocytoma cells where it is then rapidly hydrolyzed. The transport process is analogous to the process for prostaglandin E2 and unsaturated fatty acids. In the case of anandamide, the process is rapid, temperature dependent, sodium ion independent, and exhibits "saturation" kinetics with a reported Km and Vmax. A sampling of prostaglandins, tetra- and trienoic acids, biogenic amines, and amino acids, capable of inhibiting membrane transporters, did not affect the cellular accumulation of tritiated anandamide. Inhibition of transport was demonstrated by N-(4-hydroxyphenyl)-arachidonamide, known as AM 404, and to a lesser extent by its 3-hydroxyphenyl congener. The results of examining a large number of substrates related to anandamide suggest that one or more cis double bonds in the anandamide "tail" are necessary for recognition, as well as a secondary amide function with a "head" group substituted by a polar hydroxalkyl or hydroxyaryl group. The investigators have also shown that tritiated anandamide, AM 404, and the endogenous compound arachidonylglycerol have comparable transport rates as measured by the decrease in radioactivity from the cellular medium over time. Several analogs lacking one, two, three, or all four of the cis double bonds in anandamide are transported relatively slowly or not at all. It has been suggested that one set of structural features may be needed for recognition by the transporter and another set for the actual transport. The development of transport inhibitors for anandamide has the potential utility of prolonging its antinociceptive effects by inhibiting its rapid inactivation due to transport and hydrolysis. Piomelli, D., Beltramo, M., Glasnapp, S., Lin, S.Y., Goutopoulos, A., Xie, X.-Q., Makriyannis, A. Proceedings of the National Academy of Sciences, 96, pp. 5802-5805, 1999.

Rewarding Effects of Drugs of Abuse and Neurotrophic Factors

The rewarding effects of drugs of abuse such as cocaine and morphine are mediated by the activation of dopamine neurons that make synaptic connections with neurons in the nucleus accumbens. Previous work by Dr. Eric Nestler and his colleagues at Yale University showed that the administration of brain derived neurotrophic factor (BDNF), a neurotrophin that supports the survival of dopamine neurons, prevents many of the biochemical and morphological changes associated with chronically administered cocaine and morphine. Injection of BDNF directly into the brain region known as the ventral tegmental area (VTA) blocks the induction of the rate-limited enzyme, tyrosine hydroxylase, in the synthesis of the neurotransmitter dopamine (DA). In addition, BDNF blocked the increase in protein kinase A (PKA) in the nucleus accumbens (Nac) produced by chronic morphine or cocaine administrations. In addition, BDNF prevents morphine from reducing the size of cell bodies in the DA neurons. Dr. Nestler suggests that BDNF prevents cocaine and morphine induced biochemical and morphological changes in the DA system though a converging biochemical pathway. If BDNF regulates the cellular signaling pathways activated by cocaine, then BDNF should alter behavioral responses to cocaine. To test this hypothesis, Dr. Nestler examined the effect of BDNF on locomotor activity and conditioned reward to cocaine. Conditioned rewards are cues that have become rewarding and provide incentive to seek a drug by virtue of being paired with the drug. The pairing of these discrete cues with cocaine could be an important mechanism involved in drug craving and relapse in addicts.

Injections of BDNF into the VTA or Nac enhanced both locomotor activity and the development of locomotor sensitization to cocaine. Mice expressing half as much endogenous BDNF showed delayed development of locomotor sensitization. Furthermore, intra-Nac injections of BDNF enhanced the ability of a stimulus to act as a conditioned reward to cocaine for up to a month following BDNF infusion. Dr. Nestler suggests that BDNF enhances the behavioral responses to cocaine by increasing dopamine transmission in response to cocaine. The delay in locomotor sensitization to cocaine in the BDNF heterozygous mutant mice also suggests that signaling pathways regulated by BDNF are required for long-term adaptation to cocaine. Horger, B.A., Iyasere, C.A., Berhow, M.T., Messer, C.J., Nestler, E.J., Taylor, J.R. Journal of Neuroscience, 19(10), pp. 4110-4122, 1999.

Orphanin FQ and Hypothalamic-Pituitary Axis

Orphanin FQ (OFQ) is a novel heptadecapeptide whose structure resembles that of dynorphin A1-17 . Its receptor shares appreciable homology with m- , d- , and k- opioid receptors, and is highly expressed in the hypothalamus. A recent article by Dr. Martin Kelly and his group demonstrates that OFQ activates a receptor that is not opioid in nature but is coupled to an inwardly-rectifying K+ channel, leading to an inhibition of b-endorphin neurons, and dopamine neurosecretory cells within the arcuate nucleus of the mediobasal hypothalamus. Collectively, these findings suggest that OFQ is not only an antiopioid peptide, but that it also modulates the hypothalamic-pituitary axis. Wagner, E.J., Ronnekleiv, O.K., Grandy, D.K., and Kelly, M.J. Neuroendocrinology, 67, pp. 73-82, 1998.

Identification and Characterization of Three New Alternatively Spliced m-Opioid Receptor Isoforms

Using genomic cloning in mice, Drs. Gavril Pasternak and Ying-Xian Pan and their research team at the Memorial Sloan-Kettering Cancer Center have identified four new m-opioid receptor (MOR)-1 exons, indicating that the gene now contains at least nine exons spanning more than 200 kilobases. Replacement of exon 4 by combinations of the new exons yields three new receptors. When expressed in Chinese hamster ovary (CHO) cells, all three variants displayed high affinity for m-opioid ligands, but k and d drugs were inactive. However, there were subtle, but significant, differences in the binding profiles of the three opioid receptor variants among themselves and from MOR-1. Immunohistochemically, the major variant, MOR-1C, displayed a regional distribution quite distinct from that of MOR-1. Region-specific processing also was seen at the mRNA level. Antisense probes designed to block each of the four new exons revealed that they were all involved in morphine analgesia. Together with two other variants generated from alternative splicing of exon 4, there are now six distinct MOR-1 receptors. The continued discovery of new opioid receptor isoforms with subtle but distinct differences in binding profiles, regional distribution, and region-specific processing and of their involvement in morphine analgesia suggests that these opioid receptor subtypes are pharmacologically relevant receptors. These findings are significant in that they provide new targets for designing analgesics that may be more effective with fewer undesirable side effects. Pan, Y-X, Xu, J., Bolan, E., Abbadie, C., Chang, A., Zuckerman, A., Rossi, G., and Pasternak, G.W. Molecular Pharmacology, 56, pp. 1-8, 1999.

Endomorphin-2 is an Endogenous Opioid in Primary Sensory Afferent Fibers

Drs. Sheryl B. Martin-Schild and James E. Zadina and their research group at Tulane University and Veterans Affairs Medical Center presented evidence that the recently discovered endogenous mu-opioid selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like immunoreactivity was found to be colocalized in a subset of substance P (a primary peptide neurotransmitter involved in the transmission of painful stimuli)- and mu opiate receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical or chemical methods virtually abolished endomorphin-2-like immunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and post-synaptic mu receptors and as a major modulator of pain perception. Martin-Schild, S.B., Gerall, A.A., Kastin, A. J., and Zadina, J.E. Peptides, 19(10), pp. 1783-1789, 1998.

Endogenously Formed Cannabinoids Control Synaptic Potentiation in Rat Hippocampus

Dr. Paul Schweitzer is studying the involvement of the endogenous cannabinoid anandamide system in brain neural synaptic activity and plasticity in the rat. It is known that administered cannabinoids impair learning and memory and that in the laboratory, superfusion of cannabinoid agonists in the hippocampus prevents long-term potentiation (LTP) of neuronal activity, a putative mechanism for learning and memory. Dr. Schweitzer hypothesized that if endogenously formed cannabinoids prevent or attenuate the potentiation process, a cannabinoid receptor antagonist would increase LTP. However, he did not observe increased LTP in the presence of a cannabinoid antagonist. He reasoned that the widely used LTP paradigm, which he also was using, was too extreme, such that the LTP could not be further increased under these conditions (a "ceiling" effect). To test this idea, Dr. Schweitzer used two moderate stimulation paradigms believed to be more relevant to physiological conditions. He found that LTP was enhanced in the presence of the cannabinoid antagonist in both of the moderate stimulation paradigms, indicating that endogenously formed cannabinoids control the enhancement of synaptic activity in the hippocampus. These results suggest that the endogenous cannabinoid system may serve as a "brake" to limit the level of potentiation and modulate synaptic neuronal activity in normal brain functioning. Schweitzer, P. International Cannabis Research Society Abstracts, p. 38, 1998.

Blockade of NMDA Receptors by Drugs of Abuse Causes Neurons to Die in the Infant Rat Brain

Several abused, addicting drugs [ethanol, phencyclidine (PCP, "angel dust"), ketamine (special K), and nitrous oxide] characteristically block the NMDA (N-methyl-D-aspartate) receptor in the brain. Since the proper development of this receptor and its activation by glutamate are essential for the formation of these synapses in the developing brain, the effects of these drugs on the fetal brain is an important area of study. Dr. John W. Olney, and his group examined the effect in rats of NMDA receptor blockade on the developing brain using dizocilpine, a specific NMDA receptor blocker. Blockade of NMDA glutamate receptors for four hours or more during early neonatal development triggered widespread neuronal death in the developing rat brain. Especially affected were layer II of parietal, frontal, and cingulate cortices, and laterodorsal thalamus. The mechanism of cell death was shown to be apoptosis, also known as the process of programmed cell death. Apoptosis is a process that occurs naturally during normal development of the brain; the brief exposure to dizocilpine magnified this process up to 40-fold. PCP and ketamine each triggered the same apoptotic response as dizocilpine. These findings may have relevance to human neurodevelopmental disorders caused by prenatal or postnatal exposure to drugs that block NMDA receptors. If peak vulnerability of the human forebrain to the pro-apoptotic action of NMDA antagonists corresponds to the developmental events in the rat, the window of vulnerability for humans would include the entire third trimester of pregnancy. Understanding the mechanisms underlying brain damage in the offspring of drug-abusing mothers may lead to strategies for preventing and treating this disorder. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Všckler, J., Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L., and Olney, J.W. Science, 283, pp. 70-74, 1999.

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