Am J Physiol Endocrinol Metab 295: E497–E504, 2008.
First published June 17, 2008; doi:10.1152/ajpendo.00045.2008.
Effects of arecoline on testosterone release in rats
Shyi-Wu Wang,1* Guey-Shyang Hwang,2,3 Te-Jung Chen,2 and Paulus S. Wang2*
1
Department of Physiology and Pharmacology, Chang Gung University, Taoyuan3; 2Department of Physiology, School
of Medicine, National Yang-Ming University, Taipei; and 3Department of Nursing, Chang Gung Institute of Technology,
Taoyuan, Taiwan, Republic of China
Submitted 23 January 2008; accepted in final form 16 June 2008
arecoline; Leydig cell; L-type calcium channel; testosterone
ARECOLINE (1,2,5,6-tetrahydrol-1-methyl-3-pyridinecarboxylic
acid methyl ester) (Fig. 1) is an alkaloid extracted from betel
nuts (Areca catechu L.). Four major alkaloids are found in
betel nut: arecoline (7.5 mg/g wt), arecaidine (1.5 mg/g wt),
guvacoline (2.0 mg/g wt), and guvacine (2.9 mg/g wt) (56).
Epidemiological studies showed a strong correlation between
oral cancer and betel nut-chewing habit (16, 22, 50). Arecoline
and/or its alkaloids have been shown to be characterized by
carcinogenicity (16, 34, 45), immunotoxicity (42), genotoxicity (30, 46), and teratogenicity (46) in animal model systems.
In addition, arecoline has been shown to be mutagenic in
mammalian cells, especially in oral mucosal fibroblasts (5, 44).
The increased frequency of micronucleated cells, chromosomal
aberrations, and sister chromatid exchanges in exfoliated cells
of the buccal mucosa was observed in areca-nut consumers (9,
11). In addition, study showed that arecoline enhances the
* S.-W. Wang and P. S. Wang contributed equally to this work.
Address for reprint requests and other correspondence: S.-W. Wang, Dept.
of Physiology, School of Medicine, Chang Gung Univ., Kweisan, Taoyuan,
33333, Taiwan, ROC (e-mail: [email protected]).
http://www.ajpendo.org
frequency of chromosomal aberrations and micronuclei in
mouse bone marrow cells in vivo (12).
Arecoline mimics the actions of acetylcholine and exerts its
effects at both muscarinic and nicotinic receptors (3). Studies
showed that arecoline readily penetrates the blood-brain barrier
(35) and exerts its excitatory action by binding to M2-muscarinic receptors on the cell membrane of neurons of the locus
coeruleus (61). Calogero et al. (4) showed that the cholinergic
agonist arecoline stimulates the hypothalamic-pituitary-adrenal
(HPA) axis in the rat, and this effect is mediated mainly by the
release of endogenous corticotropin-releasing hormone (CRH).
Arecoline, injected intravenously to catheterized, freely moving male Sprague-Dawley rats stimulated plasma ACTH and
corticosterone release in a dose-dependent fashion. Arecoline
stimulates the HPA axis via secretion of CRH.
It has been confirmed by the dose-dependent ability of
arecoline to cause hypothalamic CRH secretion in vitro and its
failure to elicit ACTH and corticosterone secretion by dispersed anterior pituicytes and adrenocortical cells in culture
(4). Arecoline at concentrations of 0.2⬃2 ␮M significantly
elevated the number of micronucleated cells in a dose-dependent manner (24). In addition, significant prolongation of cell
cycles was observed by treatment with arecoline (⬃2.0 ␮M) in
Chinese hamster ovarian cells (24). Moreover, morphology
abnormality and unscheduled DNA synthesis were observed in
the arecoline-treated germ cells of the mouse (47). Intravenous
arecoline administration (3 mg/person) was followed by the
increases in plasma epinephrine and ACTH, which were maximal at 10 and 20 min, respectively (32). Although there is no
evidence proving that arecoline affects the thyroid function, the
speed of induction of rapid eye movement sleep induced by
arecoline had a modestly significant negative correlation with
free thyroxine (T4) index (20). Panda and Kar (29) showed that
a low dose of betel leaf extract was found to increase the
triiodothyronine (T3) concentration and T3-to-T4 ratio along
with a decrease in T4 concentration. By contrast, increased T4
and decreased T3 and T3-to-T4 ratios were observed in higherdose betel leaf extract. In Alzheimer’s patients, elevated
plasma ACTH and cortisol were observed in acute high-dose
but not in low-dose arecoline administration (2). These studies
indicated that arecoline might affect the endocrine system.
However, it is still unclear whether arecoline affects reproductive function.
To examine the possible physiological effects of arecoline,
we studied the acute effects of arecoline on endocrine systems.
The purpose of this study was to 1) determine the effect of
arecoline on human chorionic gonadotropin (hCG)-stimulated
The costs of publication of this article were defrayed in part by the payment
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0193-1849/08 $8.00 Copyright © 2008 the American Physiological Society
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Wang S-W, Hwang G-S, Chen T-J, Wang PS. Effects of
arecoline on testosterone release in rats. Am J Physiol Endocrinol
Metab 295: E497–E504, 2008. First published June 17, 2008;
doi:10.1152/ajpendo.00045.2008.—Arecoline is one of the major
components of betel nuts, which have been consumed as chewing gum
in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human
chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 ␮g/kg) plus
hCG via a jugular catheter. Blood samples were collected at several
time intervals subsequent to the challenge. Rat anterior pituitary was
treated with gonadotropin-releasing hormone in vitro with or without
arecoline, and then the concentrations of luteinizing hormone (LH) in
the medium were measured. Rat Leydig cells were purified by Percoll
density gradient centrifugation and incubated with arecoline, hCG,
forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or
tetrandrine at 34°C for 1 h. A single intravenous injection of arecoline
resulted in an increase of the hCG-induced level of plasma T.
Administration of arecoline (10⫺8 to 10⫺6 M) in vitro increased T
production in Leydig cells. The stimulatory effect of arecoline on T
release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10⫺6
M), or 8-Br-cAMP (10⫺5 M). By contrast, nifedipine, nimodipine, or
tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute
regulatory (StAR) protein expression compared with vehicle. These
results suggested that arecoline stimulates testosterone production by
acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17␤hydroxysteroid dehydrogenase and enhancing the expression of StAR.
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ARECOLINE ON TESTOSTERONE RELEASE
testosterone production in rat Leydig cells, and to 2) determine
the mechanisms of this effect at the molecular level. In addition, we also investigated the direct effects of arecoline on
purified Leydig cells in vitro. The data presented here demonstrate that arecoline stimulates both basal and hCG-evoked
testosterone production in rat Leydig cells. The data also
indicate that arecoline stimulates the protein expression of
StAR protein.
MATERIALS AND METHODS
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Animals. Male Sprague-Dawley rats weighing 300 –350 g were
housed in a temperature-controlled room (22 ⫾ 1°C) with 14 h of
artificial illumination daily (0600 –2000) and food and water ad
libitum. The use of the animals was approved by the Institutional
Animal Care and Use Committee of the National Yang-Ming University. All animals received humane care in compliance with the
principles of laboratory animal care and the Guide for Care and Use
of Laboratory Animals published by the National Science Council
(Taiwan, Republic of China).
Materials. BSA, HEPES, Hanks’ balanced salt solution, Medium
199, sodium bicarbonate, penicillin-G, streptomycin sulfate, heparin,
collagenase, arecoline, hCG, forskolin, 8-bromo-cGMP (8-Br-cAMP),
and testosterone were purchased from Sigma Chemical (St. Louis,
MO). Trilostane (4,5-epoxy-17-hydroxy-3-oxoandrostane-2-carbonitrile), an inhibitor of 3␤-hydroxysteroid dehydrogenase (HSD), was
provided by Sanofi-Synthelabo (Malvern, PA). Anti-cytochrome
P-450scc (P450scc) antibody was provided by Dr. Bon-Chu Chung
(Academia Sinica, Taipei, Taiwan, ROC), and anti-steroidogenic
acute regulatory (StAR) antibody was provided by Dr. D. M. Stocco
(Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX). [3H]testosterone and
[3H]pregnenolone were obtained from Amersham Life Science Limited (Buckinghamshire, UK).
Effect of arecoline on plasma testosterone in rats. Male rats were
divided into four groups with seven to eight rats in each group. Each
animal was anesthetized with ether and catheterized via the right
jugular vein (53, 57). After 20 h, they were infused with 1 ml of
saline, arecoline (1 ␮g/kg; Sigma Chemical), hCG (5 IU/kg), or
hCG ⫹ arecoline via a jugular catheter. Blood samples (0.3 ml each)
were collected from the jugular catheter at 0, 30, 60, 120, 180, 240,
300, and 360 min after infusion.
Plasma was separated by centrifugation at 10,000 g for 1 min and
stored at ⫺20°C. To measure the concentrations of testosterone, 0.1
ml plasma was mixed with 0.5 ml diethyl ether, agitated for 20 min,
centrifuged at 1,000 g for 5 min, and then quick-frozen in a mixture
of acetone and dry ice. The organic phase was collected, dried, and
reconstituted in a buffer solution containing 0.1% gelatin in PBS (pH
7.5). The concentrations of testosterone in the reconstituted extracts
were measured by RIA.
Effect of arecoline on luteinizing hormone release in vitro. Male
rats were decapitated, and the anterior pituitary (AP) glands were
bisected, preincubated, and then incubated with Locke’s solution in
the presence of arecoline (10⫺7 to 10⫺6 M) with or without 10 nM
gonadotropin-releasing hormone (GnRH) at 37°C for 30 min. At the
end of incubation, media were collected for measurement of luteinizing hormone (LH) by RIA.
Preparation of rat Leydig cells. Animals were killed, and the testes
were collected and decapsulated. Testicular interstitial cells were
isolated with the collagenase dispersion method as previously described (6). The procedure used for preparing Leydig cells has been
described elsewhere (15). Testicular interstitial cells were centrifuged
at 200 g for 10 min at 4°C. The cell pellet volume was suspended in
the incubation medium (1% BSA in medium 199 with 25 mM
HEPES, 2.2 g/ml sodium bicarbonate, 100 IU/ ml penicillin-G, 50
␮g/ml streptomycin sulfate, 2,550 USP K U/l heparin, pH 7.4, and
aerated with 95% O2 and 5% CO2) to 5 ml and then added gently to
the upper layer of the continuous Percoll gradient. The continuous
Percoll gradient (20 ml/dispersion) was made by adding 9 parts of
Percoll to 11 parts of 1.8 concentrated incubation medium before
centrifugation at 20,000 g for 60 min at 4°C. The mixture of testicular
cells was loaded on the Percoll gradient and centrifuged at 800 g for
20 min at 4°C. Leydig cells were located in the 3- to 7-ml layer from
the bottom. The Leydig cell layer was collected, diluted to 10 ml in
incubation medium, and then centrifuged at 200 g for 10 min at room
temperature.
After repeating the washing steps, the cell pellet was suspended to
10 ml in incubation medium. The cell concentration (2 ⫻ 105 cells/ml)
and viability (95%) were determined by using a hemacytometer and
the trypan blue method. To measure the abundance of Leydig cells in
our preparation, the 3␤-HSD staining method was used (4, 24). The
cells (2 ⫻ 105 cells/ml) were incubated with a solution containing 0.2
mg/ml of nitro blue tetrazolium (Sigma) in 0.05 M PBS, pH 7.4, at
34°C for 90 min. When the blue formazan deposit sites of 3␤-HSD
activities were developed, the abundance of Leydig cells was determined by using a hemacytometer. Macrophages were determined by
flow cytometry with fluorescein isothiocynate-conjugated monoclonal
antibody (ED1, IgG 1; Biosource International, Foster City, CA). Our
preparation contained ⬃87% Leydig cells and very few macrophages.
Effects of arecoline on testosterone release by rat Leydig cells.
Cells at a concentration of 1 ⫻ 105 cells/ml were preincubated at 34°C
for 1 h in a controlled atmosphere (95% O2 and 5% CO2) and shaken
at 100 cycles/min. The supernatant fluid was decanted after centrifuging the tubes at 100 g for 10 min. The cells were then incubated
with arecoline (0, 10⫺8, 10⫺7, or 10⫺6 M) in 200 ␮l of fresh medium.
Following 1 h incubation, 1 ml of ice-cold 0.1% gelatin-PBS, pH 7.5,
was added to stop the incubation. The medium was centrifuged at 100
g for 10 min, and the supernatant was stored at ⫺20°C until analyzed
for testosterone by RIA.
Aliquots (1 ml) of the cell suspensions (1 ⫻ 105 cells/ml) were
challenged with arecoline (10⫺7 M) in the presence of hCG (0.001
IU/ml), forskolin (an adenylyl cyclase activator, 10⫺6 M), or 8-BrcAMP (a membrane-permeable analog of cAMP, 10⫺5 M) in 200 ␮l
of fresh medium. At the end of incubation, the media were collected
for testosterone RIA.
Effects of arecoline on the functions of 17␤-HSD in rat Leydig
cells. In this study, Leydig cells were incubated for 1 h with or without
arecoline (10⫺7 M) in the presence of immediate precursors of
testosterone, androstenedione (10⫺7 and 10⫺5 M). At the end of
incubation, the media were collected for testosterone RIA. The level
of testosterone was an index of the activity of 17␤-HSD in rat Leydig
cells.
Effects of arecoline on the activities of calcium channels in rat
Leydig cells. Rat Leydig cells were incubated for 1 h with or without
arecoline (10⫺7 M) in the presence or absence of tetrandrine (a
blocker for both L-type and T-type calcium channels, 10⫺5 M),
nifedipine (a blocker for L-type calcium channel, 10⫺5 M), and
nimodipine (a blocker for L-type calcium channel, 10⫺5 M). At the
end of incubation, the media were collected for testosterone RIA.
Effects of arecoline on pregnenolone production stimulated with
25-OH-cholesterol (25-OH-C) in rat Leydig cells. Pregnenolone is the
product of P450scc by conversion of cholesterol. Intracellular pregnenolone is metabolized by 3␤-HSD (Fig. 1). To investigate the
production of pregnenolone, we used trilostane (an inhibitor of 3␤HSD) to inhibit the catabolism of pregnenolone. Leydig cells (1 ⫻ 105
cells/ml) were preincubated with incubation medium for 1 h at 34°C.
Cells were primed with trilostane (10⫺6 M) for 30 min and then
incubated for 1 h with arecoline (10⫺7 M). At the end of incubation,
the media were collected for pregnenolone RIA.
For kinetic analysis of P450scc, Leydig cells (1 ⫻ 105 cells/ml)
were primed with trilostane (10⫺5 M) for 30 min and then incubated
for 1 h with arecoline (10⫺7 M) in the presence of 25-OH-C (10⫺6 to
ARECOLINE ON TESTOSTERONE RELEASE
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(48). A difference between two means was considered statistically
significant when P ⬍ 0.05.
RESULTS
Fig. 1. Chemical structure and molecular weight of arecoline.
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Fig. 2. Effects of arecoline on the basal (top) and human chorionic gonadotropin (hCG)-stimulated (bottom) level of plasma testosterone in male rats.
Rats were given a single intravenous injection of vehicle arecoline, hCG, or
hCG ⫹s arecoline via a right jugular catheter and bled at different time
intervals after injection. The concentration of testosterone was measured by
RIA. **P ⬍ 0.01 compared with vehicle or hCG group. P ⬍ 0.05 (⫹) and 0.01
(⫹⫹) compared with time 0 min. Each value represents the mean ⫾ SE.
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10⫺4 M). At the end of incubation, the media were collected for
pregnenolone RIA.
Hormone RIAs. Testosterone concentrations in media were determined by RIA as described previously (53, 57). The sensitivity of the
testosterone RIA was 2 pg/assay tube. The intra-assay and interassay
coefficients of variation were 4.1% (n ⫽ 6) and 4.7% (n ⫽ 10),
respectively.
The concentrations of pregnenolone were determined by RIA, as
described previously (7). The sensitivity of the pregnenolone RIA was
16 pg/assay tube. The intra-assay and interassay coefficients of variation were 2.3% (n ⫽ 6) and 3.7% (n ⫽ 4), respectively.
The concentrations of LH were determined by RIA as described
previously (7). The sensitivity of the LH RIA was 0.1 ng/assay tube.
The intra-assay and interassay coefficients of variation were 2.0%
(n ⫽ 6) and 4.5% (n ⫽ 8), respectively.
Western blot analysis. The Western blotting method has been
described elsewhere (6, 25). Leydig cells (1 ⫻ 106 cells/ml) were
incubated with or without arecoline (10⫺7 to 10⫺5 M) for 1 h. At the
end of incubation, cells were washed two times with ice-cold saline
and then homogenized, and the protein was extracted in 50 ␮l of
homogenization buffer (1.5% sodium lauroylsacrosine, 2.5 mM Tris
base, 1 mM EDTA, and 0.1% phenylmethylsulfonyl fluoride, pH 7.8).
The cell extract was centrifuged at 12,000 g for 10 min at 4°C. The
supernatant was separated, and protein concentration was determined
by modifying the Bradford protein assay method (62). SDS-PAGE
sample buffer (0.06 M Tris base, 2% SDS, 0.0005% bromphenol blue,
6% sucrose, and 50 ␮M dithiothreitol) was added to the samples, and,
after boiling for 5 min, the samples were stored at ⫺20°C until they
were used.
Samples (20 ␮g) were electrophoresed on a 12% minigel by
standard SDS-PAGE procedures, along with prestained molecular
weight markers (Bio-Rad, Hercules, CA). Gels were electrophoresed
at 75 V for 15 min and then 150 V for 30 min. The protein bands were
transferred to polyvinylidene difluoride membranes (NEN Life Sciences Products, Boston, MA) with a semidry transfer cell (Bio-Rad)
for 45 min in a blotting solution. The membrane was washed in 0.8%
NaCl, 0.02 M Tris base, and 0.3% Tween 20 (pH 7.6; TBS-T buffer)
for 5 min and blocked by a 120-min incubation in blocking buffer
(TBS-T buffer containing 5% nonfat dry milk). The membrane was
then incubated overnight with anti-StAR (1:1,000), anti-P450scc (1:
2,000), and anti-␤-actin (1:8,000). After three washes for 5 min each
with TBS-T buffer, the membranes were incubated for 2 h with
horseradish peroxidase-conjugated secondary antibody (1:6,000).
Specific signals were detected by enhanced chemiluminescence (ECL
Western blotting detection reagents; Amersham Life Science Limited).
Statistical analysis. All values were given as the means ⫾ SE. The
treatment means were tested for homogeneity by one-way ANOVA,
and the differences between specific means were tested for significance by Duncan’s multiple-range test or by paired Student’s t-test
Effect of arecoline on the concentration of plasma testosterone. The basal level of plasma testosterone was 0.4 – 0.5 ng/ml
(Fig. 2, top). A single intravenous injection of arecoline (1
␮g/kg) gradually increased (P ⬍ 0.01) the concentration of
plasma testosterone. A plateau of plasma testosterone concentration (0.9 –1.0 ng/ml) was reached 5– 6 h following injection
of arecoline.
A single intravenous injection of hCG (5 IU/kg) stimulated
the concentration of plasma testosterone by 3.7-fold at 60 min
following challenge (Fig. 2, bottom). The concentration of
plasma testosterone returned to the basal level 4 h following
hCG challenge. After administration of arecoline plus hCG (1
h), the secretion of testosterone increased markedly by 7.2-fold
compared with the basal level. The peak and postpeak levels of
plasma testosterone were significantly higher (P ⬍ 0.01) in rats
treated with arecoline plus hCG than in hCG-treated animals
(Fig. 2, bottom).
Effect of arecoline on LH release in vitro. Incubation of rat
AP with 10 nM GnRH increased (3.2-fold, P ⬍ 0.01) the
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ARECOLINE ON TESTOSTERONE RELEASE
Fig. 3. Effects of arecoline on the basal and gonadotropin-releasing hormone
(GnRH)-stimulated luteinizing hormone (LH) release by male rat anterior
pituitary (AP) gland in vitro. The concentration of LH in medium was
measured by RIA. **P ⬍ 0.01 compared with vehicle group. Each value
represents the mean ⫾ SE.
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Fig. 4. Effects of arecoline in vitro on testosterone release by rat Leydig cells.
Each column represents the mean ⫾ SE. P ⬍ 0.05 (*) and P ⬍ 0.01 (**)
compared with 0 M arecoline.
trilostane only or trilostane plus arecoline was observed
(P ⬍ 0.05; Fig. 9).
Protein expression of StAR and P450scc. Bands at 54 kDa
(P450scc) and 30 kDa (StAR) were detected in rat Leydig cells
(Fig. 10A). The ␤-actin signal (45 kDa) was employed as an
internal control. Six replications showed the similar pattern.
The Leydig cells were challenged with 10⫺7 or 10⫺6 M
arecoline for 1 h, and then the protein expression of StAR was
increased by the addition of arecoline (Fig. 10B). No difference
in the protein expression of P450scc was observed after the
addition of arecoline (Fig. 10, A and B).
DISCUSSION
Betel is a masticatory substance, and betel chewing is a
popular oral habit in Southern Taiwan. Numerous studies
showed a significant correlation of betel chewing with the
incidence of oral cancer or oral submucous fibrosis (16, 17, 31,
36, 43, 51, 58). Arecoline is one of the major ingredients in
betel nuts. The other three are arecaidine, guvacoline, and
isoguvacine (55, 56). Each chewer in Taiwan consumed on
average 14 –23 betel quid a day (47), and each betel quid
contains 7.5 mg/g of arecoline (55). Although there is no report
concerning the plasma arecoline concentration, in vitro study
showed that the arecoline cytotoxicity to gingival keratinocytes
was observed at concentrations of 0.8 –1.2 mM (18). Therefore, chewing betel quids is thought to be a bad habit associated with malignant cancer, especially the oral cancer. Although a number of studies showed betel chewing is strongly
related to oral cancer, betel has been used for the treatment of
diarrhea, edema, throat inflammations, and tapeworm (51). In
this study, we demonstrated that arecoline, with or without
hCG, significantly stimulated testosterone secretion both
in vivo and in vitro.
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release of LH in vitro (Fig. 3). Neither basal nor GnRHstimulated release of LH was significantly altered by the
administration of arecoline (10⫺7 to 10⫺6 M).
Effect of arecoline on the basal and cAMP-evoked release of
testosterone in vitro. Administration of arecoline at the doses
ranging from 10⫺8 M to 10⫺6 M dose dependently increased
the basal release of testosterone by rat Leydig cells. (Fig. 4).
Incubation of rat Leydig cells with 8-Br-cAMP (a permeable
analog of cAMP, 10⫺5 M), forskolin (an adenylyl cyclase
activator, 10⫺6 M), or hCG (0.001 IU/ml) significantly increased (P ⬍ 0.001) the release of testosterone in vitro.
Coincubation of arecoline (10⫺7 M) increased not only the
basal but also the 8-Br-cAMP, forskolin, and hCG-evoked
production of testosterone (P ⬍ 0.01, Fig. 5).
Effect of arecoline on the activity of 17␤-HSD. Incubation of
rat Leydig cells with 10⫺7 and 10⫺5 M of androstenedione (the
immediate precursor of testosterone) dose dependently increased the production of testosterone (Fig. 6). Administration
of 10⫺7 M arecoline significantly enhanced the release of
testosterone in response to androstenedione (Fig. 6).
Effect of arecoline on testosterone release in response to
calcium channel blockers. Although the spontaneous release of
testosterone was not significantly altered by the administration
of tetrandrine (a blocker of L-type and T-type calcium channels, 10⫺5 M), nifedipine (an L-type calcium channel blocker,
10⫺5 M), or nimodipine (an L-type calcium channel blocker,
10⫺5 M), the arecoline-evoked production of testosterone was
significantly reduced by all of these calcium channel blockers
(P ⬍ 0.01, Fig. 7).
Effect of arecoline on the activity of P450scc. To examine the
activity of P450scc (an enzyme converts cholesterol into pregnenolone), an inhibitor of 3␤-HSD (an enzyme converts pregnenolone into progesterone), trilostane, was employed to incubate with rat Leydig cells. Incubation of trilostane (10⫺6 M)
with Leydig cells significantly increases the production of
pregnenolone (P ⬍ 0.01, Fig. 8). Administration of 10⫺7 M
arecoline increased both basal and trilostane-evoked levels
of pregnenolone released by rat Leydig cells (P ⬍ 0.01; Fig.
8). In addition, increased pregnenolone release from Leydig
cells treated with different concentrations of 25-OH-C with
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LH, enhances either the testosterone production in mouse
interstitial cells (60) or plasma testosterone concentration in
rats (41). Therefore, the stimulatory effect of arecoline on
testosterone secretion was LH independent. In addition, we are
Fig. 5. Effects of arecoline on testosterone release by rat Leydig cells incubated with vehicle, b-bromo-cAMP (8-Br-cAMP; 10⫺5 M), forskolin (10⫺6 M),
or hCG (0.001 IU/ml). Each column represents the mean ⫾ SE (n ⫽ 6 rats).
P ⬍ 0.01 compared with 0 M arecoline (**) and compared with the vehicle
group (⫹⫹). Please note the log scale in the y-axis.
Our results reveal a dose-dependent effect of arecoline, one
of the major components in betel nuts, on the changes of
testosterone concentration in male rats. In addition, the effects
of arecoline on the increased testosterone secretion from isolated Leydig cells were due to, at least in part, the increased
steroidogenesis. This conclusion is supported by the following
observations. 1) Significantly increased plasma testosterone in
rats was observed 60 min after arecoline injection. 2) The
hCG-induced plasma testosterone could be enhanced by arecoline at 60 min. 3) Arecoline did not alter the GnRH-induced
LH secretion in vitro. 4) Calcium channel blocker blunted the
stimulatory effect of arecoline on the secretion of testosterone
in vitro. 5) An increase of intracellular StAR protein was
observed after arecoline treatment.
In this study, arecoline did not alter the LH secretion from
the bisected AP gland treated with GnRH. However, the
secretion of testosterone from Leydig cells was stimulated
significantly by arecoline alone. We note that there has been no
study of plasma concentration of arecoline in patients with
betel chewing. Also, no investigation has shown the plasma
concentration of testosterone in men who chew betel nuts.
Recent studies also indicated that arecoline, with or without
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Fig. 7. Effects of arecoline on testosterone release by rat Leydig cells in
response to calcium channel blockers. Leydig cells were incubated with
vehicle, calcium channel blocker, or calcium channel blocker ⫹ arecoline for
1 h. **P ⬍ 0.01 compared with vehicle group in the presence of arecoline.
Each value represents the mean ⫾ SE.
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Fig. 6. Effects of arecoline on androstenedione-stimulated testosterone secretion in vitro by rat Leydig cells. Leydig cells pretreated with arecoline or
steroidogenic precursor ⫹ arecoline for 1 h. The medium concentration of
testosterone was measured by RIA. P ⬍ 0.01 compared with the vehicle group
(**) compared with the group treated without androstenedione (⫹⫹). Each
value represents the mean ⫾ SE.
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ARECOLINE ON TESTOSTERONE RELEASE
the first, as we know, to demonstrate that arecoline stimulates
testosterone secretion from isolated Leydig cells. However, it
is still unknown at present whether the action of arecoline on
Leydig cells is through muscarinic and nicotinic receptors
expressed in other cells and tissues (8, 19, 59).
In Leydig cells, testosterone is synthesized by several metabolic steps, collectively known as steroidogenesis. We examined the effects of arecoline on the activities of 17␤-HSD in
Leydig cells by challenging the cells in vitro with the immediate precursor of testosterone, androstenedione (⌬4, substrate
of 17␤-HSD). Because androstenedione can be converted to
testosterone by 17␤-HSD, and production of testosterone stimulated by androstenedione was further enhanced in the presence of arecoline (Fig. 6), we suggested that the activity of
17␤-HSD was increased by the administration of arecoline.
It is well known that the conversion of cholesterol into
pregnenolone is catalyzed by P450scc enzyme (13). In addition,
trilostane is an inhibitor of the 3␤-HSD enzyme system with no
inherent steroidal activity, and it inhibits the production of
progesterone and results in an accumulation of pregnenolone,
Fig. 9. Effects of arecoline (10⫺7 M) on 25-OH-cholesterol (25-OH-C)stimulated pregnenolone release by Leydig cells in the presence of a 3␤hydroxysteroid dehydrogenase (3␤-HSD, an enzyme for converting pregnenolone to progesterone) blocker, trilostane (10⫺5 M). *P ⬍ 0.05 compared
with the trilostane group. UD, undetectable.
AJP-Endocrinol Metab • VOL
Fig. 10. Effects of arecoline on cytochrome P-450scc (P450scc; A) and steroidogenic acute regulatory (StAR) protein (B) expression in rat Leydig cells.
Leydig cells were treated with arecoline for 1 h. The expressions of P450scc
and StAR protein were analyzed by Western blot. *P⬍0.05 compared with the
vehicle group.
which is a sensitive index for the activity of 3␤-HSD (40, 54).
In the presence of trilostane, administration of arecoline increases the production of pregnenolone, suggesting that the
activity of P450scc was increased by arecoline (Figs. 8 and 9).
In addition, the stimulatory effect of arecoline was observed at
both lower (10⫺7 M) and higher (10⫺5 M) concentrations of
androstenedione (Fig. 6) and other testosterone precursors
(data not shown). Also, the accumulation of pregnenolone
was observed in the media treated with arecoline. These
results indicate that arecoline affects the activities of several
enzymes, at least including P450scc and 17␤-HSD, required
for the steroidogenesis.
The biosynthesis of steroid hormones in the testes, in response to the steroidogenic stimuli including hCG, starts with
the transfer of cholesterol from the outer to the inner mitochondrial membrane, which is facilitated by StAR (28, 49).
Moreover, the P450scc enzyme is located in the inner mitochondrial membrane (13, 27). Subsequent steroid hormones,
including progesterone, testosterone, and estrogen, are synthesized in the endoplasmic reticulum. Therefore, inhibition of
StAR activity is associated with the transfer of cholesterol and
the inhibition of the steroidogenic response. Our results
showed that the expression of StAR protein was enhanced by
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Fig. 8. Effects of arecoline on the basal and trilostane-stimulated pregnenolone release by rat Leydig cells. The concentration of pregnenolone was
measured by RIA. P ⬍ 0.01 compared with 0 M arecoline (**) and compared
with the vehicle group (⫹⫹). Each value represents the mean ⫾ SE.
ARECOLINE ON TESTOSTERONE RELEASE
ACKNOWLEDGMENTS
The anti-P450scc antibody was kindly provided by Dr. B. C. Chung,
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C. In
addition, the anti-StAR antibody was kindly provided by Dr. D. M. Stocco,
Department of Cell Biology and Biochemistry, Texas Tech University Health
Science Center, Lubbock, TX. The technical assistance provided by Sung-Yun
Chen is also greatly appreciated.
AJP-Endocrinol Metab • VOL
GRANTS
This study was supported by Grant NSC 90-2320-B-182-049 from the
National Science Council.
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