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- Original Article -
Relaxation mechanisms of neferine on the rabbit corpus
cavernosum tissue in vitro
Jun Chen1,2,, Jun
Qi1,, Fang Chen1, Ji-Hong
Liu2, Tao Wang2, Jun
Yang2, Chun-Ping Yin3
1Department of Urology, Xinhua Hospital, School of
Medicine, Shanghai Jiaotong University, Shanghai 200092, China
2Department of Urology, 3Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, China
Abstract
Aim: To investigate the relaxation mechanisms of neferine (Nef) on the rabbit corpus cavernosum tissue
in vitro. Methods: Strips of rabbit corpus cavernosum were mounted in organ chambers. The effects of Nef were examined
on isolated muscle strips precontracted with phenylephrine (PE) alone, in the presence of
NW-nitro-L-arginine (LNNA, a nitric oxide synthase inhibitor),
1-H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor),
indomethacin (cyclooxygenase inhibitor), tetraethylammonium
(Ca2+-activated K+ channel
blocker), 4-aminopiridine (4-AP ,voltage dependent
K+ channel blocker) and glibenclamide (ATP sensitive
K+channel blocker). The effects of Nef on KCl-induced contraction of isolated muscle strips were also investigated. The procedure of calcium
absence-calcium addition was designed to observe the effect of Nef on two components of the contractile responses to PE
based on the source of Ca2+ (extracellular
vs. intracellular). Results:
Corpus cavernosum strips relaxed in response to Nef
(10_9_10_4 mol/L) in a concentration-dependent manner with an
IC50 of
4.60 × 10_6 mol/L. However, they
were not affected by LNNA, ODQ, indomethacin or
K+-channel blockers. Nef
(10_6 mol/L, 10_5 mol/L) concentration
dependently reduced the maximal contraction response of isolated strips induced by KCl to 79.3% ± 5.5% and
61.5% ± 3.2%, respectively
(P < 0.01). In the calcium absence_calcium addition procedure, Nef
10-5 mol/L inhibited both
intracellular calcium-dependent and extracellular calcium-dependent contraction induced by PE (2 ×
10-5 mol/L) (P < 0.05).
The inhibition ratios were 26.2% ± 5.4% and
48.3% ± 7.6%, respectively.
Conclusion: The results of the present
study suggest that Nef possesses a relaxant effect on rabbit corpus cavernosum tissues, which is attributable to the
inhibition of extracellular Ca2+ influx and the inhibition of release of intracellular stored
Ca2+, but not mediated by the release of nitric oxide, prostaglandins or by the activation of potassium
channels.(Asian J Androl 2007 Nov; 9:
795_800)
Keywords: neferine; corpus cavernosum; relaxation
Correspondence to: Prof. Fang Chen, Department of Urology, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University,
Shanghai 200092, China.
Tel: +86-21-6515-1555 Fax: +86-21-6579-5173
E-mail: chenfang007@hotmail.com
Prof. Jun Qi, Department of Urology, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China.
Tel: +86-21-6579-0000 ext.7805 Fax: +86-21-6579-5173
E-mail: jasonqi@sh163.net
These two authors contributed equally to this work.
Received 2006-12-27 Accepted 2007-06-04
DOI: 10.1111/j.1745-7262.2007.00321.x
1 Introduction
Erectile dysfunction (ED) is a common problem
affecting approximately 50% of men aged 40_70 years
[1]. Current pharmacological treatment for ED includes
the oral, intracavern
osal and intraurethral
administration of erectogenic drugs. Oral pharmacotherapy is the
most effective therapy for ED with 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 (intracaver-nosal and intraurethral treatments) is a second line
therapy in cases of failure or contraindica-tions for oral
pharmacotherapy [2]. Although many drugs are now available for treating ED, finding a new drug for
treating ED and understanding its mechanism of action is
still important.
Many traditional Chinese medicines are effective as
treatments for ED. Because of the complex chemical
ingredients, it remains unclear which ingredients exactly,
and by which mechanisms, have the chemical effect in
the treatment of ED. Some extracts from traditional
Chinese medicines, of the alkaloids, coumarin and saponin
series, relax the smooth muscle of corpus cavernosum
[3_8], which provides an open window for developing
new drugs for the treatment of ED.
Neferine (Nef) is a bis-benzylisoquinoline alkaloid
extracted (isolated) from the green seed embryo of
Nelumbo nucifera Gaertn, which is effective in preventing the onset of reentrant ventricular
tachyarrhy-thmias [9_10]. Nef can inhibit very low density
lipoprotein oxidation [11] and platelet aggregation [12],
protect vascular endothelial cells from damage induced by
oxygen free radicals [13] and increase sensitivity to
anticancer drugs [14]. 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 [6]. In the present study, the relaxation
mechanisms of Nef on the isolated rabbit corpus cavernosum were investigated.
2 Materials and methods
All animal experiments were carried out with the
approval of the Institute for Animal Care and Use
Committee of Xinhua Hospital (Shanghai, China).
2.1 Materials
Nef was kindly provided by Prof. Jia-Ling Wang (Department of Pharmacology, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan,
China). The purity of Nef was greater than 99.8%. It
was dissolved in HCl 0.1 mol/L, then diluted with Krebs'
solution to the desired concentration. The following
drugs were all obtained from Sigma Chemical Company (St. Louis, MO, USA):
NW-nitro-L-arginine (LNNA, a nitric oxide synthase inhibitor),
1-H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, a guanylyl cyclase
inhibitor), indomethacin (cyclooxygenase inhibitor),
tetraethylammonium (TEA, Ca2+-activated
K+ channel blocker), 4-aminopiridine (4-AP, voltage dependent
K+ channel blocker) and glibenclamide (ATP sensitive
K+channel blocker). Phenylephrine (PE) was from
Shanghai Harvest Pharmaceutical (Shanghai, China).
The Krebs' solution (in mmol/L) consists of: NaCl 118,
KCl 4.7, CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25 and glucose 11 (pH 7.4). In calcium-free
solution, CaCl2 was omitted from the Krebs' solution with the
addition of 0.05 mmol/L egtaic acid (EGTA).
2.2 Tissue procurement
Adult male (4_6 months) New Zealand white
rabbits (2.5_3.0 kg) were killed with pentobarbital sodium
(50 mg/kg). The penis was surgically removed
en bloc with care being taken to keep the tunica albuginea intact in
all animals. The corpus spongiosum and urethra were
excised. The corpus cavernosum tissue was then
carefully dissected free from the surrounding tunica albuginea.
Each rabbit provided two strips of corpus cavernosum
tissue. The strips of corpus cavernosum with dimensions
of approximately 2 × 2 × 6 mm were mounted in 10 mL
Organ-bath chambers containing Krebs buffer solution
(pH 7.4) at 37ºC, and aerated with 95% oxygen and 5%
CO2. The strips were suspended with silk ties to a
force-displacement transducer (ML T0201/D, AD Instruments,
Sydney, Australia). Changes in isometric tension were
measured by Powerlab/4sp (AD Instruments, Sydney, Australia). The strips of corpus cavernosum were
stretched to a resting force of 2.0 g and were
equilibrated for at least 60 min. During this period, the tissue
was washed with fresh solution at 37ºC, for a
consecutive 15 min, and tension was adjusted if necessary.
2.3 Organ bath experiments
2.3.1 Effects of Nef on PE-induced rabbit corpus
cavernosum smooth muscle responses alone, in the
presence of LNNA, ODQ, indomethacin, TEA, 4-AP and
glibenclamide
Strips were precontracted with 10_5 mol/L PE.
After muscle strips were stabilized, relaxation responses
to cumulative concentrations of Nef
(10_9_10_4 mol/L) were assessed. When the influence of antagonists on
the Nef-induced relaxation was evaluated, inhibitors
(LNNA 10_4 mol/L, ODQ
10_4 mol/L, indomethacin
10_4 mol/L, TEA 10_4 mol/L, 4-AP
10_4 mol/L or glibencla-mide
10_5 mol/L) were added to the organ bath 20 min
before addition of PE in different experiments.
2.3.2 Effects of Nef on KCl-induced rabbit corpus
cavernosum smooth muscle responses
The addition of cumulative concentrations of
KCl (2 × 10_2,
4 × 10_2,
6 × 10_2,
8 × 10_2,
10 × 10_2 mol/L)
was performed and concentration response was recorded.
After washout by Krebs solution and reequilibration, each
cavernosal strip was incubated with different
concentrations (10_6 mol/L,
10_5 mol/L) of Nef and equilibrated
for 20 min. Cumulative concentration responses of KCl
were repeatedly recorded.
2.3.3 Effects of Nef on the two contractile elements of
isolated corpus cavernosum smooth muscle strips induced
by PE
In Ca2+-free medium, PE induced rapid transient
contraction, presumably because of release of
intracellular Ca2+; and the sustained contraction when
extracellular Ca2+ was present presumably the result of
Ca2+ influx [15]. After being washed with
Ca2+-free Krebs solution three times and being equilibrated for 20 min,
the strips were contracted by
2 × 10_5 mol/L PE (due
to release of intracellular Ca2+). After a stable higher
tension baseline was established,
2.5 × 10_3 mol/L
CaCl2 was added, strips contracted again and a peak was
reached (as a result of entry of Ca2+). This was
followed by thorough washout by Krebs solution and
refilling of intracellular Ca2+ stores by repeated
stimulation of 6 × 10_2 mol/L KCl in
Ca2+ containing Krebs solution three times. After washing with
Ca2+-free Krebs solution,
10_5 mol/L Nef was added and incubated for
20 min, and then the above-mentioned experiment was
repeated. The effects of Nef on the two contractile
elements of isolated strips induced by PE were observed.
2.3.4 Data analysis
All data were expressed as mean ± SEM. Responses
were expressed as a percentage of active muscle tone
induced by PE or KCl. Statistical analysis was performed
with one-way analysis of variance using SPSS 12.0
software (SPSS Ltd., Chicago, IL, USA).
P < 0.05 was considered a significant difference.
3 Results
3.1 Effects of Nef on PE-induced rabbit corpus
cavernosum smooth muscle responses alone, in the
presence of LNNA, ODQ, indomethacin, TEA, 4-AP and
glibenclamide
Corpus cavernosum strips relaxed in response to Nef
(10_9_10_4 mol/L) in a concentration-dependent manner
with an IC50 of
4.60 × 10_6 mol/L
(n = 8, P < 0.05)
(Figure 1). However, the response of isolated muscle
strips was not affected by nitric oxide synthase inhibitor
(LNNA), guanylyl cyclase inhibitor (ODQ),
cyclooxy-genase inhibitor (indomethacin),
Ca2+-activated K+ channel blockers (TEA), voltage dependent
K+ channel blocker (4-AP) and ATP sensitive
K+channel blocker (glibenclamide)
(n = 8, P > 0.05) (Figure 2).
3.2 Effects of Nef on KCl-induced contraction of
isolated corpus cavernosum smooth muscle strips
Nef (10_6 mol/L,
10_5 mol/L) concentration dependently reduced the maximal contraction response of
isolated strips induced by KCl to 79.3% ± 5.5%
and 61.5% ± 3.2%, respectively
(n = 8, P < 0.01) (Figure 3).
3.3 Effects of Nef on the two contractile elements of
isolated corpus cavernosum smooth muscle strips induced
by phenylephrine
Nef 10_5 mol/L inhibited both intracellular
calcium-dependent and extracellular calcium-dependent
contraction induced by PE
(2 × 10_5 mol/L)
(n = 6, P <
0.05). The inhibition ratios were 26.2% ± 5.4% and
48.3% ± 7.6%, respectively (Figure 4).
4 Discussion
A number of mechanisms have been identified for
the local regulation of penile smooth muscle contractility
and, therefore, penile erection. Molecules participating
in these pathways can be considered targets for the
development of new drugs to treat ED.
Many studies have demonstrated that nitric oxide
(NO) is the most important factor for immediate
relaxation of penile vessels and corpus cavernosum. During
sexual stimulation, NO releases from the non-adrenergic
non-cholinergic nerve terminals and endothelium within
the erectile tissue of the penis. It activates guanylyl
cyclase, resulting in an increased conversion of guanosine
triphosphate to cyclic guanosine monophosphate (cGMP). cGMP in turn activates protein kinase G, which
phosphorylates certain proteins and ion channels. This
process facilitates the reduction of intracellular calcium,
enabling the smooth muscle to relax, resulting in
erection [16, 17]. To investigate whether relaxation induced
by Nef was attributable to an interaction with NO-cGMP
pathway, corpus cavernosum tissues were pretreated with
LNNA (a nitric oxide synthase inhibitor) and ODQ (a
guanylyl cyclase inhibitor), respectively. The treatment
of corpus cavernosum tissues with these inhibitors did
not significantly affect the relaxant activity of Nef. These
findings indicate that the relaxant action of Nef on the
rabbit corpus cavernosum is not mediated by NO-cGMP
pathway.
Prostaglandin (especially PGI2 and
PGE1) is derived from arachidonic acid by the action of cyclooxygenases.
Prostaglandin inhibits the release of noradrenaline from
penile adrenergic nerves and increases intracellular cAMP
levels in corpus cavernosum smooth muscle [18, 19].
This process promotes muscle relaxation through
intracellular reduction of calcium concentration, resulting in
erection. Therefore, the possibility that relaxation induced
by Nef was a result of an interaction with prostaglandins
was further investigated. However, the treatment of
corpus cavernosum tissues with cyclooxygenase inhibitor
(indomethacin) did not affect the relaxant activity of Nef.
Therefore, the corpus cavernosum tissues relaxation
effect of Nef was not related to prostaglandins.
Potassium channels (particularly
Ca2+-activated K+ channel, voltage dependent
K+ channel, and ATP sensitive
K+channel) play an important role in the modulation
of corpus cavernosum smooth muscle cell tone.
Potassium channel opening is be expected to cause
hyperpo-larzation, inhibit Ca2+ channel activity, lower intracellular
Ca2+ concentration and result in relaxation [20_23].
However, in the present experiment, we found that the
treatment of corpus cavernosum tissues with TEA
(Ca2+-activated K+ channel
blocker), 4-AP (voltage dependent
K+ channel blocker) and glibenclamide (ATP sensitive
K+channel blocker) did not change the relaxant activity
of Nef. These findings suggest that the relaxant effect of
Nef is unrelated to Ca2+-activated, voltage dependent and
ATP sensitive K+ channels.
It is understood that the contractile activity of corpus
cavernosum smooth muscles is regulated by the
concentration of free Ca2+ in the cytosol, which depends on
voltage-dependent Ca2+ influx, receptor-operated
Ca2+ influx, and release of intracellularly stored
Ca2+ [24]. High extracellular
K+ induced the membrane depolarization rapidly,
which opened the voltage-dependent Ca2+ channel and
brought about the influx of the extracellular
Ca2+. PE elicited
[Ca2+]i elevation by: (i) activating
α1-adrenoceptor-operated
Ca2+ channel to induce extracellular
Ca2+ entry; and (ii) activating G protein and then facilitating the
formation of IP3, which acts on IP3 receptor and induces
intracellular Ca2+ release. In the present study, the
contraction of corpus cavernosum tissues induced by PE or
KCl was reduced by Nef. Furthermore, Nef also concentration-dependently relaxed PE-induced contraction
of isolated muscle strips in a calcium-free solution. The
results suggest that Nef relax corpus cavernosum
tissues by inhibiting both extracellular
Ca2+ influx and release of intracellular stored
Ca2+.
In conclusion, the results of the present study
suggest that Nef possesses a relaxant effect on rabbit
corpus cavernosum tissues, which is attributable to the
inhibition of extracellular Ca2+ influx and the inhibition of
release of intracellular stored Ca2+, but is not mediated
by the release of nitric oxide, prostaglandins or by the
activation of potassium channels. Further experiments
are required to establish whether cAMP signal
transduction pathway, Rho-A/Rho-kinase pathway and cell-cell
communication contributes to Nef induced relaxation or
not. Because in the experiment on muscle strips
in vitro, the influence of arterial inflow, venous outflow,
autonomic innervation and a multitude of local and total body
factors were negligible, further pharmacological research
and toxicology tests are needed in vivo to clarify whether
Nef can be developed into a drug for ED.
Acknowledgment
The authors thank Prof. Jia-Ling Wang for kindly
supplying the Nef. The technical assistance from Mr Jun
Pan (Department of Urology, Xinhua Hospital, School
of Medicine, Shanghai Jiaotong University, Shanghai,
China) is also greatly appreciated. This study was
sponsored in part by the National Natural Science Foundation
of China (No. 30471736).
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