Effect of dextrometorphan and dextrorphan on nicotine and neuronal nicotinic receptors

31 Pages·2004·0.6 MB·English

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Effect of dextrometorphan and dextrorphan on nicotine and neuronal
nicotinic receptors: In vitro and in vivo selectivity
M. I. DAMAJ, P. FLOOD, K. K. HO E.L. MAY AND B. R. MARTIN

Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia
Commonwealth University, Richmond, VA 23298-0613 (M.I.D., E.L.M., B.R.M); and
Department of Anesthesiology, Columbia University, New York, NY 10032 (P.F., K.K.H.).

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Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: EFFECTS OF DEXTROMETORPHAN ON NICOTINIC RECEPTOR
SUBTYPES

* Address all correspondence to:
Dr. M. Imad Damaj
Department of Pharmacology and Toxicology, Medical College of Virginia,
Virginia Commonwealth University,
Box 980613
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Richmond, VA 23298-0613.
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Text pages: 25 t A
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Abstract: 204 words ry
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Introduction: 750 words 0
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Discussion: 930 words

ABBREVIATIONS : nAChR, Acetylcholine nicotinic receptor; CNS, Central nervous system;
%MPE, maximum possible effect; CL, confidence limits for potency; s.c., subcutaneous
injection; i.p., intraperitoneal injection; AD , antagonist dose 50%; IC , Inhibitory
50 50
concentration 50%; NMDA, N-methyl-D-aspartate.
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Abstract _ The effects of dextrometorphan and its metabolite dextrorphan on nicotine-induced
antinociception in two acute thermal pain assays after systematic administration were evaluated
in mice and compared to that of mecamylamine. Dextrometorphan and dextrorphan were found
to block nicotine’s antinociception in the tail-flick and hot-plate tests with different potencies
(dextrometorphan is 10-times more potent than its metabolite). This blockade was not due to
antagonism of NMDA receptors and/or interaction with opiate receptors, since selective drugs of
these receptors failed to block nicotine’s analgesic effects. Our results with the tail-flick and hot-
plate tests showed an interesting in vivo functional selectivity for dextrometorphan over
dextrorphan. In oocytes expressing various neuronal nAChRs, dextrometorphan and dextrorphan D
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blocked nicotine activation of expressed α3β4, α4β2 and α7 subtypes with a small degree of nloa
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selectivity. However, the in vivo antagonistic potency of dextrometorphan and dextrorphan in fro
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the pain tests does not correlate well with their in vitro blockade potency at expressed nAChRs jp
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subtypes. Furthermore, the apparent in vivo selectivity of dextrometorphan over dextrorphan is e
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not related to its in vitro potency and does suggest the involvement of other mechanisms. In that rna
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respect, dextrometorphan seems to behave as another mecamylamine, a non-competitive rg
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nicotinic receptor antagonist with a preferential activity to α3β4* neuronal nAChRs subtypes. SP
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Dextrometorphan is structurally related to the morphinan opioid levorphanol and is
widely known as a centrally acting non-narcotic antitussive agent. Although dextromethorphan
produces little analgesia by itself, it was found to potentiate the antinociceptive effects of opiates
and reduce tolerance and physical dependence to morphine (Mao et al., 1996). In addition, Glick
et al (2001) have shown that both dextrometorphan and its major metabolite, dextrorphan,
decreased nicotine, metamphetamine and morphine self-administration in rats after s.c.
administration. These multiple pharmacological and behavioral effects may be related to the
ability of dextrometorphan and dextrorphan to interact with various receptor systems.
Dextrometorphan has little or no opioid activity but binds with high affinity to σ sites (Klein and D
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Musacchio, 1989). It binds along with dextrorphan, with low affinity to the Phencyclidine (PCP) lo
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site of the NMDA receptor with dextrorphan having 10-fold higher affinity at the site (Franklin fro
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and Murray, 1992). In addition, dextrometorphan and dextrorphan are both antagonists at jp
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NMDA glutamate receptors (Murray and Leid, 1984). They were also recently shown to block e
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α3β4 neuronal nicotinic receptors in a non-competitive fashion (Hernandez et al., 2001) with rnals
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dextrometorphan 3-fold less potent than dextrorphan. These reports suggest that rg
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dextrometorphan and/or dextrorphan may hold promise for understanding nicotine pharmacology P
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and nicotine dependence. However, further studies are needed to investigate the modulation of o
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nicotine’s pharmacological effects by dextrometorphan and its metabolite, and to compare their ls o
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effects on various neuronal nAChRs. Although a wide range of pharmacological effects are n
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elicited after nicotine administration, we were particularly interested in nicotine’s antinociceptive 15
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action. 3
Activation of cholinergic pathways by nicotine elicits antinociceptive effects in a variety
of species (Aceto et al., 1986; Mattila et al., 1968; Phan et al., 1973). There is strong evidence
that the antinociceptive effect of nicotine can occur via activation of nAChRs expressed in the
CNS and peripheral sensory neurons (Iwamoto and Marion, 1993; Iwamoto, 1989, 1991; Bitner
et al., 1998; Aceto et al., 1986; Christensen and Smith, 1990; Damaj et al., 1998; Khan et al.,
1997; Flores et al., 1996). The antinociceptive effect of nicotine is of short duration (5 - 45 min)
and occurs at relatively high doses (0.5 – 2 mg/kg). In addition, tolerance develops after
repeated administration as measured in acute thermal pain models (Damaj and Martin, 1996). It
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is still unclear, however, which nicotinic receptor populations are responsible for the
antinociceptive effects of nicotine. In large part, this ambiguity results from a lack of available
subtype-specific agonists and antagonists. Nonetheless, multiple experimental approaches such
as transgenic mice and antisense studies have proved insightful. These mechanistic studies
conducted to date have largely focused on the role of α β * receptors in tests of acute, thermal
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nociception. The reported results suggest that α β receptor subtypes are components of the
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nicotinic response in the hot-plate test and to a lesser extent in the tail-flick assay Marubio et al.
1999; Bitner et al. 2000. The role of non-α β nAChRs in nicotinic analgesia is still however
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largely unknown. Recent studies have reported that the α antagonists failed to block the D
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antinociceptive effects of various nicotinic agonists in the tail-flick test after central lo
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administration in rats and mice indicating little involvement of α7 subtypes (Damaj et al., 1998; fro
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Rao et al., 1996). jp
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In the present study, we investigated the extent to which dextrometorphan and its e
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metabolite could alter the antinociceptive activity of nicotine in two acute thermal pain assays, rna
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the tail-flick and the hot-plate tests. The potential antagonism of nicotine-induced rg
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antinociception by dextrometorphan and dextrorphan was assessed after systemic (s.c. and i.p. P
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injections) administration. The route of administration of dextrometorphan can have a o
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substantial impact on its metabolism. Much higher levels of dextrorphan are generated after an ls o
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i.p. injection of dextrometorphan compared to the levels after s.c. injection (Wu et al., 1995). n
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The comparison of different routes of administration will be helpful in understanding the relative 15
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extent to which dextrometorphan and dextrorphan mediates the effects of dextrometorphan on 3
nicotine’s antinociceptive activity. The effects of dextrometorphan and its metabolite were
compared to that of mecamylamine, a non-competitive nicotinic antagonist with a preferential
activity at α β receptor subtypes (Papke et al., 2001). In addition, the selectivity of their
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nicotinic response was examined by testing the effects of various dextrometorphan structural
analogs, opiate- and/or NMDA receptor modulators, on nicotine-induced antinociception in
mice.
In vivo studies were complemented with the in vitro profiling of dextrometorphan and
dextrorphan on various neuronal nicotinic receptor subtypes. The antagonistic activity of
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dextrometorphan and dextrorphan was assessed at the α β and α β and α nicotinic receptor
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subtypes expressed in oocytes.
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Materials and Methods

Animals. Male ICR mice (20-25g) obtained from Harlan Laboratories (Indianapolis, IN) were
used throughout the study. Animals were housed in groups of six and had free access to food
and water. Animals were housed in an AALAC approved facility, and the study was approved
by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Drugs. Mecamylamine hydrochloride was supplied as a gift from Merck, Sharp and Dohme &
Co. (West Point, PA). (-)-Nicotine was obtained from Aldrich Chemical Company, Inc.
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(Milwaukee, WI) and converted to the ditartrate salt as described by Aceto et al. (1979).
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Dizocilpine (MK-801), dextromethorphan and dextrorphan were purchased from RBI (Natick, e
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MA). ). Acetylcholine and other chemicals used in the oocyte buffer solutions were obtained
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from Sigma-Aldrich (St. Louis, MO). Naloxone, (+)-morphine, codeine isomers and levorphanol t.as
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were supplied by the National Institute on Drug Abuse (Washington, DC). All drugs were u
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dissolved in physiological saline (0.9% sodium chloride). All doses are expressed as the free .o
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base of the drug. t A
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Antinociceptive tests. a
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1. Tail-flick test. Antinociception was assessed by the tail-flick method of D'Amour and J
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Smith (1941) as modified by Dewey et al. (1970). A control response (2-4 sec) was determined ry
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for each mouse before treatment, and a test latency was determined after drug administration. In 0
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order to minimize tissue damage, a maximum latency of 10 sec was imposed. Antinociceptive
response was calculated as percent maximum possible effect (% MPE), where %MPE = [(test-
control)/(10-control)] x 100.
2. Hot-plate Test. Mice were placed into a 10 cm wide glass cylinder on a hot plate
(Thermojust Apparatus) maintained at 55.0 ˚C. Two control latencies at least ten min apart were
determined for each mouse. The normal latency (reaction time) was 8 to 12 seconds.
Antinociceptive response was calculated as percent maximum possible effect (% MPE), where
%MPE = [(test-control)/(40-control) x 100]. The reaction time was scored when the animal
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jumped or licked its paws. In order to minimize tissue damage, a maximum latency of 40 sec
was imposed.
Groups of eight to twelve animals were used for each dose and for each treatment.
Antagonism studies were carried out by pretreating the mice with either saline, dextrometorphan
or dextrorphan 15 min before nicotine. This 15 min time was determined in separate
experiments to be the optimum pretreatment time for dextrometorphan and dextrorphan (data not
shown). The animals were tested 5 min after administration of nicotine. Opiate agonists and
antagonists as well as NMDA antagonists were evaluated for their ability to antagonize nicotine
in the tail-flick test. Codeine isomers, levorphanol (a structural analog of dextrometorphan with
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opiate activity) and (+)-morphine were tested at the highest inactive doses in the tail-flick test. w
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Naloxone (a non-selective opiate antagonist) was given at 1 mg/kg, a dose that is largely reported e
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to block morphine’s analgesic effects. m
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Oocytes Expression Studies. u
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1. Molecular Biology: The rat cDNAs for the α , β , α and β nAChRs were in ls.o
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pcDNAI/neo Polymerase T7, sk-Polymerase T3, psp64 Polymerase SP6 and psp65 Polymerase t A
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SP6 vectors, respectively. The human α nAChR clone was in a pMXT expression vector. The T
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vectors were linearized and used as templates for run-off transcription from the SP6 promoter rna
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using standard techniques. J
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2. Oocyte extraction and injection: Xenopus laevis oocytes were extracted from ry
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anesthetized females and placed in ND-96 medium (NaCl 96 mM, KCl 2 mM, MgCl2 1 mM, , 202
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CaCl H O 1.8 mM, HEPES 5 mM, Na-pyruvate 2.5 mM, theophylline 0.5 mM, and 10 mg/L of
2 2
gentamicin, adjusted to pH 7.5). The oocyte clusters were incubated in 0.2% collagenase (type
IA, Sigma-Aldrich) in ND-96 medium for defolliculation. Oocytes were agitated at 18.5°C for 4
hours and afterwards were rinsed with Barth’s medium (NaCl 88 mM, KCl 1 mM, NaHCO 2.4
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mM, HEPES 15 mM, pH 7.6). The oocytes were left to recover for 24 hr in L-15 oocyte
medium before injection of cRNA. L-15 oocyte media was obtained from Specialty Media
(Phillipsburg, NJ). Approximately 10 ng of cRNA was injected into individual oocytes in
volumes of ~100 nl using a manual injector (Nanoject; Drummond Scientific, Broomall, PA).
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For α β and α β nicotinic receptors, cRNA for the 2 subunits was injected mixed in a 1:1
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ratio. The oocytes were incubated at 17°C for 3-7 days in ND-96 medium prior to
electrophysiological recording.
3. Electrophysiology: Oocytes preparation and recording was performed as previously
described by Coates and Flood (2001). Current recordings were made from whole oocytes at
room temperature (19-23°C) using a Gene-Clamp 500 two-microelectrode voltage-clamp

amplifier with an active ground circuit (Axon Instruments, Inc., Foster City, CA). The recording
electrodes were pulled from glass capillaries (Drummond, Broomall, PA) to obtain a resistance
between 1 and 5 MΩ when filled with 3M KCl. The Ringer’s solution (115 mM NaCl, 2.5 mM D
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KCl, 1.8 mM BaCl 10 mM HEPES, 1 µM atropine, pH 7.4) used for recordings, contained nlo
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atropine to prevent muscarinic receptor stimulation and barium in place of calcium to avoid fro
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current amplification by calcium activated chloride currents. Oocytes were clamped at a holding jp
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potential of -60mV unless otherwise indicated and held in a 125 µl cylindrical channel. All pe
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drugs were applied by perfusion at a flow rate of 4ml/min. The oocyte was pre-exposed to rna
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dextromethorphan or dextrorphan for 2 min unless otherwise noted and tested with a 2 sec rg
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agonist application in the continued presence of dextromethorphan. Activation was complete P
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during the application period. To minimize the contribution of nAChR desensitization, three o
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min passed between Acetylcholine applications to α7 nAChRs and 5 min passed between ls o
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agonist applications to α β and α β nAChRs. Using this time interval steady state recordings an
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could be obtained in control experiments. A baseline control response to the agonist was 15
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measured before and after each agonist-antagonist co-application. The response to 3
dextromethorphan was compared to the average of the preceding and following agonist
response.
4. Statistical Analysis of the curves: Inhibitory concentration-response relationships for
the inhibition were constructed by calculating the current recorded in the presence of antagonist
as a percentage of that elicited by the agonist alone. The resulting data were fitted to a modified
Hill equation, y= y /(1+(x/IC )n ), where IC is the concentration of antagonist that elicited
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50% of the maximal inhibition, y is the maximal current elicited by the agonist, and n is the
max
Hill coefficient. The data points obtained at each concentration were averaged and the
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calculated mean and standard error were fit to a modified Hill equation. Clampex 7 (Axon
Instruments, Inc., Foster City, CA) was used for data acquisition and Microcal Origin 5.0
(Microcal, Northampton, MA) was used for graphics and statistical calculation. P < 0.05 was
considered significant and data were represented as mean ± SEM.

Statistical analysis. Statistical analysis of all analgesic studies was performed using either t-test
or analysis of variance (ANOVA) with Tukey’s test post hoc test when appropriate. All
differences were considered significant at p < 0.05. AD values with 95% CL for behavioral
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data were calculated by unweighted least-squares linear regression as described by Tallarida and D
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Murray (1987). lo
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