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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