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National Institute on Drug Abuse

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

May, 1999

Research Findings

Basic Research

Dopamine and Reward

The operation of a circumscribed reward system in which dopamine accumulation in the nucleus accumbens is appreciated as a reward is a key concept underlying substance abuse research. This concept evolved from two observations: 1) most drugs of abuse and natural rewards elicit the accumulation of dopamine in the accumbens, and 2) animals will self-administer electrical impulses to areas of their brains that closely correspond to catecholaminergic projections. The results of a newly published study raise questions about this interpretation of the actions of dopamine. Wightman and his colleagues used voltametry to measure the extracellular dopamine in the nucleus accumbens after intracranial electrical stimulation in rats. The stimulation was delivered either by the rats themselves, or by the experimenter without regard to the rats' behavior. Wightman found that: 1) rats were unable to learn to self-stimulate if the stimulation produced no dopamine release; 2) experimenter-delivered stimulation reliably increased the dopamine accumulation in the accumbens in animals that could learn the task; and 3) unexpectedly, once the task was learned, little dopamine accumulated as the animal self-administered the stimulation. By using a method with better temporal resolution than that available with microdialysis, Wightman demonstrated that the release of dopamine may not be critical in reinforcement once the task is learned. These findings also suggest that the dopaminergic response may evolve as an alerting function in response to novelty or as a predictor of reward. Garris, P.A., Kilpatrick, M., Bunin, M.A., Michael, D., Walker, Q.D., and Wightman, R.M. Nature 398, pp. 67-69, 1999.

Neuronal Nitric Oxide Is Involved in Cocaine Reward

Dr. Yossef Itzhak led studies to investigate the role of nitric oxide (NO) in the rewarding property of cocaine. Mice treated with cocaine every other day and saline on the alternate days for 8 days developed conditioned place preference (CPP) for the cocaine-paired compartment of the cage. Treatment with the neuronal NO synthase (nNOS) inhibitor 7-nitroindazole prior to each cocaine injection completely blocked cocaine CPP. Further, homozygote nNOS(-/-) knockout mice (i.e. mice lacking the nNOS gene) did not develop preference for the cocaine-paired compartment following the administration of cocaine, while wild-type nNOS(+/+) mice developed marked cocaine CPP. Thus, both pharmacologic and genetic manipulations of nNOS suggest that NO is involved in the rewarding property of cocaine. Itzhak, Y., Martin, J.L., Black, M.D., and Huang, P.L. NeuroReport, 9(11), pp. 2485-2488, 1998.

The Neuroendocrine Protein 7B2 Is Required for Peptide Hormone Processing In Vivo and Provides a Novel Mechanism for Pituitary Cushing's Disease

The neuroendocrine protein 7B2 is widely distributed within endocrine and neural tissues. It is absolutely required for the production of active prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. NIDA grantee Iris Lindberg of Louisiana State University Medical Center and her coworkers, in collaboration with researchers at Harvard, report the creation of 7B2 knockout mice. These animals have no PC2 activity, thus providing an in vivo confirmation of the requirement of PC2 for the 7B2 protein. Like PC2 knockout mice, the 7B2 knockout mice exhibit deficient processing of islet hormones and altered pancreatic islet morphology, and are hypoglycemic, hyperproinsulinemic and hypoglucagonemic. In contrast to the PC2 knockout mice, however, these mice show markedly elevated circulating adrenal corticotrophic hormone (ACTH) and corticosterone levels, with expansion of the adrenal cortex. Before they are 9 weeks old 7B2 knockout mice die of severe Cushing's disease as a consequence of ACTH hypersecretion from the intermediate lobe of the pituitary. Therefore, 7B2, required for activation of the processing enzyme PC2, has additional important functions in the regulation of secretion of pituitary hormones, and 7B2 knockout mice can provide a new avenue for the exploration of the regulation of peptide secretion. Westphal, C., Muller, L., Zhou, A., Zhu, X., Bonner-Weir, S., Schambelan, M., Steiner, D., Lindberg, I., and Leder, P. Cell, 96, pp. 689-700, 1999.

Orphanin-FQ/Nociceptin (OFQ/N) Modulates the Activity of Suprachiasmatic Nucleus Neurons

Circadian rhythms characterized by regular daily fluctuations in behavioral and physiological processes are regulated by the suprachiasmatic nucleus (SCN), a structure located above the optic chiasm of the ventral hypothalamus. Alterations in the duration of light modify circadian rhythms by influencing the patterns of activity of the SCN through NMDA receptor-mediated calcium influx. Increases in intracellular calcium activate nitric oxide synthase to produce nitric oxide, which activates cGMP and alters neuronal activity. Researchers at Oregon Health Sciences University have discovered the presence of the Orphanin-FQ/Nociceptin receptor, a receptor sharing homology with the opioid receptors and its ligand orphanin FQ in the SCN. To test whether OFQ/N plays an important role in regulating circadian rhythms, OFQ/N was unilaterally injected into the SCN of Syrian hamsters. OFQ/N inhibited the light-induced phase shifts of the onset of wheel running activity but did not directly phase shift the circadian clock. OFQ/N appears to attenuate the light induced phase shifts of the circadian clock by directly inhibiting NMDA receptor mediated calcium influx, and indirectly inhibiting the activation of NMDA receptors in the SCN by activating a potassium current that hyperpolarizes SCN neurons. Hyperpolarization prevents glutamate from activating the NMDA receptor. The effects of OFQ/N were blocked by OFQ/N antagonists but not by naloxone suggesting that OFQ/N acts through a unique non-opioid receptor. Allen, C.N., Jiang, Z.G., Teshima, K., Darland, R., Ikeda, M., Nelson, C.S., Quigley, D.I., Yoshioka, T., Allen, R.G., Rea, M.A., Grandy, D.K. Journal of Neuroscience, 19, pp. 2152-2160, 1999.

Endomorphin Conformation

An analysis of the conformations of the endogenous mu-selective ligand endomorphin-1 has been carried out by a collaborative study involving NIDA-funded researchers Dr. David M. Ferguson and Dr. Maria G. Paterlini of the University of Minnesota. Results of solution nuclear magnetic resonance (NMR) and molecular modeling simulation suggest the existence of a major "extended" conformation lacking internal hydrogen bonds. After aligning the respective tyrosines (the so-called "message" region involved in producing the ligand's opioid effect) of endomorphin-1 with the mu-selective ligands PL-017 and TIPP, reasonably good overlap was seen in the "address" regions (the third amino acid of each) believed to be involved in conferring mu-selectivity. The same region of overlap was not seen in comparing endomorphin-1 with the delta-selective ligand DPDPE and the mu/delta ligand TIPP-Nh3. Further studies will elucidate the structure-function relationships in the endomorphins. Podlogar, B.L., Paterlini, M.G., Ferguson, D.M., Leo, G.C., Demeter, D.A., Brown, F.K., and Reitz, A.B., FEBS Letters 439(1-2), pp. 13-20, 1998.

Peptide Prodrugs

In a recent collection of reports, Dr. Ronald Borchardt and his associates have described efforts to improve the membrane permeability and stability of opioid peptides Leu-enkephalin and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). This was accomplished by converting them into various cyclic prodrugs, and examining their passage across an in-vitro cellular model of the intestinal mucosa, and their stability toward chemical and enzymatic cleavage. In the case of coumarinic acid-based prodrugs, enhanced bi-directional permeation was demonstrated in the cellular model as compared to the peptides themselves. It was possible to demonstrate separately in buffered solution the formation of a non-cyclic intermediate, and subsequent quantitative release of the parent peptide and coumarin. In the case of acyloxyalkoxy-based prodrugs, the permeation was largely unidirectional, suggesting that these prodrugs were a substrate for an efflux transporter, such as P-glycoprotein. In all cases, considerably shorter half lives for the prodrugs were found upon exposure to rat liver and plasma enzymes, which fairly rapidly cleave the prodrug to the peptide and subsequently hydrolyze the peptide. This work points out some of the practical considerations in the design of delivery systems capable of transporting and delivering peptides of therapeutic interest, particularly to enhance their oral bioavailability. Bak, A., Gudmundsson, O.S., Pauletti, G.M., Shan, D., Wang, W., Siahaan, T.J., Friis, G.J., Wang, B., Shan, D., Zhang, H., and Borchardt, R.T. Pharmaceutical Research, 16, pp. 7-29, 1999.

Anandamide May be Involved in the Response of the Immune System to Cannabinoids

Dr. Burstein and his colleagues have examined whether the capacity of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line. Lipopolysaccharide (LPS), platelet-activating factor (PAF), and cannabinoids such D9-tetra-hydrocannabinol (THC) and ANA were all found to be agonists for the release of AA and led to increased ANA synthesis in macrophage cells for the mouse cell line RAW264.7. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. These data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis. Pestonjamasp, V.K., and Burstein, S.H. Biochimica et Biophysica Acta 55325, pp. 1-12, 1998.

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") Induced Acute Changes in Dopamine (DA) Transporter Function

The acute effects of the amphetamine designer drug, MDMA, on dopamine (DA) transporter function in rat striatum were investigated and compared to other psychostimulants known to influence monoaminergic systems. A single MDMA injection (10-20 mg/kg; sub-cutaneously (s.c.)) caused a dose-related decrease in [3H]dopamine uptake into striatal synaptosomes prepared one hour after MDMA injection. This rapid effect on [3H]dopamine uptake returned to control levels 24 hours after treatment. A single administration of other amphetamine analogs, such as methamphetamine (15 mg/kg; s.c.), p-chloroamphetamine (10 mg/kg; intra-peritoneally (i.p.)) or methcathinone (30 mg/kg; s.c.), also rapidly decreased striatal [3H]dopamine uptake. In contrast, a single or multiple administrations of cocaine (30 mg/kg; i.p.) had no effect on [3H]dopamine transport into striatal synaptosomes. These changes in dopamine transporter activity by the amphetamine analogs may occur via reactive oxygen species-mediated mechanisms. The results obtained implicate novel mechanisms by which the DA transporter can be regulated pharmacologically as well as physiologically, and further demonstrate a newly observed effect of amphetamine analogs on dopaminergic neurons. Since the normal function of the DA transporter is to regulate the action of released DA, disruption of DA transporter function can lead to deleterious effects such as changes in dopaminergic transmission and behavior. Metzger, R.R., Hanson, G.R., Gibb, J.W., Fleckenstein, A.E. Europ J Pharmacol, 349, pp. 205-210, 1998.

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