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National Institute on Drug Abuse

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

September, 1996


Research Findings


Basic Research


Chronic Morphine and Cocaine Produces Sustained Activation of The Extracellular Regulated Signal Regulated Kinase (ERK) Pathway

The laboratory of Eric Nestler at Yale University reported in the August 1 issue of The Journal of Neuroscience, Vol 16, pages 4707-4715 that chronic, but not acute, morphine or cocaine treatment selectively increases extracellular signal regulated kinase (ERK) activity in the ventral tegmental area (VTA) but not in substantia nigra, frontal cortex, or nucleus accumbens. The increase in ERK activity and associated induction of tyrosine hydroxylase (TH) produced by chronic morphine or cocaine treatment is prevented by brain derived growth factor (BDNF) and glutamate antagonists. To determine whether ERK kinases play a direct role in the increases in tyrosine hydroxylases in the VTA, antisense ERK1 oligonucleotides were infused into the VTA. Intra-VTA infusion of ERK1 antisense, like BDNF and glutamate antagonists prevented increases in both ERK activity and tyrosine hydroxylase. These findings suggest that sustained increase in ERK phosphorylation and activity contributes to drug induced increases in tyrosine hydroxylase (TH), and perhaps other drug-induced adaptations, elicited selectively in the VTA.


Opiate Exposure Leads to Increase in Synapsin I mRNA in Defined Central Nervous System Regions

Previous research has shown that chronic opiate exposure of spinal cord-dorsal root ganglion (SC-DRG) co-cultures leads to a time- and dose-dependent increase in the immunoreactive levels of synapsin I (a synaptic vesicle-associated protein important for neurotransmitter release). More recently, it was found by Dr. Zvi Vogel and colleagues at the Weizmann Institute of Science that chronic opiate treatment also affects the expression of synapsin I in the CNS of intact animals. A 3-fold increase in synapsin mRNA was observed (by Northern blots) in SC of rats chronically treated for 4 days with morphine. In situ hybridization with digoxygenin-labeled selective antisense cRNA probes revealed an increase of 3-7-fold in synapsin mRNA in selective areas of the CNS, including SC, locus coeruleus and amygdala (areas known to be involved in opiate activity and dependence), but not in most other brain areas, including the hippocampus and cerebellum. These findings raise the important question of the role of synapsin in opiate actions, tolerance and dependence.
Matus-Leibovitch et al., Mol. Brain Res. 34: pp. 221-230, 1995.


Opiate Regulation of Potassium Channel mRNAs

K+ channels are important synaptic proteins which regulate membrane potential of nerve terminals. Opiates affect the activity of various K+ channels (both activation and inhibition of K+ channels have been reported). Utilizing in situ hybridization, RNAse protection, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical techniques, it was found that motor neurons are highly enriched in the mRNAs of Kv1.5 and Kv1.6 voltage-gated K+ channels, and in Kv1.5 channel protein. A significant increase (2-2.5 fold) in the mRNA and protein of these channels was observed in spinal cord of morphine-treated rats, compared to controls. These results suggest an important role for opiate neurotransmission and for Kv1.5 and Kv1.6 K+ channels in regulating motor activity. Matus-Leibovitch et al., Mol Brain Res, In Press.


Ligands for the Cannabinoid Receptor

Pravadoline, a "non-classical" cannabinoid receptor ligand, is an alkylindole that displays antinociceptive activity. In a recent study, Yamada et al., reported that a naphthyl derivative of this compound which contains the electrophilic isothiocyanate group binds to the cannabinoid receptor with high affinity, and after "washing out" the ligand, it produces an irreversible loss of the receptor binding capacity, possibly by chemical reaction between the isothiocyanate group and nucleophilic amino groups such as lysine or histidine within the binding site. It remains to be shown whether the binding site or sites for this class of compounds is the same as for classical cannabinoids. Yamada, K., Rice, K.C., Flippen Anderson, J.L., Eissenstat, M.A., Ward, S. J., Johnson, M.R., and Howlett, A.C. Journal of Medicinal Chemistry, 39, pp. 1967-1974, 1996.


New Peptidomimetic

It has been previously reported that the peptides FMRFamide (isolated from mollusk) and NPFF (isolated from bovine brain) have antiopiate pharmacological action, possibly via specific NPFF receptors. In a recent NIDA-funded study, a conformationally constrained peptidomimetic analog of FMRF containing E-2,3 methanomethionine and E-2,3 methanophenylalanine proved to be approximately 200 times more potent in a morphine abstinence model, principally because of its enhanced resistance to peptidase degradation as compared to FMRF. Malin, D.H., Lake, J.R., McDermitt, L.S., Smith, D.A., Witherspoon, W.E., Jones, J.A., Schumann, M.D., Payza, K., Ho, K.K., and Burgess, K. Peptides, 17, pp. 83-86, 1996.


Tolerance Development Reduced to Opioid Receptors

DPDPE in animal models results in a tolerance to this delta receptor ligand's analgesic capability. A NIDA-funded study has recently provided evidence that this development of tolerance may be reduced by the chronic administration of MK-801 (a competitive NMDA antagonist) or LY 235959 (a noncompetitive NMDA antagonist). The antagonists may be activating the opioid systems or regulating the processing of opioid peptides. Zhao, G.M., and Bhargava, H.N. Peptides, 17, pp. 233-236, 1996.


Cellular Trafficking of Opioid Receptors

Mark Von Zastrow's laboratory at the University of California, San Francisco reported in the August 9 issue of The Journal of Biological Chemistry, Vol 271, pages 19021-19024 that morphine activates opioid receptors without causing their rapid internalization. In this report the authors examined the endocytic trafficking of epitope tagged d and m receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by enkephalins as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to be internalized rapidly in transferin-containing endosomes. Remarkably, morphine does not stimulate the rapid internalization of either d or m opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonists ligands which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G-coupled receptor.


Opioid Peptide Biosynthesis: Regulatory Mechanisms

Enkephalins and endorphin like other biologically active peptides are cleaved from a larger poly-peptide by intracellular processing enzymes. The intracellular processing enzymes responsible for the formation of biologically active opioid peptides in nervous tissue are the new PC1 and PC2 subtilisin processing enzymes. NIDA Grantee Iris Lindberg of Louisiana State University Medical Center and her coworkers recently identified the first endogenous inhibitor of PC enzymes called 7B2. They have now obtained evidence using site-directed mutagenesis that the proline-rich region (residues 888-95, PDPPNPCP) is responsible for the binding of the 21 kDa portion of 7B2 to PC2. This region bears similarities to src homology 3 (Sh4) domain, known to mediate protein-protein interactions. Four different assays were used to assess the functional aspects of mutated 7B2s and the roles of the prolines in the proline-rich region (coimmunoprecipitation, facilitation of proC2 activation, protection from thermal inactivation, and acquisition of enzymatic activity).

Since proteolytic cleavage represents the first step of the enkephalin biosynthetic pathway, it is likely that regulatory mechanisms which control opioid peptide production may involve these crucial proteolytic enzymes. Deficiencies in the biosynthetic capacity for opioid peptides may be responsible for the addictive properties of opiate drugs in certain individuals; thus the study of enzymatic mechanisms regulating endogenous opioid production is of extreme importance. A thorough understanding of regulatory mechanisms in opioid peptide synthesis might one day lead to enzyme-based drugs serving as therapeutic agents in opiate addiction. J. Biol. Chem., In Press.


The Role of Opiate Systems in Immune System Regulation

Expressing the "neural" opiate receptor at high levels in a prototype human immune cell, this group was able to demonstrate a link between opiates and the basic Calcium transport system function. Until recently, functionality as well as binding for these receptors has been difficult to measure due to the low level of expression of these receptors in immune cells. Information gained from this research should enable scientists to better evaluate the importance of opiate systems and immunity regulation. Sharp, B.M., Shahabi, N.A., Heagy, W., McAllen, K., Bell, M., Huntoon, C., and McKean, D. J. Dual Signal Transduction through Delta Opioid Receptors in a Transfected Human T-Cell Line. Proc Natl Acad Sci USA, 93, pp. 8294-8299, 1996.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T-cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([ca2+]I ) and/or cAMP are affected by opiates acting through delta type opioid receptors (DOR), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T cell line. The DOR agonists, deltorphin and [D-Ala2~D-Leus]-enkephalin (DADLE), elevated [ca2+]I, measured by flow cytofluorometry using the calcium sensitive dye, Fluo-3. At concentrations from 10-11to 10-7 M, both agonists dose-dependently increased [ca2+]I from 60 nM to peak concentrations of 400 nM within 30 sec (ED50 of approximately 5xl0-9 M). Naltrindole, a selective DOR antagonist, abolished the increase in [ca2+]I and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA which reduced the initial deltorphin-induced elevation of [ca2+]I by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately 10-11 M) and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify 2 pertussis toxin-sensitive G-protein mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation. Sharp, B.M., Shahabi, N.A., Heagy, W., McAllen, K., Bell, M., Huntoon, C. and McKean, D.J. 1996 Proceeding of the National Academy of Sciences, USA, In Press.


Effects of Cocaine on Pharmacodynamics and Pharmacokinetics

Drs. Charles Mactutus and Rosemary Booze at the University of Kentucky have been studying the effects of cocaine on pharmacodynamics and pharmacokinetics in adult Sprague Dawley rats. They have reported that repeated cocaine administration (0.5 to 3.0 mg/kg/day IV) resulted in a significant dose-dependent increase in striatal D3 receptors and a significant decrease in D3 receptors in the nucleus accumbens. Sensitization to cocaine was also seen, with the time to peak being dose dependent following the rank order of 0.5>1.0>3.0 mg/kg. D2 receptors were unchanged in both striatum and nucleus accumbens. These data suggest that the D3 receptors in the striatum and nucleus accumbens may be differentially involved in the locomotor stimulation (striatal D3) and reinforcing aspects (nucleus accumbens D2) of repeated cocaine administration. Synapse 23: pp. 152-163, 1996. In aged Fisher-344XBrown Norway rats, these investigators reported that D3 receptors in the striatum and the nucleus accumbens were significantly increased. European J. Pharmacology, In Press.

In other studies, Drs. Mactutus and Booze have concentrated on the pharmacokinetic effects of a single injection of cocaine at 0.5 to 3.0 mg/kg, IV. Arterial plasma concentrations of cocaine and metabolites (benzoylecgonine [BE], ecgonine methyl ester [EME], and norcocaine [NC]) were determined in adult Sprague Dawley rats. Peak plasma concentrations of cocaine occurred at 30 seconds (the first time point measured) and were dose dependent. The distribution half-life (T1/2a) was less than 1 minute for all groups, but inversely related to dose. More importantly, the elimination half-life (T1/2ß) (12-13 minutes), the mean residence time (MRT) (14.5-16 minutes), the volume of distribution at steady state (Vdss) (2.8-3.3 L/kg), and total clearance (Cltot) (195 204 ml/min/kg) were all independent of dose. Although the metabolic profile of IV cocaine was similarly ordered for all dose groups (BE>EME>NC), a quantitative shift in metabolite profile was evident as a function of increasing dose. This metabolic shift, perhaps attributable to saturation of plasma esterases, suggests that the recently reported pharmacodynamic effects positively correlated with IV cocaine are unlikely attributable to norcocaine. In summary, the IV pharmacokinetic profile of cocaine in adult rats is distinct from that observed via the IP, PO, or SC routes of administration. Neurotoxicology and Teratology, In Press.


Effects of NMDA Receptor Antagonists on Opioid Tolerance and Withdrawal

Dr. Tony Yaksh and colleagues at the University of California have studied the effects of administration of NMDA receptor antagonists on opioid tolerance and withdrawal in rats and have found that infusion of MK-801 in combination with morphine resulted in the development of considerably less tolerance of morphine's analgesic properties. MK-801 had no effect on pain processing by itself. These data suggest that activation of NMDA receptors may serve a permissive action in the development of tolerance to morphine's analgesic properties. Similar findings were observed when a different class of spinal analgesics (alpha-2 agonists) were studied, demonstrating broad generality of the involvement of NMDA receptors in tolerance. Anesthesiology, In Press.


THC and Pregnancy

NIDA supported research to be published soon reports that the mouse uterus during early pregnancy has the capacity to synthesize and degrade anandamide. These findings coupled with earlier findings of cannabinoid receptors in the preimplantation mouse embryo and uterus suggest that these tissues could be targets for cannabinomimetic ligands. The researchers also observed an inverse relationship between the synthase and amidase activity at the implantation or inter-implantation sites in the uterus during the peri-implantation period suggesting embryonic influence in regulating these activities. Although the physiological significance of these findings is not yet clear, it is possible that aberrant synthesis of anandamide and/or expression of the cannabinoid receptors in the uterus and/or embryo could contribute to early pregnancy. Paria, B.C., Deutsch, D.D., and Dey, S. K. Molecular Reproduction and Development, In Press.


Renal Function and Drugs of Abuse

The most predominant renal lesion seen in heroin addicts as well as in patients with HIV infection is glomerulosclerosis; however, the mechanism underlying this injury is not understood. A recent report from Dr. Pravin Singhal's laboratory showed that morphine-induced macrophage secretory products stimulated proliferation of mesangial cells derived from rats and mice. Synthesis of extracellular matrix components, laminin and collagen was also enhanced. Furthermore, these actions of morphine on mesangial cells appeared to be mediated through TGF-ß. These findings suggest a possible role for macrophages in the development of glomerular lesions in patients with drug addiction. Singhal, P.C., et al., Kidney Intl. 49: pp. 94-102, 1996.

In another recent paper, these researchers report that macrophage gp-160 interaction products enhance matrix synthesis as well as modulate mesangial cell proliferation which may contribute to expansion of the mesangium. These results, according to the investigators, support the hypothesis that HIV-1 proteins have the potential to cause expansion of the mesangium, and that replication of virus in renal cells is not a prerequisite for the development of glomerular lesions in patients with HIV infection. Singhal, P.C., et al., Amer J. Pathology 147: pp. 1780-1789, 1995.


Potential Treatment for PCP Toxicity

NIDA grantee Dr. Michael Owens from the University of Arkansas College of Medicine has recently published work in the Journal of Pharmacology and Experimental Therapeutics in which he reports having produced anti-PCP monoclonal antibody fragments (Fab) which can clear 90% of injected PCP from rat brain within 10 minutes. Rat behavior returns to normal within 30 minutes of treatment with anti-PCP Fab. This makes anti-PCP Fab a candidate for development as an emergency room treatment for PCP toxicity.


Activation of Limbic Regions During Cue-Induced Cocaine Craving

Using PET, Dr. Anna Rose Childress from the University of Pennsylvania showed that participants with histories of cocaine use demonstrated significant increases in regional cerebral blood flow (rCBF) in temporal pole, amygdala, and anterior cigulate when shown a video with cocaine-related stimuli but not when shown a neutral video. Systematic activation did not occur when shown a neutral video. Results suggest that limbic activation may underlie certain aspects of cue-induced craving. d and m receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by enkephalins as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to be internalized rapidly in transferin-containing endosomes. Remarkably, morphine does not stimulate the rapid internalization of either d or m opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonists ligands which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G-coupled receptor.


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