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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 296:132–140, 2001
Vol. 296, No. 1
2803/872056
Printed in U.S.A.
Influence of Gender and Sex Hormones on Nicotine Acute
Pharmacological Effects in Mice
M. IMAD DAMAJ
Department of Pharmacology and Toxicology, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia
Received April 12, 2000; accepted September 10, 2000
This paper is available online at http://jpet.aspetjournals.org
In spite of heightened education and prevention strategies,
cigarette smoking remains a major health risk. Nicotine is
believed to be the primary reason that people consume tobacco products. Indeed, substantial evidence now shows that
nicotine is the addictive substance found in tobacco. In 1974,
31% of U.S. women and 43% of U.S. men were tobacco smokers. Current estimates indicate that the difference between
men and women has narrowed considerably to a comparable
rate (21–25%) (CDC, 1998). Furthermore, poorer outcome of
women in smoking cessation trials, especially those involving
nicotine replacement, has also been reported. In other words,
nicotine replacement is less effective for smoking cessation
than in men. There are several potential causes for this shift
toward more women smokers. There are several possible
explanations for this trend. Factors such as women’s greater
concern about weight gain, greater difficulty with negative
mood (and higher prevalence of affective disorders), and
greater need for social support to quit smoking could be of
influence. Reduced availability of social support for cessation
in women and greater impact of advertising on promoting
smoking in women versus men may also have an influence.
Broad cultural influences also have been advanced as an
explanation for sex differences in smoking prevalence. Although cultural factors are clearly important in explaining
the historical differences between the smoking patterns of
This work was supported by National Institute on Drug Abuse Grant
DA-05274.
indicate that sex hormones can modulate the effects of nicotine
and nicotinic receptors in a differential manner. Progesterone
and 17␤-estradiol were found to block nicotine’s antinociception in mice. Testosterone failed to do so. In addition, progesterone and 17␤-estradiol blocked nicotine activation of ␣4␤2
neuronal acetylcholine nicotinic receptors expressed in oocytes. Our findings contribute to our search for receptor mechanisms in drug dependence and in the discovery of better
pharmacological agents for nicotine dependence.
American men and women earlier this century and in nonWestern countries, they do not seem to be a likely explanation. Indeed, cultural restrictions in smoking behavior specific to women in Western societies are no longer in existence.
One obvious possibility is the involvement of biological differences between the sexes might contribute to this gender
difference.
Animal studies have found that male and female rodents
have different sensitivities to the effects of nicotine. For
example, female rats are less sensitive than male rats to the
discriminative stimulus effects of nicotine (Schechter and
Rosecrans, 1971), consistent with findings in humans (Perkins et al., 1999). Female mice were less sensitive to nicotineinduced suppression of Y-maze activity (Hatchell and Collins, 1980) and increase in active avoidance learning (Yilmaz
et al., 1997). In addition, sex differences in receptor upregulation after chronic exposure to nicotine was also reported with only male rats showing up-regulation of brain
[3H]cytisine binding sites (Koylu et al., 1997). Conversely,
female rats were more sensitive to nicotine’s effects on prepulse inhibition (Faraday et al., 1999) and food intake (Grunberg et al., 1984). Female rats were also more sensitive to
nicotine-induced antinociception compared with males in
acute and chronic pain models (Craft and Milholland, 1998;
Chiari et al., 1999; Lavand’homme and Eisenach, 1999). In
addition, sensitization to nicotine-induced increase in locomotor activity was found to be greater in female rats after
ABBREVIATIONS: nAChR, acetylcholine nicotinic receptor; %MPE, percentage maximum possible effect; i.t., intrathecal; CL, confidence limit;
AD50, antagonist dose 50%; ACh, acetylcholine.
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ABSTRACT
The present study conducted a comprehensive examination of
the putative sex differences in the potency of nicotine between
male and female ICR mice using several pharmacological and
behavioral tests. Among the responses to nicotine where significant sex differences were observed are the antinociceptive
and the anxiolytic effects of nicotine. Female mice were found
less sensitive to the acute effects of nicotine in these tests after
s.c. administration. Similar gender differences were found after
i.t. injection. Influence of gonadal hormones could underlie sex
differences observed in our studies. Indeed, our data clearly
Influence of Gender on Nicotine’s Effects
Materials and Methods
Animals
Male and female ICR mice (20 –25 g) matched for age were obtained from Harlan Laboratories (Indianapolis, IN). They were
housed in groups of six and had free access to food and water.
Animals were housed in an American Association of Laboratory
Animal Care-approved facility, and the study was approved by the
Institutional Animal Care and Use Committee of Virginia Commomwealth University.
Drugs
(⫺)-Nicotine was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI) and converted to the ditartrate salt as described by
Aceto et al. (1979). (⫺)-Epibatidine (hemi oxalate salt) was supplied
by Dr. S. Fletcher (Merck Sharp and Dohme & Co., Essex, UK).
Mecamylamine hydrochloride was supplied as a gift from Merck,
Sharp and Dohme & Co. (West Point, PA). Testosterone, 17␤-estradiol, and progesterone water-soluble (cyclodextrin-encapsulated) and
oil-soluble (testosterone decanoate) forms were purchased from
Sigma Chemical Co. (St. Louis, MO); neostigmine was purchased
from Research Biochemicals International (Natick, MA). Oil-soluble
hormones were diluted in sesame oil (Sigma Chemical Co.). All other
drugs were dissolved in physiological saline (0.9% sodium chloride)
and given in a total volume of 1 ml/100 g of body weight for s.c.
injections. All doses are expressed as the free base of the drug.
Intrathecal Injections
Intrathecal injections were performed free-hand between the L5
and L6 lumbar space in unanesthetized male mice according to the
method of Hylden and Wilcox (1980). The injection was performed
using a 30-gauge needle attached to a glass microsyringe. The injection volume in all cases was 5 ␮l. The accurate placement of the
needle was evidenced by a quick “flick” of the mouse’s tail.
Antinociceptive Tests
Tail-Flick Test. Antinociception was assessed by the tail-flick
method of D’Amour and Smith (1941) as modified by Dewey et al.
(1970). A control response (2– 4 s) was determined for each mouse
before treatment, and a test latency was determined after drug
administration. To minimize tissue damage, a maximum latency of
10 s was imposed. Antinociceptive response was calculated as percentage maximum possible effect (%MPE), where %MPE ⫽ [(test ⫺
control)/(10 ⫺ control)] ⫻ 100. Groups of 8 to 12 animals were used
for each dose and for each treatment. The mice were tested 5 min
after either s.c. or i.t. injections of nicotine.
Hot-Plate Test. The method is a modification of that described by
Eddy and Leimbach (1953) and Atwell and Jacobson (1978). Mice
were placed into a 10-cm-wide glass cylinder on a hot-plate (Thermojust Apparatus, Richmond, VA) maintained at 55.0°C. Two control latencies at least 10 min apart were determined for each mouse.
The normal latency (reaction time) was 6 to 10 s. Antinociceptive
response was calculated as %MPE, where %MPE ⫽ [(test ⫺ control)/
(40 ⫺ control) ⫻ 100]. The reaction time was scored when the animal
jumped or licked its paws. Eight mice per dose were injected s.c. with
nicotine and tested 5 min after injection.
The effects of testosterone, 17␤-estradiol, and progesterone on
nicotine’s antinociceptive effects were examined. Female (17␤-estradiol and progesterone) and male (testosterone) mice were pretreated
with different doses of hormones given i.p. and then challenged with
s.c. nicotine at different times. After determining the time-course
effect of hormones, the potency of these hormones in blocking nicotine-induced antinociception was performed at the maximum time.
Behavioral Testing
Locomotor Activity. Mice were placed into individual Omnitech
photocell activity cages (28 ⫻ 16.5 cm) 5 min after s.c. administration
of either 0.9% saline or nicotine. Interruptions of the photocell beams
(two banks of eight cells each) were then recorded for the next 10
min. Data are expressed as number of photocell interruptions.
Body Temperature. Rectal temperature was measured by a
thermistor probe (inserted 24 mm) and digital thermometer (Yellow
Springs Instrument Co., Yellow Springs, OH). Readings were taken
just before and at 30 min after the s.c. injection of either saline or
nicotine. The difference in rectal temperature before and after treatment was calculated for each mouse. The ambient temperature of the
laboratory was 21–24°C from day to day.
Seizure Activity. After a s.c. injection of nicotine at a dose of 9
mg/kg, each animal was placed in a 30- ⫻ 30-cm Plexiglas cage and
observed for 5 min. Whether a clonic seizure occurred within a 5-min
time period was noted for each animal after s.c. administration of
different drugs. This amount of time was chosen because seizures
occur very quickly after nicotine administration. Results are expressed as percentage seizure.
Elevated Plus-Maze. An elevated plus-maze, prepared with gray
Plexiglas, consisted of two open arms (23 ⫻ 6.0 cm) and two enclosed
arms (23 ⫻ 6 ⫻ 15 cm) that extended from a central platform. It was
mounted on a base raised 60 cm above the floor. Fluorescent lights
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chronic i.v. administration of nicotine (Booze et al., 1999), but
not after chronic s.c. injection (Kanyt et al., 1999). Such
complexity and variability in the response of male and female
rodents to nicotine may be due to differences in the dose and
elimination profile of nicotine, age and specie of the test
animal, route or duration of administration, time of evaluation, or the behavioral task used.
Another biological factor that could underlie sex differences is the influence of gonadal hormones. Levels of sex
hormones change through the menstrual (human) or estrous
(rodent) cycle. Human studies examining the effects of the
phase of the menstrual cycle on cigarette smoking and on the
psychoactive actions of nicotine are conflicting. However,
most studies report that menstrual cycle phase may influence self-reported withdrawal symptoms in women (Benowitz and Hatsukami, 1998). Animal studies suggest that ovarian hormones could influence nicotine acute effects and
reinforcement. Dluzen and Anderson (1997) reported that
estrogen treatment to ovariectomized female rats increased
the in vitro nicotine-induced release of dopamine from striatal slices, whereas the opposite effect was seen in castrated
males.
Therefore, the aim of the present study was to conduct a
comprehensive examination of the putative sex differences in
the potency of nicotine between male and female mice using
several pharmacological and behavioral tests. For that, the
potency of nicotine in various pharmacological effects measures (antinociception, hypothermia, seizures, and motor activity, plus-maze activity) in animals was examined after
systemic and intrathecal administration. A wide range of
nicotinic effects is important to consider because it is believed
that various nicotinic receptor subtypes mediate different
pharmacological effects of nicotine. Because gonadal hormones may also mediate such sex differences, the effects of
testosterone, 17␤-estradiol, and progesterone on nicotine’s
effects were examined. The in vivo effect of hormones was
correlated with in vitro studies using the oocyte expression
system. For that, the effect of sex hormones on the functional
activity of the neuronal nAChRs ␣4␤2 (the major nicotinic
receptor subtype in the brain) expressed receptors was studied.
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Damaj
(350-lux intensity) located in the ceiling of the room provided the
only source of light to the apparatus. The animals were placed in the
center of the maze and the following variables were scored: 1) time
spent in the open arms, 2) time spent in the closed arms, and 3) total
number of crossings between arms. These variables are automatically recorded by a photocell beam system. The test lasted 5 min and
the apparatus was thoroughly cleaned after removal of each animal.
Readings were taken 15 min after the s.c. injection of either saline or
nicotine. Anxiolytic response was calculated as the percentage of
time spent in the open arm, where % time ⫽ [(test ⫺ 300 s) ⫻ 100].
Oocyte Expression Studies
Results
Antinociception Studies
Tail-Flick Test. Nicotine time course of action after an
equipotent dose in male (2 mg/kg) and female (3.5 mg/kg)
mice was evaluated in the tail-flick after s.c. injection. As
seen in Fig. 1A, no significant difference was seen between
male and female mice. Nicotine’s effect disappeared completely within 30 min after s.c. administration in both sexes.
Dose-response relationships were then established for nicotine in male and female mice by measuring antinociception at
the time of maximal effect (5 min) (Fig. 1B). Nicotine was 3
times less potent in females compared with males. Nicotine
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Oocyte Preparation. Oocytes preparation was performed according to the method of Mirshahi and Woodward (1995) with minor
modifications. Briefly, oocytes were isolated from female adult oocyte-positive Xenopus laevis frogs. Frogs were anesthetized in a 0.2%
3-aminobenzoic acid ethyl ester solution (Sigma Chemical Co.) for 30
min and a fraction of the ovarian lobes was removed. The eggs were
rinsed in Ca2⫹-free ND96 solution, treated with collagenase type IA
(Sigma Chemical Co.) for 1 h to remove the follicle layer, and then
rinsed again. Healthy stage V-VI oocytes were selected and maintained for up to 14 days after surgery in 0.5⫻ L-15 media (Sigma
Chemical Co.).
mRNA Preparation and Microinjection. ␣4 and ␤2 rat subunit
cDNA contained within a pcDNAIneo vector were kindly supplied by
Dr. James Patrick (Baylor College of Medicine, Houston, TX). The
template was linearized downstream of coding sequence and mRNA
was synthesized using an in vitro transcription kit from Ambion
(Austin, TX). The quantity and quality of message were determined
via optical density (spectrophotometer; Beckman Instruments Inc.,
Chaumburg, IL) and denaturing formaldehyde gel analysis. Oocytes
were injected with either 51 ng (41 nl) of ␣4 and ␤2 mRNA mixed in
a 1:1 ratio using a Variable Nanoject (Drummond Scientific Co.,
Broomall, PA). Oocytes were incubated in 0.5⫻ L-15 media IA supplemented with penicillin, streptomycin, and gentimycin for 4 to 6
days at 19°C before recording.
Electrophysiological Recordings. Oocytes were placed within
a Plexiglas chamber (total volume 0.2 ml) and continually perfused
(10 ml/min) with buffer consisting of 115 mM NaCl, 1.8 mM CaCl2,
2.5 mM KCl, 1.0 ␮M atropine, and 10.0 mM HEPES at pH 7.2.
Oocytes were impaled with two microelectrodes containing 3 M KCl
(0.3–3 M⍀) and voltage-clamped at ⫺70 mV using an Axon Geneclamp amplifier (Axon Instruments Inc., Foster City, CA). Oocytes
were stimulated for 10 s with various concentrations of acetylcholine
using a six-port injection valve. Except where noted, applications
were separated by 5-min periods of washout. Currents were filtered
at 10 Hz and collected by a Macintosh Centris 650 with a 16-bit
analog digital interface board, and data were analyzed using Pulse
Control voltage-clamp software running under the Igor Pro graphic
platform (Wavemetrics, Lake Oswego, OR). Water-soluble forms of
hormones were applied at different concentrations and concentration-response curves were normalized to the current induced by 1
␮M ACh. The normalizing concentration of ACh was applied before
and after drug application to each oocyte to check for desensitization.
Data were rejected if responses to the normalizing dose fell below
75% of the original response. IC50 values were determined using data
from four to six oocytes.
and Murray (1987). If confidence limit values did not overlap, then
the shift in the dose-response curve was considered significant.
Statistical Analysis
Data were analyzed statistically by an analysis of variance followed by the Fisher protected least-significant difference multiple
comparison test. For time course studies, Dunnett’s test was used.
The null hypothesis was rejected at the 0.05 level. IC50 (antagonist
concentration 50%) and ED50 (effective dose 50%) values with 95%
confidence limits (CL) were calculated by unweighted least-squares
linear regression as described by Tallarida and Murray (1987). Tests
for parallelism were calculated according to the method of Tallarida
Fig. 1. Antinociceptive effects of nicotine in male (䡺) and female (F)
female mice using the tail-flick test after s.c. administration. A, time
course of antinociception induced by nicotine (2 and 3.5 mg/kg for male
and female, respectively) after s.c. administration in mice. B, nicotine
dose-response curves in both sexes 5 min after injection. C, comparison of
mecamylamine blockade of antinociception induced by nicotine in male
and female mice. Mecamylamine at different doses was injected s.c. 10
min before an equipotent dose of nicotine (ED84) was administered s.c.
and mice were tested 5 min after the second injection. Each point represents the mean ⫾ S.E. of 12 to 16 mice.
Influence of Gender on Nicotine’s Effects
Fig. 3. Antinociceptive effects of nicotine in male (䡺) and female (F) mice
using the hot-plate test after s.c. administration. Nicotine dose-response
curves in both sexes were determined 5 min after injection. Each point
represents the mean ⫾ S.E. of 12 to 16 mice.
(Fig. 4A). Nicotine produced a dose-responsive increase in the
tail-flick latency in both male and female mice with ED50
(⫾CL) values of 11.4 (9.5–13.7) and 23 (15.1–34.2) ␮g/animal,
respectively. To determine whether the sex differences extended to cholinesterase inhibitors, neostigmine was evaluated by i.t. injection. The ED50 values for neostigmine-induced antinociception in male and female mice were 0.004
and 0.003 ␮g/animal, respectively. Therefore, the effects of
elevating endogenous ACh were not influenced by gender.
Other Behavioral Effects of Nicotine
To further characterize sex differences, additional experiments were conducted to determine nicotine potency in other
behavioral responses.
Effect on Plus-Maze Activity. Nicotine increased the
time spent in the open arm of the maze when administered
s.c. to male and female mice (Fig. 5A). Nicotine potency was
higher in male mice compared with female mice with ED50
(⫾CL) values of 0.40 (0.2– 0.6) and 0.95 (0.8 –1.2) mg/kg,
respectively. No gender differences were seen for total crossings between the different arms in the plus-maze test (data
not shown).
Effect on Body Temperature, Seizure Activity, and
Locomotor Activity. Unlike the antinociceptive and anxiolytic effects, nicotine potency in male and female mice was
similar in inducing hypothermia, hypomotility, and seizures
production after s.c. administration (Fig. 5, B–D; Table 1). No
sex differences were observed in baseline values in the spontaneous activity and body temperature (data not shown).
In Vivo Interaction of Sex Hormones with Nicotine with
Expressed Neuronal Nicotinic Receptors
Fig. 2. Antinociceptive effects of epibatidine in male (䡺) and female (F)
mice using the tail-flick test after s.c. administration. Epibatidine doseresponse curves were determined in both sexes 10 min after injection.
Each point represents the mean ⫾ S.E. of 12 to 16 mice.
The interaction between nicotine and different sex hormones (testosterone, 17␤-estradiol, and progesterone) was
studied in the tail-flick test after s.c. administration of nicotine. Time course studies were performed to determine the
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produced a dose-responsive increase in the tail-flick latency
in both male and female mice with ED50 (⫾CL) values of 1.0
(0.6 –1.3) and 2.9 (1.4 –5.8) mg/kg, respectively. The basal
latencies between male and female mice were not statistically different (2.5 ⫾ 0.2 and 2.7 ⫾ 0.2 s for male and female,
respectively). Pretreatment with the centrally active noncompetitive nicotinic receptor antagonist mecamylamine (1
mg/kg s.c.) 10 min before nicotine blocked its antinociceptive
activity in both male and female mice (Fig. 1C). Although
mecamylamine seems to be more potent in blocking nicotine’s
effects in female mice [AD50 (⫾CL) ⫽ 0.09 (0.01– 0.4) compared with male AD50 (⫾CL) ⫽ 0.2 (0.04 –1.1)], the difference
was not significant because confidence limits of the two
curves overlapped. A similar sex difference was also observed
with epibatidine, a very potent nicotinic agonist. Epibatidine
produced a dose-responsive increase in the tail-flick latency
(Fig. 2) in both male and female mice with ED50 (⫾CL) values
of 0.8 (0.5–1.1) and 1.7 (1.2–2.6) ␮g/kg, respectively.
Hot-Plate Test. Nicotine was then evaluated in both male
and female mice in another acute pain test, the hot-plate
assay. Dose-response relationships were established for nicotine in male and female mice by measuring antinociception
at the time of maximal effect (5 min) (Fig. 3). Similar to that
observed with the tail-flick test, female mice were less sensitive to the effect of nicotine compared with males. The
difference of potency between the two sexes was less (1.8
times) than the one determined for the tail-flick test. Nicotine produced a dose-responsive increase in the hot-plate
latency in both male and female mice with ED50 (⫾CL)
values of 0.5 (0.4 – 0.6) and 0.9 (0.8 –1.2) mg/kg, respectively.
The basal latencies between male and female mice were not
statistically different (12.9 ⫾ 0.8 and 11.5 ⫾ 3.2 s for male
and female, respectively).
Antinociceptive Effects after i.t. Injection in the
Tail-Flick Test. Nicotine potency was then evaluated in the
tail-flick after i.t. injection in male and female mice. Similar
to that observed with s.c. administration, female mice were
less sensitive to the effect of nicotine compared with males
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Damaj
time of maximal blockade and then dose-response curves for
each hormone in blocking nicotine-induced antinociception
were carried out to determine the blockade potency.
Effect of Progesterone. Progesterone was evaluated for
its ability to antagonize a 3.5-mg/kg dose of nicotine in female
mice using the tail-flick procedure after i.p. injection. As
shown in Fig. 6A, progesterone time dependently blocked
nicotine-induced antinociception. The duration of action of
progesterone in the tail-flick test was time-dependent with
maximum blockade occurring between 3 and 4 h after injection of a dose of 20 mg/kg. Nicotine’s effect started to recover
within 6 h after pretreatment with progesterone. Additionally, as showed in Fig. 6B, progesterone dose dependently
blocked nicotine-induced antinociception with an AD50
(⫾CL) of 4.8 (2.8 – 8.4) mg/kg when given s.c. 4 h before
nicotine.
Experiments were then carried out to determine whether
repeated exposure to progesterone could enhance its blockade potency against nicotine. Female mice were injected with
either vehicle or progesterone (10 mg/kg) twice a day for 4
days. Twelve hours after the last injection, the potency of
progesterone in blocking a challenge of nicotine was assessed. As shown in Fig. 7, progesterone potency in blocking
nicotine-induced antinociception was enhanced as shown by
a 3-fold shift to the left of its dose-response curve. The AD50
values (and 95% CL) for vehicle-treated and progesteronetreated animals were 2.4 (1.6 –7.5) and 0.8 (0.2–1.4) mg/kg,
respectively. Chronic administration of vehicle did increase
the potency of progesterone as a nicotinic blocker [4.8 (2.8 –
8.4) versus 2.4 (1.6 –7.5) mg/kg]. This difference was not
significant because confidence limits of the two curves overlapped.
Effect of 17␤-Estradiol and Testosterone. Similar to
progesterone, 17␤-estradiol time and dose dependently
blocked nicotine-induced antinociception in female mice. The
duration of effect of 17␤-estradiol (20 ␮g/kg i.p.) was briefer
than that of progesterone, with a maximum effect at 3 h after
injection and a full recovery of nicotine’s effects 6 h later (Fig.
8A). Additionally, as showed in Fig. 8B, 17␤-estradiol dose
dependently blocked nicotine-induced antinociception with
an AD50 (⫾CL) of 5.5 (4.0 – 6.6) ␮g/kg when given s.c. 4 h
before nicotine. In contrast to progesterone and 17␤-estradiol, testosterone (up to 500 mg/kg i.p.) did not significantly
decrease nicotine-induced antinociception at the times (Fig.
9A) and doses tested (Fig. 9B).
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Fig. 4. Antinociceptive effects of direct and indirect cholinergic agonists
in male (䡺) and female (F) mice using the tail-flick test after i.t. administration. A, nicotine dose-response curves in both sexes 5 min after
injection. B, neostigmine dose-response curves in both sexes 5 min after
injection. Each point represents the mean ⫾ S.E. of 12 to 16 mice.
Fig. 5. Potency of nicotine in male (䡺) and female (F) female mice in
different behavioral tests after s.c. injection. A, nicotine-induced increase
in open arm time using the plus-maze test. Mice were tested 15 min after
nicotine injection. B, nicotine-induced hypothermia. Mice were tested 30
min after nicotine injection. C, nicotine-induced hypomotility. Mice were
tested 5 min after nicotine injection. D, nicotine-induced seizures. Mice
were tested 5 min after nicotine injection. Each point represents the
mean ⫾ S.E. of 8 to 12 mice.
Influence of Gender on Nicotine’s Effects
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TABLE 1
Summary of the potency of nicotine in male and female mice in different behavioral tests
ED50 values (⫾CL) were calculated from the dose-response curve of the respective sex and expressed as milligrams per kilogram. Each dose group included 12 to 16 animals.
Pharmacological Effect or Test
Male ED50
Potency Ratio (F/M)a
Female ED50
mg/kg
Tail-flick after s.c.
Tail-flick after i.t.
Hot-plate
Hypothermia
Hypomotility
Seizures
Plus-maze
a
b
1.0 (0.6–1.3)
11.4 (9.5–13.7)b
0.50 (0.4–0.6)
1.1 (0.5–2.2)
0.30 (0.05–0.8)
5.2 (4.8–5.7)
0.4 (0.2–0.6)
2.9 (1.4–5.8)
23 (15.1–34.2)b
0.9 (0.8–1.2)
0.85 (0.6–1.9)
0.35 (0.15–0.8)
5.8 (4.4–7.4)
0.95 (0.8–1.2)
2.9
2.0
1.8
0.80
1.2
1.1
2.4
Potency ratio ⫽ female ED50 value/male ED50 value.
ED50 values (⫾CL) after i.t. administration are expressed as micrograms per animal.
Fig. 6. Blockade of nicotine-induced antinociception in the tail-flick test
by progesterone after i.p. injection in female mice. A, time course of
progesterone effect on nicotine-induced antinociception (3 mg/kg s.c.)
after i.p. administration of 20 mg/kg in mice. B, dose-response blockade of
progesterone after i.p. administration in mice. Progesterone at different
doses was administered i.p. 4 h before nicotine (3.0 mg/kg s.c.) and mice
were tested 5 min later. Each point represents the mean ⫾ S.E. of 8 to 12
mice. *p ⬍ 0.05 compared with correspondent zero time point.
Interaction of Sex Hormones with Neuronal Nicotinic
Receptors Expressed in Oocytes
Because progesterone and 17␤-estradiol blocked nicotine’s
behavioral effects, their potency as a blocker at expressed
neuronal nicotinic receptors was investigated. Progesterone
and 17␤-estradiol at 50 ␮M elicited little current when applied for 10 s to oocytes expressing the ␣4␤2 subunit combinations (⫺5 ⫾ 2 nA). Although they did not activate these
expressed receptors, they antagonized the effects of nicotine
in a concentration-related manner. The concentration that
blocked 50% of the nicotinic current was determined to be 1.8
(1.1–2.9) and 25 (15– 43) ␮M for progesterone and 17␤-estradiol, respectively (Fig. 10, A and B). At the lower concentration of 17␤-estradiol (0.01 ␮M), there was a small (12% increase) enhancement in the nicotinic current when the two
drugs were coapplied (Fig. 10B). This enhancement disappeared at 0.1 ␮M 17␤-estradiol. The effect of progesterone
and 17␤-estradiol on ␣4␤2 receptors was reversible after
5-min wash period (Fig. 10, A and B).
Discussion
The primary findings of this study are that there are sex
differences in nicotine’s acute pharmacological effects, but
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Fig. 7. Dose-response curve of progesterone blocking nicotine-induced
antinociception in female mice chronically treated with progesterone (F)
or vehicle (f) using the tail-flick test. Mice were injected with either
vehicle (sesame oil i.p.) or progesterone (10 mg/kg i.p.) twice a day for 4
days. Twelve hours after the last injection, the potency of progesterone in
blocking a challenge of nicotine (3.0 mg/kg s.c.) was assessed. Each point
represents the mean ⫾ S.E. of 8 to 12 mice.
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Damaj
Fig. 8. Blockade of nicotine-induced antinociception in the tail-flick test
by 17␤-estradiol after i.p. injection in female mice. A, time course of
17␤-estradiol’s effect on nicotine-induced antinociception (3 mg/kg s.c.)
after i.p. administration of 20 ␮g/kg in mice. B, dose-response blockade of
17␤-estradiol after i.p. administration in mice. 17␤-Estradiol at different
doses was administered i.p. 3 h before nicotine (3.0 mg/kg s.c.) and mice
were tested 5 min later. Each point represents the mean ⫾ S.E. of 8 to 12
mice. *p ⬍ 0.05 compared with correspondent zero time point.
these differences are response-dependent. This study also
confirms that sex hormones are functional blockers of nicotinic receptors in in vivo and in vitro tests. Previous work
suggested that gender was an important factor that influences certain behavioral effects of nicotine, such as those on
feeding behavior (Grunberg et al., 1984), locomotor sensitization (Booze et al., 1999), and cognition (Kanyt et al., 1999).
Among the responses to nicotine where significant sex differences were observed in our studies are the antinociceptive
and the anxiolytic effects of nicotine. Female mice were found
less sensitive to the acute effects of nicotine in these tests.
Although gender differences in the plus-maze test have been
not described before, the difference in nicotine-induced antinociception agrees well with a recent human study where
women were less sensitive to nicotine than men (Jamner et
al., 1998).
This sex difference is not limited to nicotine, but other
nicotinic agonists such as epibatidine showed a similar difference. In addition, the fact that a similar sex difference was
seen after spinal injection would also confirm the involvement of spinal mechanisms in mediating such a difference.
However, no central administration of nicotine was performed in the present study. Furthermore, the lack of sex
difference in the case of neostigmine suggests that sex interacted significantly with nicotinic and not muscarinic drugs
because neostigmine-induced antinociception is mediated
through muscarinic and not nicotinic receptors (Yaksh et al.,
1985; Naguib and Yaksh, 1994).
Proposed Mechanisms Underlying Sex Differences
in Nicotine Analgesia. One obvious possibility is that nicotine bioavailability might differ between the sexes. Al-
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Fig. 9. Lack of testosterone’s effects on nicotine-induced antinociception
in the tail-flick test after i.p. injection in male mice. A, effect of testosterone (250 mg/kg i.p.) at different times after injection on nicotineinduced antinociception (3 mg/kg s.c.) in mice. B, effect of testosterone at
different doses on nicotine-induced antinociception (3 mg/kg s.c.) 4 h
before nicotine. Mice were tested 5 min later. Each point represents the
mean ⫾ S.E. of 8 to 12 mice.
Influence of Gender on Nicotine’s Effects
though a sex difference in nicotine kinetics is probably not a
major influence in humans, there is evidence of sex difference
in nicotine metabolism in rats, where studies showed that
male rats eliminate nicotine faster than females (Kyerematen et al., 1988; Nwoso and Crooks, 1988). Although
plasma and brain concentrations of nicotine were not measured in our studies, our data argue against an exclusive role
for pharmacokinetics in accounting for the differential analgesic response of male and female mice to nicotine. Indeed,
the following arguments suggest that kinetic factors are
probably playing a minor role: 1) the fact that sex differences
were not seen in a uniform manner in all behavior responses
measured; 2) sex differences were seen with more than one
nicotinic agonist (epibatidine and nicotine); and 3) sex differences were seen in nicotine potency in the tail-flick test after
i.t. administration.
Another possibility put forward to explain sex differences
in nicotine’s effects relates to the influence of pharmacodynamic factors. Recent results suggest that females require
higher doses than males to produce nicotine receptor upregulation (Collins et al., 1988; Koylu et al., 1997; Musachio
et al., 1997). Sex differences in receptor up-regulation after
chronic exposure to nicotine was reported with only male rats
showing up-regulation of brain [3H]cytisine binding sites
(Koylu et al., 1997). In this study female rats showed a
slightly higher baseline density of nicotinic receptors than
males.
Several recent reports (Craft and Milholland, 1998; Chiari
et al., 1999; Lavand’homme and Eisenach, 1999) that describe sex differences in nicotine-induced antinociception do
not agree with the present study. These reports have shown
that male rats are less sensitive to nicotine than females in
acute and chronic pain models. It is possible that species
differences in nicotine metabolism, receptor affinity, density,
or functional regulation could explain such contrast between
these results and our data. The results with mecamylamine,
where no significant difference in its potency in blocking
nicotine in both male and female mice was observed, may
indicate that regulation of nicotinic receptors involved in
nicotine-induced antinociception does not play a major role in
the gender difference observed.
Another factor that could underlie sex differences is the
influence of gonadal hormones. Our data clearly indicate that
sex hormones can modulate the effects of nicotine and nicotinic receptors in a differential manner. There have been a
few reports on the influence of hormones on nicotine’s pharmacological effects, and our results agree with their findings.
Ke and Lukas (1996) reported that progesterone and estradiol blocked 86Rb⫹ efflux in cells expressing muscle and ganglionic nicotinic receptors, and that testosterone failed to
inhibit nicotine’s effects even at high concentration. Additionally, progesterone and its A-ring reduced metabolites
were reported to be noncompetitive inhibitors on thalamic
86
Rb⫹ efflux assay that likely measures the functional activity of ␣4␤2-type nicotinic receptors (Bullock et al., 1997). A
previous report (Valera et al., 1992) showed that progesterone and testosterone were noncompetitive nicotinic blockers
in oocyte expressing chick ␣4␤2 nicotinic receptors, with an
IC50 of 9 and 46 ␮M, respectively. Our results extended these
observations and we found that progesterone and 17␤-estradiol are functional blockers of nicotinic receptors in in vivo
and in vitro conditions, with progesterone being a more potent antagonist of ␣4␤2 receptors (1.8 versus 25 ␮M). Testosterone failed to block nicotine’s behavioral effects at very
high doses, which correlates well with previous studies. We
also report that chronic exposure to progesterone enhances
its in vivo effects on nicotine consistent with what is reported
in in vitro tests (Ke and Lukas, 1996). The increase in progesterone potency after repeated injections could be also due
to drug accumulation. But how important might these effects
of hormones be on nicotinic receptor function? Our data with
progesterone indicate that nicotinic receptor function could
be affected by this steroid. Progesterone levels in rat plasma
can reach levels between 2.5 and 20 ␮M at different stages
(Ichikawa et al., 1974; Concas et al., 1999). Under some
conditions such as stress and pregnancy, blood levels of progesterone in humans can reach 1 ␮M (Hart et al., 1985). In
addition, local concentrations of these hormones at receptor
sites could be higher because of their lipid-soluble nature
(Concas et al., 1999).
In summary, evidence is accumulating that sex differences
in nicotine’s effects are important factors to consider in our
effort to develop better prevention and treatment strategies
in smoking cessation. Indeed, a number of studies have
shown that women find it more difficult than do men to quit
smoking cigarettes (for review, see Perkins et al., 1999). Our
results with nicotine analgesia are important in that regard
for several reasons. A number of studies have pointed to
nicotine’s analgesic effects as a potential reinforcer of smok-
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Fig. 10. Concentration-dependence of progesterone (A) and 17␤-estradiol
(B) inhibition of ␣4␤2 nAChRs subtype expressed in oocytes. Responses of
neuronal ␣4␤2 receptor to 1 ␮M nicotine in the absence and presence of
different concentrations of progesterone or 17␤-estradiol when they were
preapplied at the tested concentration for 20 s and then followed by a
coapplication of the hormone and nicotine. Each application is separated
by 5-min washout. Oocytes were held at ⫺70 mV. ACh “after” represents
the current activated by an application of ACh after hormone washout.
139
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Damaj
ing behavior (Fertig and Pomerleau, 1986; Perkins et al.,
1994; Jamner et al., 1998) because of the ability of nicotine to
reduce pain and discomfort. In addition, Jamner et al. (1998)
reported a significant gender difference with men showing a
decrease in electrical pain threshold and tolerance after application of nicotine patches but they had no effect in women.
Furthermore tolerance was reported to develop to nicotine’s
antinociceptive effects in animals. Our findings contribute to
our search for receptor mechanisms in drug dependence and
in the discovery of better pharmacological agents for nicotine
dependence.
Acknowledgments
We greatly appreciate the technical assistance of Dr. Tie Han and
Gray Patrick.
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