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- Original Article -
Effects of plant extract neferine on cyclic adenosine monophosphate and cyclic guanosine monophosphate
levels in rabbit corpus cavernosum in vitro
Jun Chen1, Ji-Hong Liu1, Tao
Wang1, Heng-Jun Xiao2, Chun-Ping
Yin3, Jun Yang1
1Department of Urology, Tongji Hospital,
3Department of Pharmacy, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, China
2Department of Urology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510630, China
Abstract
Aim: To further investigate the relaxation mechanism of neferine (Nef), a bis-benzylisoquinoline alkaloid extracted
(isolated) from the green seed embryo of Nelumbo nucifera Gaertn in China, on rabbit corpus cavernosum tissue
in vitro. Methods: The effects of Nef on the concentrations of cyclic adenosine monophosphate (cAMP) and
cyclic guanosine monophosphate (cGMP) in isolated and incubated rabbit corpus cavernosum tissue were
recorded using 125I radioimmunoassay. Results: The basal concentration of cAMP in corpus cavernosum tissue
was
5.67 ± 0.97 pmol/mg. Nef increased the cAMP concentration in a dose-dependent manner
(P < 0.05), but this effect was not inhibited by an adenylate cyclase inhibitor (cis-N-[2-phenylcyclopentyl]azacyclotridec-1-en-2-amine,
MDL-12, 330A) (P > 0.05). The accumulation of cAMP induced by prostaglandin
E1 (PGE1, a stimulator of cAMP
production) was also augmented by Nef in a dose-dependent manner
(P < 0.05). The basal concentration of cGMP in
corpus cavernosum tissue is 0.44 ± 0.09 pmol/mg. Nef did not affect this concentration of cGMP, either in the presence
or in the absence of a guanyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ)
(P > 0.05). Also, sodium nitroprusside (SNP, a stimulator of cGMP production)-induced cGMP production was not enhanced by Nef
(P > 0.05). Conclusion: Nef, with its relaxation mechanism, can enhance the concentration of cAMP in rabbit corpus
cavernosum tissue, probably by inhibiting phosphodiesterase
activity. (Asian J Androl 2008 Mar; 10: 307_312)
Keywords: neferine; cyclic adenosine monophosphate; cyclic guanosine monophosphate; rabbit corpus cavernosum
Correspondence to:
Prof. Ji-Hong Liu, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China.
Tel: +86-27-8366-2278 Fax: +86-27-8360-8783
E-mail: jhliu@tjh.tjmu.edu.cn
and Dr Jun Chen, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China.
Tel: +86-21-6502-1292
E-mail: jchen121121@hotmail.com
Received 2006-12-22 Accepted 2007-07-26
DOI: 10.1111/j.1745-7262.2008.00342.x
1 Introduction
Erectile dysfunction (ED) is a common problem with
a prevalence of approximately 50% in men aged 40_70 years [1]. Current pharmacological treatment for ED
includes the oral, intracavernosal and intraurethral
administration of erectogenic drugs. Oral pharmacotherapy
is the most effective therapy for ED and has the highest
patient preference. Oral phosphodiesterase type 5
(PDE5) inhibitors (sildenafil, tadalafil and vardenafil)
are superior in effectiveness to centrally acting drugs
(apomorphine and yohimbine). Local pharmacotherapy
(intracavernosal and intraurethral treatments) is a
second line therapy in cases of failure or contraindications
for oral pharmacotherapy [2]. Although many drugs are
now available for treating ED, finding a new drug for
treating ED and understand its mechanism of action are
still important research goals.
Many traditional Chinese medicines are effective in
treating ED. Because of the complex che
mical ingredients,
it remains unclear which ingredient exactly, and by which
mechanisms, has a positive effect in treating ED.
Recent studies have found that some extracts from
traditional Chinese medicines, containing alkaloids, coumarin
and saponin compounds, can relax the smooth muscle
of corpus cavernosum [3_15], which provides an open
window for developing new drugs for the treatment of
ED.
Penile erection is the process that takes place under
the regulation of the neural and endocrine system through the dilation of the penile arteries and corpus
cavernosum, thereby increasing the blood flow into the
penis. During sexual stimulation, nitric oxide (NO) is
released, by the action of nitric oxide synthase (NOS),
from non-adrenergic, non-cholinergic nerves,
parasympathetic nerves and endothelium. Guanylate cyclase is
activated by NO, which converts cyclic guanosine monophosphate (GTP) into cyclic guanosine
monophosphate (cGMP). In addition, adenylate cyclase is also
activated by receptor mechanisms (such as by prostacyclin, prostaglandin E1 and E2, and
β-adrenoceptor agonists) or non-receptor mechanisms (such
as activated by forskolin) leading to increases in
cyclic adenosine monophosphate (cAMP) levels. By
inhibiting calcium channels, both cGMP and cAMP decrease intracellular
Ca2+, causing the relaxation of
cavernous smooth muscle, and thus erection [16_18].
Therapeutically, drugs that enhance cGMP and/or cAMP accumulation could be applied in the treatment
of ED.
Neferine (Nef) is a bis-benzylisoquinoline alkaloid
extracted (isolated) from the green seed embryo of
Nelumbo nucifera Gaertn in China, which is effective in
preventing from the onset of reentrant ventricular
tachyarrhythmias [19, 20]. Moreover, Nef can inhibit
very low density lipoprotein oxidation [21] and platelet
aggregation [22], protect vascular endothelial cells from
damage induced by oxygen free radicals [23], and
increase sensitivity to anticancer drugs [24]. The
empirical formula of Nef is
C38H44N2O6
, with a relative molecular weight of 624. The molecular structure of Nef is
showed in Figure 1. In the course of our studies on the
development of naturally occurring agents for the
treatment of ED, we found that Nef induced relaxation on the
phenylephrine (PE)-precontracted corpus cavernosum
[14]. In the present study, the effects of Nef on cAMP
and cGMP levels in isolated and incubated rabbit corpus
cavernosum are measured using 125I radioimmunoassay
to clarify the relaxation mechanisms of Nef on the
corpus cavernosum smooth muscle.
2 Materials and methods
All animal experiments were carried out with the
approval of the Institute for Animal Care and Use
Committee at Tongji Hospital (Wuhan, China).
2.1 Reagents
Neferine was kindly provided by Professor Jia-Ling
Wang (Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology,
Wuha, China). The purity of Nef was greater than 98%.
cGMP and cAMP 125I radioimmunoassay kits were
purchased from Shanghai Chinese Medicine University
(Shanghai, China). Dulbecco's Modified Eagle Medium
(DMEM) was acquired from Gibco (Grand Island, NY, USA); MDL-12,330A
(cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine) and ODQ
(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) from Sigma
Chemical; sodium nitroprusside (SNP) from the Board
of Beijing Double-Crane Pharmaceuticals (Beijing, China)
and PGE1 (prostaglandin
E1) from Nanyang Pukang Groups (Nayang, China).
2.2 Rabbit corpus cavernosum preparation
Male New Zealand white rabbits (aged 4_6 months,
approximately 2.5_3.0 kg body weight) were killed by
air injection and their penises were excised soon afterwards. After the epidermis, connective tissue and
albuginea were removed, and each penis was cut
longitudinally into two strips and then transversely into eight
segments. The segments of corpora cavernosa from all
the rabbits were pooled and incubated in DMEM at 37ºC
for 1 h with regular changes of medium to allow the
tissues to recover from preparative handling. The
solution was continuously gassed with 95% oxygen and 5%
carbon dioxide (carbogen).
2.3 Pro-releasing of cAMP and cGMP
2.3.1 Effects of Nef on cAMP and cGMP levels in
rabbit corpus cavernosum
The freshly isolated corpus cavernosum segments
were incubated in 48-well plates with
5 × 10_1 mL DMEM at 37ºC for 1 h. After DMEM was removed,
these segments were divided into three groups and the
segments in each group were subdivided into four portions. (1) Nef group:
2 × 10_1 mL DMEM was added
to the four portions. (2) MDL-12,330A (adenylyl cyclase [AC] inhibitor) + Nef group:
1.8 × 10_1 mL DMEM and MDL-12,330A (final concentration
3 × 10_5 mol/L)
2 × 10_2 mL solvent were added, respectively. (3) ODQ
(Guanylate cyclase inhibitor) + Nef group:
1.8 × 10_1 mL DMEM and ODQ (final concentration
3 × 10_5 mol/L)
2 × 10_2 mL solvent were added, respectively. After
pre-incubation of the tissue for 10 min at 37ºC, various
concentrations of Nef
2 × 10_2 mL were added (final
concentration 0, 10_7 mol/L, 10_6
mol/L, 10_5 mol/L), respectively. They were then incubated at 37ºC for
another 30 min.
2.3.2 Effects of Nef on cAMP and cGMP levels in
rabbit corpus cavernosum in the presence of sodium
nitroprusside and PGE1
Fresh corpus cavernosum segments were incubated
in 48-well plates with
5 × 10_1 mL DMEM at 37ºC for
1 h. After DMEM was removed,
1.8 × 10_1 mL DMEM and
2 × 10_2 mL solvent with various concentrations of
Nef (final concentration 0, 10_7,
10_6, 10_5 mol/L, respectively) were added. After pre-incubation of the tissue
for 10 min at 37°C, these segments were incubated with
2 × 10_2 mL
PGE1 (final concentration
10_6 mol/L) or SNP (final concentration
10_6 mol/L) for 30 min at 37ºC.
2.4 Extraction of cAMP and cGMP
The supernatants were removed; the reaction was
stopped by the addition of
5 × 10_1 mL of 1 mol/L
perchloric acid, and then the tissues were crushed and
homo-genated with 1 mL acetic acid buffer
(5 × 10_2 mol/L,
pH 4.75) in an ice bath. After centrifugation at
3 000 × g for 15 min at
4ºC, the supernatants were collected, dried
in an oven at 60ºC, and then stored at 4ºC.
2.5 cAMP and cGMP assay
The concentrations of cAMP and cGMP were measured using the
125I radioimmunoassay kits according to
the manufacturer's instructions.
2.6 Statistical analysis
Data were expressed as mean ± SEM. Statistical
analysis was performed using one-way analysis of
variance followed by Dunnett's multiple comparisons test,
and the paired t-test using SPSS software version 10.0
(SPSS, Chicago, IL, USA).
P < 0.05 was considered a significant difference.
3 Results
3.1 Effects of neferine on cAMP levels in the rabbit
corpus cavernosum in vitro
The basal level of cAMP in the rabbit corpus cavernosum was 5.67 ± 0.97 pmol/mg. Nef could
increase the contents of cAMP in corpus cavernosum
tissue in a dose-dependent manner
(P < 0.05), but MDL-12,330A did not affect this elevation of cAMP levels
(n = 6, P > 0.05) (Figure 2).
In the presence of PGE1, the cAMP level increased
to 56.42 ± 7.26 pmol/mg, representing a 9.95-fold
increase, and Nef dose-dependently increased the cAMP
levels in corpus cavernosum tissue (n = 6,
P < 0.05) (Figure 3).
3.2 Effects of Nef on cGMP levels in the rabbit corpus
cavernosum in vitro
The basic level of cGMP in the rabbit corpus cavernosum was 0.44 ± 0.09 pmol/mg. Whether ODQ
was present or not, Nef had no effect on the cGMP
accumulation cavernosum tissue (n = 6,
P > 0.05) (Figure 4).
In the presence of SNP, the cGMP level increased to
1.90 ± 0.70 pmol/mg, representing a 4.32-fold increase.
However, Nef had no effect on SNP-induced elevation
of cGMP levels (n = 6,
P > 0.05) (Figure 5).
4 Discussion
Using 125I radioimmunoassay, we found that Nef had
no effect on the cGMP accumulation in isolated and
incubated corpus cavernosum tissue, whether or not the
guanyl cyclase inhibitor (ODQ) or stimulator of cGMP
production (SNP) was present. This indicated that the
NO-cGMP pathway had nothing to do with the effect of
Nef on the relaxation of the corpus cavernous smooth
muscle.
In the present study, we found that Nef enhanced
cAMP accumulation in the corpus cavernosum. These
results demonstrate that the rise of cAMP levels might
be one of the mechanisms of Nef-induced relaxation.
cAMP is produced from adenosine triphosphate (ATP)
by AC, and is degraded to 5'-AMP by phosphodiesterases
(PDE). The levels of cAMP are regulated by the AC and
PDE activities. In the absence of any stimulation, the
basal level of cAMP in the rabbit corpus cavernosum
in vitro was 5.67 ± 0.97 pmol/mg. However, the
incubation in the presence of 3 ×
10_5 mol/L MDL-12,330A (AC inhibitor) had no effect on the basal level of cAMP. This
indicated that the ability of producing cAMP by the
rabbit corpus cavernosum in vitro was very weak when
there was not any stimulation. However, in the absence
of any stimulation, Nef could increase the contents of
cAMP in corpus cavernosum tissue in a dose-dependent
manner, and MDL-12,330A did not affect this elevation
of cAMP levels. Moreover, Nef dose-dependently increased cAMP levels in corpus cavernosum tissue in the
presence of PGE1 (a stimulator of cAMP production).
This indicated that Nef increased cAMP in the corpus
cavernosum probably by inhibiting PDE activity,
without activating AC.
PDE is a superfamily enzyme system, 11 types of
which have been reported. Some of these isoenzyme
families contain more than one gene (isogenes) and some
genes are alternatively spliced [25_27]. In early 1990s,
using anion exchange chromatography, Stief and co-workers [28] reported the separation of hydrolytic
activities of PDE isoenzymes 3, 4 and 5 from cytosolic
supernatants prepared from human cavernous smooth muscle, whereas others reported the presence of PDE 2,
3 and 5 [29]. Currently, the presence of mRNAs
specific for 14 different human PDE isoenzymes and
isoforms in human cavernous tissue has been shown by
means of reverse transcription polymerase chain
reaction (RT-PCR) and Northern blot analysis. The
expression of the following genes were detected: PDE1A,
PDE1B, PDE1C, PDE2A and PDE10A, which hydrolyze both cAMP and cGMP; the cAMP specific PDEs PDE3A,
PDE4A-D, PDE7A and PDE8A, and the cGMP specific PDEs PDE5A and PDE9A [26, 27]. However, the mere
expression of an enzyme does not yield any information
regarding its functional relevance. PDE3 and 5 are the
predominant isoenzymes in the degradation of cNMP in
human corpus cavernosum musculature [26_32]. Because a PDE isoenzyme can be expressed in several
organs or tissues, there might be side effects associated
with the systemic administration of a PDE inhibitor.
For example, PDE5 is not only expressed in corpus cavernosum smooth muscle, but also in the vascular and
central nervous system and the gastrointestinal tract.
Therefore, the main adverse events of sildenafil include
headache, visual disturbances, flushing and dyspepsia
[33]. Meanwhile, the distributions of PDE isozymes in
the corpus cavernosum vary from species to species. In
rabbit corpus cavernosum, the PDE is PDE5, 2 and 1.
PDE3, which contributes significantly to the total PDE
activity in human corpus cavernosum, is apparently
lacking in rabbit corpus cavernosum [34]. Therefore, it is
important to perform further research to determine
whether Nef enhances cAMP accumulation by inhibiting
PDE1, 2, 3, 4, 7, 8 and/or by inhibiting PDE10 in corpus
cavernosum and other organs or tissues in different
species.
Acknowledgment
The authors thank Prof. Jia-Ling Wang for kindly
supplying the neferine. The technical support from Prof.
Bo-Hua Shu is also greatly appreciated. This study was
sponsored by the National Natural Science Foundation
of China (No. 30471736) and China Postdoctoral Science Foundation (No. 20070410176).
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