Schering-Plough Research Institute, Kenilworth, New Jersey
(G.J.C.) and Harvard Medical School, McLean Hospital ADARC,
Belmont, Massachusetts (J.B.)
Squirrel monkeys were trained to discriminate i.m. injections of the
-opioid receptor agonist enadoline (0.0017 mg/kg) from saline in a
two-lever drug-discrimination procedure. Enadoline produced a reliable
discriminative stimulus that was reproduced by the -selective
agonists PD 117302, U 50,488, GR 89686A, (
)-spiradoline, ICI 204448, and EMD 61753, and by the mixed-action /µ-agonists bremazocine and
ethylketocyclazocine. The discriminative stimulus effects of
enadoline were not reproduced by the µ-selective agonist morphine,
the 
-selective agonist BW373U86, the mixed-action opioids nalbuphine
and nalorphine, or by the less active enantiomers of enadoline and
spiradoline PD 129829 and (+)-spiradoline, respectively. The selective
µ-opioid antagonist 
-funaltrexamine (10.0 mg/kg) did not
appreciably alter the dose-effect function for enadoline in any
subject. However, the nonselective and -selective opioid antagonists
quadazocine (0.03-3.0 mg/kg) and nor-BNI (3-10 mg/kg), and the
mixed-action opioid nalbuphine (0.3-30 mg/kg) served to surmountably
antagonize enadoline's discriminative stimulus effects. The antagonist
effects of nor-BNI were long-lasting and did not distinguish between
drugs purported to act at different -receptor subtypes. The present
results bolster the view that common discriminative stimulus effects of
enadoline and other opioids are mediated by -agonist actions that
are surmountably antagonized by nor-BNI in a long-lasting manner. The
enadoline-antagonist effects of nalbuphine support the idea that it
acts with low efficacy at -opioid receptors.
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Introduction |
Drug
discrimination procedures have been used extensively to
pharmacologically characterize opioids with µ-receptor-mediated actions in rodent, avian, and primate species. -Opioid agonists have
been studied less comprehensively, and the majority of studies has been
conducted with mixed-action /µ-agonists or selective -agonists
in pigeons and rats. In those studies, other - but not µ-agonists
elicited responding on the lever associated with the training drug
(Shearman and Herz, 1982
; Picker and Dykstra, 1987, 1989; Holtzman et
al., 1991; Picker et al., 1993; Picker, 1994a; Brandt and
France, 1996). In contrast, responding on the drug-associated lever was
produced only by µ- but not -agonists when subjects were trained
with µ-agonists (Shearman and Herz, 1982; Holtzman et al., 1991;
Comer et al., 1993; Picker et al., 1993). Results from limited studies
in monkeys tend to agree with results obtained in rats and pigeons. In
rhesus monkeys, for example, the discriminative stimulus effects of the
/µ-opioid EKC were reproduced by other -agonists, but not by
the µ-agonist alfentanil (Hein et al., 1981; Tang and Collins, 1985;
Dykstra et al., 1987a; France et al., 1994). Taken as a whole, these
data suggest that pharmacological specificity in the discriminative
stimulus effects of -opioid drugs is conserved across species.
The availability of antagonists that selectively block different types
of opioid receptors also has contributed to the pharmacological classification of opioids in drug discrimination studies. For instance,
the selective µ- and -antagonists -funaltrexamine (-FNA;
Portoghese et al., 1980) and naltrindole (Portoghese et al., 1988),
respectively, have helped distinguish between discriminative stimulus
effects mediated at these two types of opioid receptor (Dykstra et al.,
1987b; Comer et al., 1993). Similarly, nor-binaltorphimine (nor-BNI;
Portoghese et al., 1987), a selective and systemically active
antagonist at -opioid receptors (Endoh et al., 1992; Horan et al.,
1992), may be useful for the study of -mediated discriminative stimulus effects. In this regard, previous studies with nor-BNI have
revealed that it has a slow onset and long duration of action (Jones
and Holtzman, 1992; Butelman et al., 1993; Broadbear et al., 1994;
Jewett and Woods, 1995).
-Opioids also may bind different subtypes of -receptor (e.g.,
1, 2; Zukin et al.,
1988; Clark et al., 1989; Devlin and Shoemaker, 1990; Rothman et al.,
1990), and a small number of studies indicate that -mediated effects
of different agonists may be differently antagonized by opioid receptor
blockers. For instance, Butelman et al. (1993) recently showed that
nor-BNI antagonized the effects of U 50,488 and U 69593, but not those of enadoline, bremazocine, EKC, or MR 2033, in a thermal
antinociception assay in rhesus monkeys. Such findings are consistent
with reports of differing degrees of selectivity for -receptor
subtypes among -opioids in radioligand binding studies (Zukin et
al., 1988; Clark et al., 1989; Devlin and Shoemaker, 1990; Rothman et
al., 1990). However, the functional significance of such distinctions among -opioid receptors remains ambiguous.
The present studies were designed to further examine the effects of
-opioid agonists by studying the effects of different opioid
agonists and antagonists in squirrel monkeys trained to discriminate
injections of enadoline from saline. Enadoline, previously forwarded
for clinical application as a diuretic and, possibly, in the treatment
of drug abuse (Hunter et al., 1990; Negus et al., 1997), is reported to
have -selective agonist actions: it binds -opioid receptors in
monkey brain with approximately 60- and 6000-fold higher affinity than
observed at, respectively, µ- and -opioid receptors (France et
al., 1994). Like other -agonists, it is highly potent in decreasing
electrically evoked contractions of both mouse vas deferens and guinea
pig ileum (Hunter et al., 1990). Also as with other -opioid
agonists, higher doses of the nonselective opioid receptor blockers
naloxone and naltrexone are needed to antagonize the effects of
enadoline than of µ-opioid agonists (Harris, 1980; Picker,
1994a,b). Finally, the -antagonist nor-BNI has been shown to
selectively attenuate behavioral effects of enadoline in assays of
antinociception, consistent with blockade of its actions at -opioid
receptors (Butelman et al., 1993; Broadbear et al., 1994).
The present results show that enadoline serves as a reliable
discriminative stimulus. Its effects are reproduced by other -opioids and some, but not all, mixed-action /µ-opioids and are
surmountably antagonized by quadazocine and nalbuphine. Nor-BNI also
produced dose-related rightward shifts in the enadoline discrimination dose-effect function, indicative of surmountable antagonism. The antagonist actions of nor-BNI were especially long-lived and were observed with both enadoline and U 50,488.
| |
Materials and Methods |
Subjects.
Five male squirrel monkeys (Saimiri
sciureus), weighing 750 to 890 g were studied during daily
experimental sessions. Between sessions, monkeys lived in individual
home cages where they had unlimited access to water and received a
nutritionally balanced diet consisting of Purina monkey chow, fresh
fruit, and vegetables. All monkeys were experimentally naive at the
beginning of these studies.
Apparatus.
Experiments were conducted in ventilated, sound
attenuated chambers in which white noise masked extraneous sounds.
During sessions monkeys sat in a customized Plexiglas chair facing a panel through which two easily accessible levers were centered and
mounted 15 cm apart (model 121-05; BRS/LVE, Beltsville, MD). Above the
levers were pairs of red stimulus lights that could be illuminated to
serve as visual stimuli. Depression of either lever with a minimum
force of 0.25 N resulted in an audible click and was recorded as a
response. The chair was also fitted with a small stock to secure a
shaved portion of the monkey's tail beneath brass electrode plates.
Electrode paste ensured a low-resistance contact between the tail and
the electrodes. Brief, low-intensity electric shock (3.0 mA, 200 ms)
could be delivered through the electrodes to the tail.
Drug Discrimination.
Each monkey was trained under a
10-response fixed-ratio (FR10) schedule of stimulus-shock termination
to respond differentially on the left or right lever, depending on
whether enadoline or saline was injected intramuscularly. Under this
schedule, the completion of 10 consecutive responses on the
injection-appropriate lever within 10 s turned off red stimulus
lights, and initiated a 40-s time-out (TO) period. If 10 responses were
not completed, a mild electric shock was delivered to the shaved
portion of the tail every 10 s. If the response requirement was
not met within 40 s (four shocks), the cycle ended automatically
and the 40-s TO period began. Responding on the left lever was
associated with injections of enadoline for monkeys s322, s205, and
s220, whereas responding on the right lever was associated with
injections of enadoline for monkeys s323 and s484. Initially, the
training dose of enadoline was 0.003 mg/kg for all monkeys.
Subsequently, initial dose-effect determinations revealed that 0.0017 mg/kg enadoline produced 100% responding on the drug-associated lever
in all monkeys. Consequently, the training dose was lowered to 0.0017 mg/kg and was maintained at that level throughout the studies.
Training sessions consisted of a varying number (n = 1-4) of components. Each component was preceded by a 10-min TO period and ended after the completion of 10 FRs or 800 s, whichever
occurred first. During most training sessions, saline was injected
during the TO periods preceding all but the last component of the
session, and drug was injected during the TO preceding the last
component. Periodically saline was injected during all TOs to limit the
association between the last component and the injection of enadoline.
Training continued until criteria of 
90% of all responses and
completion of all 10 FRs on the injection-appropriate lever were
achieved for five consecutive sessions.
Test sessions were conducted no more than twice weekly and only
following training sessions during which all FRs in each component and
90% of all responses were completed on the injection-appropriate lever. Test sessions consisted of four components, each preceded by a
10-min TO period. During each component, the completion of 10 consecutive responses on either lever turned off the red stimulus lights and initiated the 40-s TO period. Except for enadoline, each
drug in substitution experiments generally was studied in a group of
four monkeys. The full dose-effect function for enadoline was
determined in all five monkeys.
Drugs were studied using a cumulative dosing procedure that has been
described previously (Spealman, 1985; Rosenzweig-Lipson and Bergman,
1993). Briefly, incremental doses of all drugs, except EKC and
morphine, were injected at the beginning of the 10-min TO periods. The
TO was shortened to 5 min for EKC, which has a short duration of action
and lengthened to 20 min for morphine, which has a slow onset to
action. For some drugs, data on five or more doses were obtained by
studying overlapping dose ranges of cumulative doses in different test
sessions. The drugs were studied up to doses that 1) substituted fully
for enadoline or 2) reduced response rates to <0.2 responses/s.
Studies involving pretreatment were completed in groups of three or
four monkeys and were conducted by administering injections of
different doses of quadazocine or nalbuphine 10 min before the test
session. Pretreatment doses of both drugs were selected on the basis of
data from preliminary experiments. Pretreatment studies also were
conducted with the selective µ-opioid antagonist -FNA, and the
selective -opioid antagonist nor-BNI (Portoghese et al., 1980,
1987). Previous studies have indicated that the antagonist effects of
these drugs are slow in onset and persist for a prolonged period of
time (Butelman et al., 1993; Broadbear et al., 1994; Jewett and Woods,
1995). Therefore, test sessions were run on consecutive days, 60 min and 24 h following administration of -FNA or nor-BNI. Studies with nor-BNI were the last to be conducted and its effects were studied
for up to 80 days by suspending training during this time and
periodically redetermining the dose-related effects of enadoline or
other -opioid agonists in individual subjects. Pretreatment doses of
-FNA and nor-BNI were selected on the basis of published data (see above).
Analysis of Drug Effects.
Data from drug
discrimination experiments were analyzed for individual monkeys by
computing the percentage of responses on the enadoline-associated lever
in each FR component, i.e., the number of responses on the enadoline
lever was divided by the total number of responses on both levers,
provided that the rate of responding in that component was 0.2
responses/s. When response rates were <0.2 responses/s, data were
recorded but not analyzed further. A drug was considered to have
substituted fully for the training dose of enadoline in individual
monkeys when responding on the enadoline-associated lever was 90%
and response rates were 0.2 responses/s in all monkeys. When effects
were consistent across monkeys, data are expressed as percentage of
responding on the enadoline-associated lever averaged for the group of
monkeys (mean ± S.E.M.). Additionally, response rates were
calculated for each session component by dividing the total number of
responses made on either lever by the total time during which stimulus
lights were illuminated. Response rates are expressed as responses per second averaged for the group of monkeys (mean ± S.E.M.). For drugs that substituted for enadoline alone or in the presence of an
antagonist, estimated ED50 values were calculated
by log-linear interpolation along the ascending portion of individual
dose-effect functions and averaged for the group of monkeys. Average
estimated ED50 values were used to 1) establish
potency relationships among drugs that substituted for enadoline, and
2) examine antagonism of the discriminative stimulus effects of
enadoline by dose-ratio analysis. For dose-ratio analysis, dose ratios
were calculated for enadoline in combination with quadazocine,
nalbuphine, and nor-BNI by dividing the estimated
ED50 for enadoline in the presence of each dose
of antagonist by the estimated ED50 for enadoline alone. For quadazocine and nalbuphine, at least three doses of each
drug were used in combination with enadoline, and dose-ratio analyses
were used to calculate pA2 values. For this
analysis, the log of the dose-ratio minus 1 was plotted as a function
of the negative log of the antagonist dose in moles per kilogram. These
points were used to derive regression lines for agonist/antagonist interactions; the apparent pA2 value was defined
as the point where the regression line intercepted the abscissa (i.e.,
where the dose-ratio equals 2). Despite some inter subject variability, the confidence interval of the slope of each calculated regression line
included unity; consequently, slopes were not constrained to 1 for
further analysis. All apparent pA2 calculations
were performed using the Pharmacological Calculation System, version 3, based on the procedures of Tallarida et al. (1979).
Drugs.
All drugs were administered intramuscularly into the
thigh or calf muscle of the seated monkey. All compounds were dissolved in sterile 0.9% saline. Drugs were obtained from the following sources: enadoline, its (+)-enantiomer PD 129829, and PD 117,302 (Parke-Davis Pharmaceuticals, Cambridge, UK and Ann Arbor, MI); GR
89686A (Glaxo Research Inc., Research Triangle Park, NC); U 50,488, the
enantiomers of spiradoline, U 63640 [(+)-spiradoline], and U 63639 [()-spiradoline] (Upjohn, Kalamazoo, MI); bremazocine (Sandoz,
Basel, Switzerland); ethylketocyclazocine and quadazocine (Sterling-Winthrop, Rensselaer, NY); BW 373U86 (Burroughs Welcome, Research Triangle Park, NC); ICI 204448 (Zeneca Pharmaceuticals, Wilmington, DE); morphine (Sigma Chemical Co., St. Louis, MO); nalorphine (National Institute on Drug Abuse, Rockville, MD); EMD 60400 (E. Merck, Darmstadt, Germany); nalbuphine (DuPont, Wilmington, DE);
butorphanol (Bristol-Meyers, Wallingford, CT); -funaltrexamine; and
nor-binaltorphimine (Research Biochemicals International, Natick, MA).
| |
Results |
Initial Training.
Monkeys met testing criteria over an average
of 66 trials (range 24-124) following the initiation of discrimination
training with 0.003 mg/kg enadoline. Injection of this dose of
enadoline consistently elevated response rates above those associated
with injections of saline. For the group of monkeys, response rates averaged 2.32 ± 0.02 responses/s following injections of
enadoline and 1.79 ± 0.01 responses/s following injections of saline.
Effects of Enadoline.
Following training, test sessions began
with determination of the effects of enadoline. Administration of
graded doses of enadoline (0.0001-0.01 mg/kg) before sequential
components of the sessions produced dose-related increases in
responding on the enadoline-associated lever, and full substitution
(90%) was observed at doses of 0.0017 and 0.003 mg/kg enadoline. The
training dose was therefore lowered to 0.0017 mg/kg for all monkeys and the effects of enadoline alone were periodically redetermined throughout the course of the present studies. Over the course of
redeterminations, full substitution eventually was observed following
administration of the cumulative dose of 0.001 mg/kg in all monkeys.
Nevertheless, the training dose was kept at 0.0017 mg/kg to maintain
consistency in the experimental protocol throughout substitution and
antagonism studies. At doses of enadoline that produced full
substitution (0.001-0.003 mg/kg), rates of responding continued to be
elevated compared with saline control values. Administration of yet
higher doses of enadoline (0.01 and 0.03 mg/kg) resulted in a slight
decrease in the level of enadoline-associated responding and reduced
response rates to an average of approximately 0.3 responses/s for the
group of monkeys (Fig. 1).
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Fig. 1.
Effect of enadoline in monkeys trained to
discriminate enadoline (0.0017 mg/kg i.m.) from saline. Abscissae,
cumulative dose, log scale; ordinates, percentage of responses on the
enadoline-associated lever (top), response rate (bottom). Responding at
or above 90% indicates full substitution. Points above C indicate
effects of training dose of enadoline ( ) and saline ( ). Data
expressed as mean ± S.E.M. for three to four monkeys at each
dose.
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Time course studies showed that responding was maintained exclusively
on the enadoline-associated lever for approximately 50 min following
injection of the training dose of 0.0017 mg/kg (Fig.
2). After 70 min, the level of
enadoline-associated responding decreased to an average of
approximately 80% for the group of monkeys. Testing at 90-min
postinjection revealed approximately 40% responding on the
enadoline-associated lever and, after 110 min, monkeys responded
exclusively on the saline-associated lever.
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Fig. 2.
Duration of action of the training dose (0.0017 mg/kg) of enadoline in monkeys trained to discriminate enadoline from
saline. Abscissa, time (min) after 0.0017 mg/kg enadoline injection;
ordinate, percentage of responding on enadoline-associated lever. Data
expressed as mean ± S.E.M. for three to four monkeys at each time
point.
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Effects of Selective Opioid Agonists.
Administration of the
-selective opioids PD 117302 (0.03-0.3 mg/kg), U 50488H (0.03-0.3
mg/kg), GR 89686A (0.0003-0.003 mg/kg), and ()-spiradoline
(0.01-0.1 mg/kg) produced dose-related increases in responding and
90% responding on the enadoline-associated lever in all monkeys.
Except for ()-spiradoline, full substitution for enadoline by these
opioids was produced at doses that, except for elevated response rates,
did not markedly disrupt fixed-ratio performance. For ()-spiradoline,
full substitution was produced at doses that disrupted the fixed-ratio
pattern of responding and reduced response rates to approximately 1.0 response/s for the group of monkeys (Fig.
3). Like -selective opioid agonists, the mixed-action /µ-opioids bremazocine (0.0003-0.003 mg/kg) and
EKC (0.003-0.03 mg/kg) substituted fully for enadoline at doses that
did not greatly affect response rates. In addition, two -selective
opioids that have been reported to penetrate the brain poorly, EMD
61753 and ICI 204448 (Shaw et al., 1989; Barber et al., 1994), produced
dose-dependent increases in enadoline-associated responding; the
highest cumulative doses, 1.0 and 3.0 mg/kg, respectively, fully
substituted for the training drug in all monkeys. As with the other
-agonists, the doses of EMD 61753 and ICI 204448 that produced
enadoline-lever responding did not markedly disrupt fixed-ratio performance (Fig. 3).
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Fig. 3.
Effect of -opioid agonists in monkeys trained to
discriminate enadoline (0.0017 mg/kg i.m.) from saline. Dotted lines
(bottom graph) bracket control rates of responding following injection
of the training dose of enadoline (top) and saline (bottom). Other
details as in Fig. 1.
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For all drugs that substituted fully for the training dose of enadoline
in these experiments, comparison of the dose estimated to produce 50%
responding on the enadoline-associated lever
(ED50) revealed the following potency
relationship: enadoline = GR 89686A > bremazocine > EKC > ()-spiradoline > PD 117302 > U 50488H > EMD 61753 > ICI 204448 (Table 1).
With few exceptions (EMD 61753 and ICI 204448, which penetrate the
brain poorly, and EKC), the rank order of potency with which
-agonists reproduced the discriminative stimulus effects of
enadoline appears to correlate well with their potency for eliciting
other -mediated effects, e.g., reduction of electrically evoked
contractions in rabbit vas deferens [r = 0.87, F(1,5) = 15.6, p = 0.01; Table 1].
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TABLE 1
Doses of -opioid agonists estimated to produce 50% enadoline lever
responding, their relative potencies in the present experiments, and
their relative potencies in studies of electrically evoked contractions
in rabbit vas deferens
ED50 values for enadoline-discrimination are calculated by
linear interpolation and, for each drug, are means ± S.E.M. of
values obtained in three to four monkeys.
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Unlike selective -opioid agonists, the µ-opioid agonist morphine
(0.3-5.6 mg/kg) and the -opioid agonist BW 373U86 (0.1-0.3 mg/kg)
did not increase responding on the enadoline-associated lever and, at
the highest doses, markedly decreased response rates. As well,
enadoline-appropriate responding was not observed following cumulative
doses of the (+)-enantiomer of enadoline PD 129829 (0.1-1.0 mg/kg) or
(+)-spiradoline (0.3-3.0 mg/kg). These enantiomers were tested to
doses up to 1000-fold greater than doses of enadoline or
()-spiradoline that substituted for the training dose of enadoline. At the highest doses, rates of responding were not disrupted by injection of either drug (data not shown).
Effects of Mixed-Action /µ-Opioids.
In contrast to EKC
and bremazocine, nalbuphine (0.1-30 mg/kg) and nalorphine (0.03-30
mg/kg), which also act at µ- and -receptors (Leander, 1983;
Schmidt et al., 1985) did not engender dose-related responding on the
enadoline-associated lever (Fig. 4). In
one monkey (s322), administration of nalbuphine or nalorphine initially produced full enadoline-associated responding following cumulative doses of 3.0 or 0.3 mg/kg, respectively. Upon redetermination of the
effects of the same doses, however, responding on the
enadoline-associated lever was no longer observed. Subsequently, 17.8 mg/kg nalbuphine was found to substitute for enadoline but again,
redetermination of the dose-effect function showed that responding was
confined almost exclusively to the saline-associated lever following
cumulative doses as high as 30 mg/kg nalbuphine (data not shown).
Although nalbuphine and nalorphine did not effectively substitute for
enadoline, the highest doses of both drugs, like other -selective
and mixed-action /µ-agonists, elevated rates of responding,
indicating that behaviorally relevant doses were studied.
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Fig. 4.
Effect of the mixed-action opioids nalorphine and
nalbuphine in monkeys trained to discriminate enadoline from saline.
Other details as in Fig. 3.
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Antagonism Studies.
Pretreatment with the nonselective opioid
antagonist quadazocine (0.03-3.0 mg/kg), but not the µ-selective
antagonist -FNA (data not shown), attenuated the discriminative
stimulus and the rate-decreasing effects of enadoline in all monkeys.
Attenuation of the discriminative stimulus effects of enadoline by
quadazocine was characterized by rightward shifts of the dose-effect
function, indicative of surmountable antagonism (Fig.
5). The degree to which the
enadoline-discrimination function was displaced rightward varied as a
function of antagonist dose: lower doses of quadazocine (0.03 and 0.3 mg/kg) produced a 2- and 4-fold increase, respectively, in the
estimated ED50 for the group of monkeys, whereas
the highest dose of quadazocine (3.0 mg/kg) produced over a 30-fold
increase in the estimated ED50 for the
discriminative stimulus effects of enadoline. A Schild plot of the
antagonist effects of quadazocine revealed an apparent
pA2 of 6.92 ± 0.49 for the group of monkeys (slope = 0.87 ± 0.34). In addition to antagonizing the
discriminative stimulus effects of enadoline, quadazocine also
antagonized the rate-decreasing effects of the highest doses of
enadoline (0.01 and 0.03 mg/kg). However, the antagonism of these
effects was not fully characterized to avoid untoward effects that
might accompany administration of yet higher doses of enadoline (>0.1
mg/kg).
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Fig. 5.
Antagonism of behavioral effects of enadoline by
increasing doses of quadazocine (0.03-3.0 mg/kg i.m.). Other details
as in Fig. 3.
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As with quadazocine, pretreatment doses of nalbuphine (0.3-30 mg/kg)
produced consistent dose-dependent rightward shifts in the
discriminative stimulus effects of enadoline in all monkeys (Fig.
6). Averaged for the group of monkeys,
the lowest dose of nalbuphine (0.3 mg/kg) produced an approximately
3-fold increase in the estimated ED50 for the
discriminative stimulus effects of enadoline. Higher doses of
nalbuphine, 3.0 and 30 mg/kg, produced correspondingly greater
rightward shifts in the dose-effect function for enadoline, with
approximately 30- and 90-fold increases, respectively, in estimated
ED50 values. Schild analysis of the antagonist
effects of nalbuphine revealed an apparent pA2
value of 5.86 ± 0.46 for the group of monkeys (mean slope = 0.77 ± 0.25). In addition to antagonizing the discriminative
stimulus effects of enadoline, nalbuphine also produced dose-dependent
rightward shifts in the rate decreasing effects of enadoline.
Comparisons of ED50 values alone and following
pretreatment indicated that 0.3 mg/kg nalbuphine had little, if any,
antagonist action, whereas 3.0 mg/kg nalbuphine produced an
approximately 4-fold increase in average ED50
values. Pretreatment with 30 mg/kg nalbuphine further antagonized the effects of high doses of enadoline on rates of responding, and, following the highest cumulative dose of enadoline (0.1 mg/kg) in the
presence of this dose of nalbuphine, averaged response rates remained
above 50% of control values.
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Fig. 6.
Antagonism of behavioral effects of enadoline by
increasing doses of nalbuphine (3.0-30 mg/kg i.m.) in monkeys trained
to discriminate enadoline from saline. Other details as in Fig. 3.
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Nor-BNI, a selective and reportedly long-acting -selective receptor
blocker in behavioral studies (Portoghese et al., 1987; Butelman et
al., 1993; Jewett and Woods, 1995) also antagonized the effects of
enadoline in the present experiments. Dose-dependent rightward shifts
in the behavioral effects of enadoline were evident in all monkeys
following treatment with 3.0 mg/kg and, subsequently, 10.0 mg/kg, of
nor-BNI. The effects of nor-BNI were relatively slow to onset and
long-lived. Whereas neither substitution for enadoline nor antagonism
were observed within 60 min following i.m. administration of 3.0 mg/kg
nor-BNI, antagonism was apparent 24 h later and persisted for more
than 20 days. Six days following treatment, the dose-effect function
for enadoline was shifted rightward by an average of more than 0.5 log
unit (0.25-0.75 log units in individual monkeys; Fig.
7). The effects of 3.0 mg/kg nor-BNI
diminished thereafter but, on average, had not completely dissipated by
day 14 (Fig. 8). Treatment with 10.0 mg/kg nor-BNI produced a yet greater antagonism of the discriminative
stimulus effects of enadoline. Six days following the second treatment, the dose-effect function for enadoline was shifted rightward by at
least 1.0 log unit in three of four monkeys (Fig. 8). In the fourth
monkey, discriminative control of performance by enadoline no longer
was fully evident by the 9th day following treatment with 10.0 mg/kg
nor-BNI and, in subsequent sessions, cumulative doses of enadoline up
to 0.1 mg/kg failed to engender appreciable responding on the
enadoline-associated lever. However, even in this subject, evidence of
antagonism persisted through day 48 following treatment with 10.0 mg/kg
nor-BNI, as doses of enadoline up to 0.01 mg/kg did not markedly
decrease responding (data not shown). In the three monkeys for which
full dose-effect data still were obtained, the averaged
ED50 value for enadoline discrimination after 10 weeks continued to be 0.75 log units higher than before treatment with
nor-BNI.
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Fig. 7.
Antagonism of behavioral effects of enadoline by
nor-BNI (3.0 and 10.0 mg/kg i.m.) in monkeys trained to discriminate
enadoline from saline. Other details as in Fig. 3.
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Fig. 8.
Duration of action of 3.0 and 10 mg/kg nor-BNI on
enadoline discriminative stimulus effects in monkeys trained to
discriminate enadoline from saline. Abscissa, time (days) after
administration; ordinate, change in the log of the ED50.
Positive change indicates that nor-BNI is antagonizing the effects of
enadoline. Data expressed as mean ± S.E.M. for two to four
monkeys at each time point.
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In additional experiments, the enadoline-like discriminative stimulus
effects of U 50,488, bremazocine, and EKC were also antagonized by
nor-BNI. Based on data obtained in three monkeys studied 18 to 25 days
after pretreatment with 10.0 mg/kg nor-BNI, the dose-effect function
for each drug was shifted rightward and average
ED50 values were increased by approximately 10- to 20-fold (Table 2).
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TABLE 2
Doses of -agonists required to produce 50% enadoline
lever-responding alone and after nor-BNI (10 mg/kg) administration
Values are data for three monkeys (mean ± 95% CI). Antagonism
data are taken from day 6 (enadoline), day 18 (U 50,488), day 23 (bremazocine), and day 25 (EKC) following nor-BNI administration.
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Discussion |
Effects of Enadoline Alone.
The present results, indicating
that the -selective opioid agonist enadoline can serve as a reliable
discriminative stimulus in squirrel monkeys, are consistent with the
findings of Brandt and France (1996) in enadoline-trained pigeons and
extend them to a primate species. The discriminative stimulus effects
of enadoline occurred at doses that moderately increased fixed-ratio
response rates maintained under a schedule of stimulus-shock
termination above values associated with vehicle injection. These
observations are in keeping with earlier findings that -opioids may
increase response rates under schedules of stimulus-shock termination
at doses that typically decrease responding maintained by food
presentation (Bergman and Warren, 1989).
-Agonist Substitution for Enadoline.
The discriminative
stimulus effects of enadoline were reproduced by other -selective
opioid agonists and mixed-action /µ-opioid agonists but not by
µ- and -selective agonists. These results generally agree with
findings in monkeys trained to discriminate the mixed-action
/µ-agonist EKC (Hein et al., 1981; Young and Stephens, 1984;
Dykstra et al., 1987a; France et al., 1994) and suggest commonality in
their mechanism of action. Furthermore, the discriminative stimulus
effects of enadoline were not reproduced by the (+)-isomers of
enadoline or spiradoline in the present studies, indicating the
stereoselectivity of the interaction of enadoline with the
-receptors mediating its effects. The relative potency with which
the -agonists used in the present study reproduced enadoline's
discriminative stimulus effects are in close agreement with the
relative potencies of these compounds in other behavioral studies
(Dykstra et al., 1987a,b; Broadbear et al., 1994; France et al., 1994;
Smith and Picker, 1995; Brandt and France, 1996), and correlate well
with their ability to reduce electrically induced contractions in
rabbit vas deferens in vitro (Hayes et al., 1990; Hunter et al., 1990;
Barber et al., 1994). This finding suggests that the intrinsic efficacy
of the compounds at -receptors influences their ability to produce
the behavioral effects observed in the present studies.
Interestingly, the discriminative stimulus effects of enadoline also
were reproduced by EMD 61743 and ICI 204448, two -selective agonists
that are reported to penetrate the brain to a limited extent (Shaw et
al., 1989; Barber et al., 1994). On the basis of their reported
-opioid affinities, the doses administered in the present studies
ensured that concentrations sufficient to reproduce enadoline's
discriminative stimulus penetrated the central nervous system
and likely were considerably higher than those required to produce
peripheral -receptor-mediated actions.
Effects of Nalorphine and Nalbuphine.
Both mixed-action
opioids nalorphine and nalbuphine have affinity for - and
µ-receptors and, depending on the species and conditions of the
experiment, may produce agonist effects through - or µ-actions
(Leander, 1983; Schmidt et al., 1985; France et al., 1994; Gerak
et al., 1994). In monkeys, the discriminative stimulus effects
of nalorphine may involve its -opioid actions, whereas the effects
of nalbuphine may more prominently involve µ-related mechanisms. For
instance, nalorphine has been shown to reproduce the discriminative
stimulus effects of EKC in rhesus monkeys, whereas nalbuphine may more
readily reproduce the µ-mediated discriminative stimulus effects of
etorphine (Hein et al., 1981; Young and Stephens, 1984; France et al.,
1994). Consistent with µ-receptor mediation of its effects,
µ-opioid agonists, but not enadoline and other -opioid agonists,
have been found to fully reproduce the effects of nalbuphine in
nalbuphine-trained monkeys (Gerak and France, 1996). In the
present study, neither nalbuphine nor nalorphine consistently
reproduced the discriminative stimulus effects of enadoline. These
findings in monkeys are consistent with those of Brandt and France
(1996), showing that neither nalorphine nor nalbuphine reproduced the
discriminative stimulus effects of enadoline in pigeons. Previous
studies have provided evidence that nalorphine may serve as a
low-efficacy -agonist (Leander, 1983; France et al., 1994) and its
ability to substitute for EKC but not enadoline may reflect a lower
efficacy requirement for substitution in EKC-trained subjects than in
enadoline-trained subjects. However, EKC, like nalorphine, is a
mixed-action opioid and, possibly, a µ-mediated aspect of its
discriminative stimulus effects may increase the likelihood of
substitution by nalorphine. Unlike nalorphine, nalbuphine typically
does not mimic the discriminative stimulus effects of EKC. In
conjunction with the lack of substitution for enadoline by nalbuphine,
its ability to surmountably antagonize the effects of enadoline (see
below) encourages the view that nalbuphine has relatively low efficacy
as a -agonist.
Despite the lack of substitution for enadoline, the highest doses of
nalbuphine and nalorphine (30 mg/kg) produced elevations in response
rate comparable with the rate-increasing effects observed with
intermediate doses of enadoline. As noted above, -opioid agonists
previously have been reported to produce increases in response rate
under schedules of stimulus-shock termination in squirrel monkeys and
the rate-increasing effects of -agonists such as nalbuphine and
nalorphine may result from similar mechanisms of action. However,
previous studies have shown that the rate-increasing effects of
-agonists are not antagonized by high doses of the nonselective
opioid receptor blocker naltrexone, suggesting that these effects
either are the product of nonopioid actions or are mediated through
naltrexone-insensitive -agonist mechanisms (Bergman and Warren,
1989).
Antagonism of Enadoline Discrimination.
In the present study,
the nonselective opioid receptor blocker quadazocine, the mixed-action
agonist nalbuphine, and the -selective antagonist nor-BNI generally
produced dose-dependent rightward shifts in the dose-effect function
for enadoline discrimination and, as well, attenuated the
rate-decreasing effects of high doses of enadoline. In contrast, a high
dose of -FNA, which previously has been shown to antagonize the
behavioral effects of µ-agonists (Picker and Dykstra, 1987b; Dykstra
et al., 1989), was without antagonistic effect. These data
indicate that the discriminative stimulus effects of enadoline, which
recently was reported to have only approximately 50-fold selectivity in
affinity for -compared with µ-receptors in monkey brain (France et
al., 1994), may be wholly ascribed to its -opioid actions.
The effects of nor-BNI were remarkably persistent (>80 days),
consistent with its previous characterization as a long-lasting -opioid antagonist (Portoghese et al., 1987; Butelman et al., 1993; Jewett and Woods, 1995). Although control experiments were not conducted to ensure that the discriminative stimulus effects of
enadoline were intact over the time training was suspended, several
observations suggest this was indeed the case. First, loss of stimulus
control most often leads to a loss of predictable dose response in
discrimination, such as occurred in one monkey. Second, dose-related
and predictable data during test sessions with enadoline were generated
consistently in the three monkeys that were studied for >80 sessions
despite the absence of explicit training sessions. In a sense, test
sessions in which surmountable antagonism to enadoline was observed
served as training sessions for these subjects. Third, antagonism of
the rate-disruptive effects of enadoline was evident throughout the
testing period, supporting the idea that the persistent rightward shift
in the dose-effect function for enadoline discrimination revealed a
true antagonism.
Previously, nor-BNI has been shown to antagonize antinociceptive
effects of U 50,488 but not those of enadoline, bremazocine, or EKC in
tail-withdrawal studies in rhesus monkeys (Butelman et al., 1993,
1998). These findings provided support in monkeys for the view that the
existence of different subtypes of -opioid receptors in radioligand
binding experiments might have functional consequences (Zukin et al.,
1988; Clark et al., 1989; Butelman et al., 1998). This
view has been strengthened by findings in monkeys that the -opioid
peptide dynorphin may differentially antagonize the antinociceptive
effects of agonists in a manner comparable with that observed in
experiments with nor-BNI and, additionally from findings that
pA2 values for antagonism by naltrexone differ
for these effects of EKC and U 50,488, on the one hand, and bremazocine
and enadoline, on the other (Butelman et al., 1995; Ko et al.,
1998). In the present experiments, however, no clear difference
was observed in the ability of nor-BNI to surmountably antagonize the
discriminative stimulus effects of these different -opioids in
enadoline-trained monkeys. Although limited in scope, these data do not
provide further evidence for the functional importance of -receptor
subtypes but, rather, are consistent with the view that the
discriminative stimulus effects of -opioids in enadoline-trained
subjects are not preferentially mediated through a single subtype of
-opioid receptor.
Despite some variability in the average slope of the Schild plot
regression, the pA2 values of 6.9 ± 0.5 for
antagonism of enadoline discrimination by quadazocine and 5.9 ± 0.4 for antagonism by nalbuphine are comparable with
pA2 values obtained with quadazocine and
nalbuphine in other studies of behavioral effects of enadoline in
monkeys and pigeons, respectively (Pitts and Dykstra, 1994; Brandt and
France, 1996) and in other studies of the antagonism of behavioral
effects of other -agonists in squirrel and rhesus monkeys (Bertalmio
and Woods, 1987; Dykstra and Massie, 1988; Dykstra, 1990). Although
nor-BNI appeared to surmountably antagonize the behavioral effects of
enadoline, its long-lasting antagonist effects suggest that its actions
are not the result of simple competition at a single receptor site,
precluding the evaluation of its effects by
pKB analysis. The mechanism by which
nor-BNI exerts its actions is not yet understood but likely differs
from mechanisms that underlie the effects of other long-acting
antagonists such as -FNA or clocinnamox, which presumably act by
reducing the µ-opioid receptor population. However, their effects
dissipate as µ-opioid receptors are restored over the course of days,
whereas the effects of nor-BNI in this and other studies appear to
persist over the course of weeks (Jewett and Woods, 1995).
We thank Dr. W. H. Morse for helpful comments on an earlier
version of this manuscript, and A. Pond for expert technical
assistance. We also thank the pharmaceutical firms listed under
Materials and Methods that generously donated
compounds for use in these studies.
This work was conducted at the New England Regional Primate
Research Center and supported by U.S. Public Health Service Grants MH07658, DA 03774, and RR00168. Animals used in this study were maintained in accordance with the guidelines of the Committee on
Animals of the Harvard Medical School and of the Guide for Care and Use
of Laboratory Animals of the Institute of Laboratory Animal Resources,
National Research Council, Department of Health, Education, and
Welfare, Publication (National Institutes of Health) 85-23 (revised 1985).
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Abstract
Introduction
