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“Mecanismo envolvido no potencial terapêutico da via
óxido nítrico-guanilato ciclase solúvel – GMP cíclico”
Dúvidas
[email protected]
Site
www.gdenucci.com
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
The NO–sGC–cGMP signal transduction pathway and potential drug targets.
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
NO–sGC–cGMP signalling in a blood vessel.
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
Main haem-dependent stimulators of soluble guanylate cyclase
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
Main haem-dependent stimulators of soluble guanylate cyclase
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
Main haem-independent activators of soluble guanylate cyclase
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
Homology model of the haem-binding domain of the human
soluble guanylate cyclase (sGC) β-subunit.
NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential
NATURE REVIEWS | DRUG DISCOVERY VOLUME 5 | SEPTEMBER 2006 | 755
Soluble guanylate cyclase (sGC) redox equilibrium.
Introduction
Bladder Function
Filling phase – the storage of urine occurs with a low pressure which implies that detrusor relaxes during this
phase. Disturbances of the storage function may result symptoms, such as urgency, frequency, and urge
incontinence, components of overactive bladder syndrome.
Abrams P. et al. 2002.
Emptying phase – Detrusor contraction and urethral relaxation provoke the elimination of urine.
Chu F., Dmochowski R. 2006.
Nitric oxide and overactive bladder
Overactive bladder (OAB) is a complex of symptoms defined by the International Continence Society
(ICS) as urinary urgency, with or without urge incontinence, usually with frequency and nocturia. It
reflects involuntary detrusor smooth muscle (DSM) contractions as a consequence of enhanced
cholinergic stimulation. The nitric oxide (NO)-cyclic GMP signaling pathway has been described to
modulate the muscular tone, neurotransmission and blood flow in DSM, but the exact role of NO in the
bladder is not completely understood. Some evidencies suggest that NO deficiency contributes to
triggering OAB.
• Intravesical and intra-arterial administration of NOS inhibitor L-NMMA in anesthetized cats decreased
the micturition volume threshold.
•Theoblad RJ. 1996.
• L-NAME (NOS inhibitor) intra-arterially administered increased the spontaneous bladder contractions
in concious rats.
Persson K., et al. 1992.
• In hypertrophied rat bladder a decrease of calcium-dependent nNOS activity has been demonstrated
Johansson R., et al. 2002.
• Intravesical administration of an NO scavenger oxyhaemoglobin produces bladder hyperactivity in
normal rats.
Pandita R.K., et al. 2002.
BAY 41-2272
BAY 41-2272 potently stimulates soluble guanylate cyclase (sGC)
through cysteine 238 and cysteine 243 region in the α1-subunit of by a
mechanism independent of NO.
Stasch J.P. et al. 2001.
BAY 41-2272 produces relaxation of ovine pulmonary artery through the
activation of sGC as well as cGMP-independent stimulation of
sarcolemmal sodium pump.
Bawankadule D.U. et al. 2005.
BAY 41-2272 causes basilar artery relaxation through the stimulation of sGC,
as well as via inhibition of Ca2+ entry in the rat basilar artery.
Teixiera C.E. et al. 2006.
BAY 41-2272 produces a potent relaxation in rabbit and human corpus cavernosum that is
partially inhibited by ODQ.
Baracat J.S. et al. 2003.
Objectives
The aim of this study was to evaluate the relaxant effect of
BAY 41-2272 in the rabbit isolated bladder and the
mechanisms underlying these responses.
• To compare the relaxant effects evoked by BAY 41-2272, sodium
nitroprusside (SNP), glyceryl trinitrate (GTN) and acidified NaNO2.
• To evaluate the effect of L-NAME (non-selective NO synthase inhibitor),
ODQ (soluble guanylate cyclase inhibitor) and sildenafil (phosphodiesterase
type-5 inhibitor) in the BAY 41-2272-induced responses.
Methods
•
New Zealand male rabbits 2-3 kg were killed by overdose of urethane (1.2 g/kg i.v.).
•
The abdomen was opened in the midline, and the bladder body was carefully removed.
•
The muscle strips (2 x 4 x 10 mm) were placed in Krebs solution of the following composition (mM): NaCl,
118; NaHCO3, 5.6; KCl, 4.7; KH2PO4, 1.2; MgSO4.7 H2O, 1.17 and CaCl2.2 H2O, 2.5.
•
Each muscle was mounted in 10-ml organ baths containing Krebs solution at 37ºC continuosly gassed with a
mixture of 95% O2 + 5% CO2 (pH 7.4) under tension 20 mN.
•
Isometric tension was recorded by force tranducers connected to a PowerLab 400 TM data acquisition system
(Software Chart 5.0, AD Intruments, MA, USA).
•
After 1 hour stabilization, viability of the bladder smooth muscle was confirmed following addition of high KCl
solution (80 mM).
•
Concentration-response curves were constructed by adding BAY 41-2272 (0.001-10 µM), SNP (0.01-10 µM)
GTN (0.01-10 µM) and acidified NaNO2 (0.01-100 µM).
•
BAY 41-2272-induced relaxations were evaluated in the absence and in the presence of ODQ (10 µM), LNAME (100 µM) or sildenafil (100 nM).
BAY 41-2272, but not SNP, GTN or acidified NaNO2, potently
relaxes the rabbit bladder
7.5
6.62 ± 0.14
6.38 ± 0.18
5.94 ± 0.16
pEC50
50
5.51 ± 0.51
5.0
2.5
0.0
BAY 41-2272
SNP
GTN
ac. NaNO2
100
150
-9
-8
-7
-6
log [drug]: M
-5
-4
Emax (% Relaxation)
Relaxation (%)
0
150
BAY 41-2272
SNP
GTN
NANO2
131.61 ± 4.52
100
*
47.41 ± 3.39
*
41.07 ± 3.57
50
0
BAY 41-2272
SNP
GTN
*
49.80 ± 4.54
NaNO2
Figure 1. Relaxant effects of BAY 41-2272, sodium nitroprusside (SNP), glyceryl trinitrate
(GTN) and acidified NaNO2 in the rabbit isolated detrusor smooth muscle (n=5). Data are
the mean ± S.E.M. * p<0,001.
BAY 41-2272-induced responses are not affected by
L-NAME, ODQ and sildenafil
pEC 50
7.5
50
6.69 ± 0.14
6.53 ± 0.10
5.99 ± 0.48
6,75 ± 0,08
Control
ODQ
L-NAME
Sildenafil
5.0
2.5
Control
ODQ
L-NAME
100
0.0
Sildenafil
150
-9
-8
-7
-6
log [BAY 41-2272]: M
-5
Emax (% Relaxation)
Relaxation (%)
0
150
131,63 ± 4,52
127,33 ± 6,90
121,80 ± 6,78
122,54 ± 3,89
100
50
0
Control
ODQ
L-NAME
Sildenafil
Figure 2. Lack of effect of L-NAME, ODQ or sildenafil on BAY 41-2272-induced
responses in the rabbit isolated detrusor smooth muscle (n=5). Data are the Mean ±
S.E.M.
Conclusion
In contrast to NO and NO donors, BAY 41-2272 causes a complete relaxation of
rabbit DSM. Our findings that the relaxant responses to BAY
41-2272 were not
altered by L-NAME, ODQ or sildenafil indicate that BAY 41-2272 acts through a
cGMP-independent mechanism to produce DSM relaxations. Therefore, the present
findings suggest that BAY 41-2272 represents a drug with therapeutic potential to
treat overactive bladder.
Effects of BAY 41-2272 on smooth muscle tone, soluble guanylyl
cyclase activity and NADPH oxidase activity/expression in
corpus cavernosum from wild-type, neuronal and endothelial
NOS null mice.
Cleber E. Teixeira, Fernanda B. M. Priviero & R. Clinton Webb
Department of Pharmacology,
Faculty of Medical Sciences,
UNICAMP, Campinas, SP, Brazil,
13087-971 (CET)
Department of Physiology, Medical
College of Georgia
Augusta, GA, 30912-3000, U.S.A.
(FBMP, RCW)
e-mail: [email protected]
Introduction
The discovery that the NO-sGC-cGMP system is one of the major effectors of cavernosal smooth
muscle relaxation and penile erection has led to the development of two classes of agents: (a)
compounds that inhibit cGMP degradation like the PDE inhibitors, and (b) compounds that elevate
cGMP levels through potentiation of cGMP synthesis. In the latter context, the main rationale for
these new therapeutic principles is based on both insufficient NO-sGC-cGMP signaling, often
associated with the use of conventional NO donors and the medical need to treat conditions
associated with oxidative stress. The bensylindazol derivate YC-1 (3(5’-hydroxymethyl-2’-furyl-1benzyl indazole) was first reported to increase intracellular cGMP content in platelets (Ko et al.,
1994) and was later shown to stimulate soluble guanylate cyclase independent of nitric oxide to
cause erectile responses (Mizusawa et al., 2002; Brioni et al., 2002). In animal models, the sGC
stimulator BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3yl]pyrimidin-4ylamine) (Stasch et al., 2001) has been confirmed to be more potent than YC-1 and to
elicit erectile responses in a conscious rabbit model of penile erection (Bischoff et al., 2003) as well
as to cause human and rabbit corpus cavernosum relaxation (Baracat et al., 2003).
Objectives
In this investigation, we examined the in vitro effects of
BAY 41-2272 in corpus cavernosum from mice with
targeted deletions of NOS isoforms (nNOS-/- or eNOS-/-), in
order to understand the contribution of each isoform to the
proerectile action of BAY 41-2272. Firstly, we studied the
effects resulting from modulation of the NO-signaling
cascade on responses evoked by BAY 41-2272 as well as
the sensitizing effects of this compound on both
endogenous and exogenous NO-mediated cavernosal
relaxation. Secondly, we investigated sGC expression and
BAY 41-2272-induced enzyme activity in cavernosal tissue
from these animals. Thirdly, we studied the effects of BAY
41-2272 on both O2- formation and NADPH oxidase
expression in cavernosal tissue treated with the
thromboxane A2 analogue, U44619.
N
H 2N
N
F
N
N
Structure of BAY 41-2272
N
Methods
Corpus cavernosum preparation. The penises were surgically removed and placed in chilled
Krebs-Henseleit buffer of the following composition (mM): NaCl, 130; NaHCO3, 14.9; dextrose,
5.5; KCl, 4.7; KH2PO4, 1.18; MgSO47H2O, 1.17 and CaCl22H2O, 1.6. Following removal of the
glans penis and urethra, the corpora cavernosa was opened from its proximal extremity towards the
penile shaft to obtain two strips (11 x 1 x 1 mm) of corpus cavernosum (CC) from each animal.
Each strip was mounted in a myograph for isometric force recording (Danish Myograph
Technology, Aarhus, Denmark) coupled to a PowerLab 8/SP™ data acquisition system (software
Chart 5.0, ADInstruments, Colorado Springs, U.S.A.). The bathing solution was maintained at 37oC
and continuously aerated with 95% O2 and 5% CO2. Tissues were allowed to equilibrate for 45 min
under a resting tension of 2 mN.
Determination of cavernosal cGMP levels. Cavernosal strips were equilibrated for 20 min in
warmed and oxygenated Krebs solution and then stimulated for 10 min with BAY 41-2272 (0.01-1
M), SNP (1 M), or their combination. Preparations were collected immediately by freezing the
segments in liquid nitrogen. Cyclic GMP was extracted and quantified using commercially available
kits (Cayman Chemical Cyclic GMP EIA kit, Ann Arbor, MI, U.S.A.) as previously described
(Teixeira et al., 2005).
Methods
Soluble guanylyl cyclase activity. Determined in the supernatant fractions of the tissue samples by
the conversion of GTP to cGMP. Briefly, 30 µg each protein sample were incubated for 10 min at
37°C in a total volume of 100 µl containing the following: 50 mM Tris-HCl (pH 7.4), 1 mM 3isobutyl-1-methylxantine, 3 mM MgCl2, 0.5 mM GTP, 3 mM DTT, 5 mM phosphocreatine and 0.25
mg/ml creatine kinase. The stimulation of enzyme activity was measured in the presence of BAY
41-2272 (0.001-1 M). The reaction was terminated by inactivation of sGC at 95°C for 10 min.
Effects of BAY 41-2272 on superoxide (O2-) formation. To assess the effect of U46619 in the
absence or in the presence of BAY 41-2272 on O2- formation, cavernosal segments were incubated
with U46619 for 1 or 8 h at 37oC in a 95% air-5% CO2 incubator. Tissues were then equilibrated in
DMEM with no phenol red for 10 min at 37oC in the incubator. Next, 20 M horseradish
cytochrome c, with or without 500 U/ml copper-zinc superoxide dismutase (SOD), was added and
incubated at 37oC in the incubator for 1 h. The reaction medium was removed and reduction of
cytochrome c determined at 550 nm and converted to mol O2-, using a DE550nm of 21.1 mmol-1cm-1
as the extinction coefficient. The reduction of cytochrome c that was inhibitable with SOD reflected
actual O2- release.
Methods
Semi-quantitative RT-PCR. An amount of 0.5 µg of total RNA was reverse-transcribed and
cDNAs were subsequently amplified in Ready-to-Go RT-PCR beads in a two-step procedure.
Primer sequences for the sGC1 gene (Genbank accession nr. AF297082) were: forward, 5’GGTCACCATGTGTGGACAGG-3’; reverse, 5’-CCAGCTCTCCACACTGCTGG-3’. Primer
sequences for the sGC1 gene (Genbank accession nr. AF020339) were: forward, 5’GCATGCATCTGGAGAAGGG-3’; reverse, 5’-CCGAGGCATCCGCTGTCC-3’. The primers for
glyceraldehyde-3-phosphate dehydrogenase, used as an internal control, (GAPDH; Genbank
accession nr. AF106860) were: forward, 5’-GGCTGCCTTCTCTTGTGACAA-3’; reverse, 5’CGCTCCTGGAGGATGGTGAT-3’.
Western blot analysis. An aliquot of 40 g protein from each sample was loaded per lane and
resolved by SDS-PAGE. Membranes were probed with antibodies against sGC1 (1:100), sGC1
(1:200), gp91phox (1:500), p47phox (1:500), p22phox (1:500) or p67phox (1:500) and incubated with a
horseradish peroxidase-conjugated second antibody. Immunoreactivity was detected by enhanced
chemiluminescence autoradiography. Results were then expressed as the densitometric ratio of
protein of interest/-actin (1:2000).
Cyclic GMP content of mouse corpus cavernosum from wild-type (WT), nNOS-/- and eNOS-/-
cGMP levels (pmol/mg protein)
WT
nNOS-/-
eNOS-/-
vehicle
0.14 ± 0.04
0.17 ± 0.03
0.04 ± 0.03
BAY 0.01 M
0.96 ± 0.15**
1.19 ± 0.19**
0.24 ± 0.10*
BAY 0.1 M
1.22 ± 0.45**
1.44 ± 0.56**
0.49 ± 0.13**
BAY 1 M
3.87 ± 0.53**
4.28 ± 0.77**
0.96 ± 0.31**
SNP 1 M
0.95 ± 0.28**
0.88 ± 0.23**
0.30 ± 0.09**
BAY 1 M + SNP
10.52 ± 1.61**,#
8.50 ± 1.18**,#
4.53 ± 0.78**,#
BAY 1 M + ODQ
0.44 ± 0.09*
0.67 ± 0.11*
0.13 ± 0.05
*p<0.05 and **p<0.01 compared to respective vehicles; #p<0.05 compared to the sum of BAY 41-2272 plus SNP values alone.
Relaxing activity of BAY 41-2272 in corpus cavernosum from WT, nNOS-/- and eNOS-/- mice
a
b
Relaxation (%)
0
30
**
60
WT (6.36+0.07)
90
nNOS-/- (6.27+0.06)
eNOS-/- (5.88+0.07)
-8
-7
-6
-5
Log [BAY 41-2272] (M)
Concentration-response curves to BAY 41-2272 (n=16) in corpus cavernosum strips from WT (closed circles), nNOS-/- (open circles)
and eNOS-/- (closed squares) mice contracted by PE. Experimental values were calculated relative to the maximal changes from the
contraction produced by PE in each tissue, which was taken as 100%. The corresponding pEC50 values are represented in the panel.
Data represent the mean ± S.E.M. of n experiments. **p<0.01 compared to curves elicited in WT and nNOS-/- strips.
Effects of ODQ and L-NAME on BAY 41-2272-induced relaxations
30
**
60
**
60
90
WT (6.34+0.09)
ODQ (5.61+0.11)
-8
30
-7
-6
-5
nNOS-/- (6.43+0.07)
ODQ (5.81+0.08)
-8
Log [BAY 41-2272] (M)
e
0
30
**
60
90
-7
-6
Log [BAY 41-2272] (M)
-6
*
60
-5
eNOS-/- (5.98+0.06)
ODQ (5.58+0.09)
-8
-5
f
30
**
60
nNOS-/- (6.43+0.05)
L-NAME (5.95+0.07)
-8
-7
-6
Log [BAY 41-2272] (M)
-7
-6
-5
Log [BAY 41-2272] (M)
0
90
WT (6.43+0.05)
L-NAME (5.99+0.06)
-8
30
Log [BAY 41-2272] (M)
Relaxation (%)
Relaxation (%)
d
-7
0
90
Relaxation (%)
90
c
0
Relaxation (%)
b
0
Relaxation (%)
Relaxation (%)
a
0
30
60
90
-5
eNOS-/- (5.88+0.08)
L-NAME (5.87+0.08)
-8
-7
-6
-5
Log [BAY 41-2272] (M)
Experimental values were obtained in the absence (closed circles) and in the presence (open circles) of ODQ (10 µM) or L-NAME (100
µM). *p<0.05 and **p<0.01 compared to values in the absence of inhibitor.
BAY 41-2272 enhances magnitude and duration of nitrergic responses
a
b
60
CTL (WT)
BAY 0.01 µM
BAY400.03 µM
BAY 0.1 µM
Duration (s)
Relaxation (%)
0
30
60
20
**
**
**
**
**
**
1
2
**
**
0
90
1
2
4
8
16
4
8
16
EFS (Hz)
EFS (Hz)
d
DuratiDuration (s) on (s)
c
0
Relaxation (%)
**
CTL (WT)
BAY 0.01 µM
BAY 0.03 µM
BAY 0.1 µM
60
CTL (eNOS -/-)
BAY 0.01 µM
BAY400.03 µM
BAY 0.1 µM
30
60
90
20
**
**
**
**
*
**
**
**
0
1
2
4
EFS (Hz)
8
16
1
2
4
EFS (Hz)
8
16
CTL (eNOS -/-)
BAY 0.01 µM
BAY 0.03 µM
BAY 0.1 µM
Effects
of
increasing
concentrations (0.01-0.1 M;
n=6) of BAY 41-2272 on the
magnitude and duration of
nitrergic relaxations evoked by
electrical field stimulation (EFS,
1-16 Hz) in mouse cavernosal
strips from WT (panels a and b)
and eNOS-/- (panels c and d) mice
contracted with phenylephrine
(PE, 10 M). Nitrergic duration
was determined as the time
elapsed from 50% relaxation to
50% recovery. Data represent the
mean ± S.E.M. of n experiments.
*p<0.05 and **p<0.01 compared
to the respective control (CTL)
values.
BAY 41-2272 enhances magnitude and duration of nitrergic responses
Representative tracing of the effects of BAY 41-2272 (0.1 M) on nitrergic relaxations of a
cavernosal strip from WT animals (4 Hz, 30-s trains). The preparation was stimulated at 30 s
intervals for 30 s. Muscle tone was raised with PE (10 M in the absence and 50 M in the
presence of BAY 41-2272).
Stimulation of cavernosal sGC activity by BAY 41-2272
sGC activity
(pmol/mg/min)
50
40
30
WT
nNOS-/eNOS-/eNOS-/nNOS-/-
20
10
0
-10
Baseline
-9
-8
-7
-6
Log [BAY 41-2272] (M)
Formation of cyclic GMP in the presence of cytosolic protein extracts of corpus cavernosum obtained from WT (closed circles),
nNOS-/- (open circles) and eNOS-/- (closed squares) mice in the absence or presence of increasing concentrations (0.001-1 M;
n=3) of BAY 41-2272. GTP (0.5 mM) was incubated in the presence of cytosolic extract (30 g protein) at 37oC (see Methods),
and cyclic nucleotide content (expressed in M) was determined thereafter. Data represent the mean ± S.E.M. of n experiments.
sGC expression in corpus cavernosum from WT, nNOS-/- and eNOS-/- mice
b
Densitometric ratio of
sGC/GAPDH
a
1.6
WT
eNOS-/eNOS-/nNOS-/nNOS-/-
1.2
0.8
0.4
0.0
a1
1
c
b1
1
d
Densitometric ratio of
sGC/ -actin
0.6
WT
eNOS-/eNOS-/nNOS-/nNOS-/-
0.4
0.2
0.0
a1
1
b1
1
Messenger RNA (panels a and b; n=6) and Western blot (panels c and d; n=6) analyses of sGC 1 and 1 subunits in corpus
cavernosum from WT, nNOS-/- and eNOS-/- mice. Expression of mRNA was analyzed by semi-quantitative RT-PCR and the
products were normalized to the GAPDH content of the samples. The immunoblots were detected with an Advance
Chemiluminescence Detection Kit and the sGC proteins normalized to the -actin content of the samples.
Inhibition of superoxide production by BAY 41-2272
10
µmol O 2 -/mg protein/h
a
#†
WT
nNOS -/-/eNOS
-/eNOS-/-
†#
†
#
**
*
**
5
*#
*#
*#
0
CTL CTL BAY BAY BAY db/db -/CTL CTL BAY BAY BAY
0.1
1 +ODQ
0.1
1 +ODQ
U46619 (10 nM)
21
µmol O 2 -/mg protein/h
b
14
CTL CTL BAY BAY BAY
0.1
1 +ODQ
U46619 (10 nM)
U46619 (10 nM)
††
##
**
nNOS-/eNOS-/-
††
##
††
##
**
**
#
##
#
##
7
##
0
CTL CTL BAY BAY BAY db/db -/CTL CTL BAY BAY BAY
0.1
1 +ODQ
0.1
1 +ODQ
U46619 (10 nM)
U46619 (10 nM)
CTL CTL BAY BAY BAY
0.1
1 +ODQ
U46619 (10 nM)
Inhibitory effect of BAY 41-2272 (0.1-1
M; n=4) on superoxide formation elicited
by incubation of cavernosal strips of WT
(closed bars), nNOS-/- (hatched bars) and
eNOS-/- (open bars) with U46619 (10 nM)
over 1 h (panel a) and 8 h (panel b). Data
represent the mean  S.E.M. of n
experiments. *p<0.05 and **p<0.01
compared to CTL values in the absence of
U46619; #p<0.05 and ##p<0.01 compared to
CTL values in the presence of U46619;
†p<0.05 and ††p<0.01 compared to BAY 1
M.
BAY 41-2272 inhibits NADPH oxidase expression in the corpus cavernosum
b
Densitometric ratio of
p22phox/ -actin
a
1.00
##
0.75
0.50
**
0.25
**
0.00
CTL
d
1 µM
1 µM
+ODQ
BAY 41-2272
Densitometric ratio of
phox
gp91 / -actin
c
0.1 µM
#
##
1.00
0.75
*
0.50
0.25
**
0.00
CTL
0.1 µM
1 µM
1 µM
+ODQ
BAY 41-2272
Inhibitory effect of BAY
41-2272 (0.1-1 M;
n=4) on the expression
of p22phox and gp91phox
in corpus cavernosum
from WT mice incubated
with U46619 (10 nM)
for 8 h. Lanes represent
U46619 alone (lane 1;
open bars) and in
combination with BAY
41-2272 0.1 M (lane 2;
grey bars), 1 M (lane 3;
closed bars) and 1 M
plus ODQ (10 M; lane
4;
hatched
bars).
*p<0.05 and **p<0.01
compared
to
CTL
#
values;
p<0.05 and
##p<0.01 compared to
BAY values alone.
Conclusions
The present study demonstrated that both expression and activity of sGC are not altered in corpus
cavernosum from mice with targeted deletions of either nNOS or eNOS. BAY 41-2272 causes
corpus cavernosum relaxation in a synergistic fashion with endogenous or exogenous NO, with
concomitant increases in intracellular cGMP levels. Our results also demonstrate that in the course
of stimulation of sGC, BAY 41-2272 has a potent inhibitory effect on O2- formation through a
reduction of both NADPH oxidase expression and activity. Taken together, such properties render
BAY 41-2272 as a promising therapeutic drug to treat erectile dysfunction, particularly in the event
of an increased intra-penile oxidative stress.
References
Brioni et al. (2002) Int J Impot Res 14: 8-14.
Baracat et al. (2003) Eur J Pharmacol 477: 163-169.
Bischoff et al. (2003) Urology 61: 464-467.
Ko et al. (1994) Blood 84: 4226-4233.
Mizusawa et al. (2002) J Urol 167: 2276-2281.
Stasch et al. (2001) Nature 410: 212-215.
Financial Support:
National Institutes of Health
(HL-71138, HL-74167)
Introdução
Introdução
Disfunção Erétil
(DE)
incapacidade
persistente
de
alcançar e manter a ereção
peniana adequada para uma
atividade sexual normal.
Aumenta significativamente com a idade
Prevalência da DE
(Feldman, 1994)
Atinge 39% dos homens a partir de 40 anos
Atinge 67% dos homens a partir de 70 anos
Ganz , 2005
Introdução
Classificação da DE masculina
(Comitê da Sociedade Internacional de Pesquisa em Impotência)
Orgânica
1. Vasculogênica
A. Arteriogênica
B. Cavernosa
C. Associada
2. Neurogênica
3. Anatômica
Psicogênica
1. Generalizada
A. dessensibilização
B. Inibição
2. Situacional
A. Relacionada ao parceiro
B. Relacionada à performance
C. Desajuste psicológico
(p.e. depressão)
4. Endocrinológica
Lisa & Rosen, 1999
Dean & Lue, 2006
Introdução
Fatores de risco envolvidos na disfunção erétil orgânica
ENVELHECIMENTO
HIPERTENSÃO
TABAGISMO
HIPERCOLESTEROLEMIA
DIABETES
Aterosclerose
Estenose arterial
Fibrose
Função endotelial e
nervosa reduzidas
INSUFICIÊNCIA ARTERIAL E ALTERAÇÕES NOS
MECANISMOS DE RELAXAMENTO DO MÚSCULO LISO
Andersson, 2003
Introdução
CONTROLE PERIFÉRICO
Grau de contração do
músculo liso cavernoso
FLACIDEZ
EREÇÃO
Equilíbrio
Fatores
Contráteis
Fatores
Relaxantes
Plexo
Pélvico
Controle
Central e Períférico
Burnett, 1992
Andersson, 2001
Rampin, 2004
Dean & Lue, 2006
Introdução
Plexo venoso
subtunical
HEMODINÂMICA E
MECANISMO DA
EREÇÃO PENIANA
Ereto
Veias
emissárias
Espaços
sinusoidais
Flácido
Veia dorsal
Túnica albugínea
Artérias helicinais
Músculo liso trabecular
Espaços sinusoidais
Plexo venoso subtunical
Corpos cavernosos
Artéria cavernosa
Corpo cavernoso humano
Saenz de Tejada et al., 1985;
Lue & Tanagho, 1987;
Introdução
Moduladores e transmissores do relaxamento da célula do
músculo liso
 Óxido nítrico (NO)
 Acetilcolina (ACh)
 Neuropeptídeos (VIP, CGRP)
 Prostanóides (PGE1, PGI2)
 ATP e adenosina
Dean & Lue, 2006
Lue et al., 1983;
Saenz de Tejada et al., 1988;
Hedlund et al.,1999; 2000a,b;
Andersson, 2003.
Introdução
PRINCIPAL VIA PARA A EREÇÃO
ESTÍMULO
SEXUAL
Fibras NANC
Endotélio cavernoso
nNOS
eNOS
sildenafil
tadalafil
vardenafil
NO
GCs
Músculo liso
vascular/
cavernoso
GTP
GMPc
PDE5
5’GMP
relaxamento
Rapoport & Murad, 1983;
Lucas et al., 2000
Introdução
Biossíntese do NO
nNOS
eNOS
NOS
HN
O
HO N
NH2
HN
NH2
NADPH
NH2
1/2 NADPH
HN
HN
O2
H2N
O2
H2O
L-Arg
NO
.
H2O
H2N
H2N
COOH
+
COOH
Nw-hydroxy-L-Arg
COOH
L-Cit
Marletta, 1988, 1993;
Feldman et al., 1993;
Fukuto & Chaudhuri, 1995;
Ignarro & Murad, 1995;
Kerwin et al., 1995;
Korth et al., 1995.
Introdução
Guanilil Ciclase Solúvel
Guanilil Ciclase Solúvel
(GCs)
• receptor mais
molécula de NO
• presente
mamíferos
no
importante
citoplasma
da
dos
• inibição da agregação plaquetária
• relaxamento de músculo liso
• vasodilatação
• transdução de sinal neuronal
• imunomodulação
Collier and Vallance, 1989
Lucas et al., 2000
Introdução
Fe2+ : reduzida
Fe3+: oxidada
HIS105: mutação produz enzima não
responsiva ao NO
Guanilil Ciclase Solúvel
Necessário para a
ativação da enzima
pelo NO
Craven & DeRubertis, 1978
α

Subunidades
necessárias para
atividade catalítica
Kamisaki et al., 1986
Complexo
HEME FERROSO-NITROSIL
forma plana  forma piramidal
Hille et al., 1979
Stone et al., 1995
Introdução
BAY 41-2272
•
Molécula introduzida na literatura em 2001, ativador específico de GCs, mais
potente que seu precursor YC-1 e desprovida de atividade inibitória
sobre
a
fosfodiesterase;
(Stasch et al., 2001; 2002a)
• Composto que se liga a GCs em um sítio alostérico da enzima, na região das cisteínas
Cys238 e Cys243 na subunidade 1;
(Martin et al., 2001; Becker et al., 2001; Stasch et al., 2001)
• Tem ação potente sobre a GCs in vitro, potencializada na presença de doador de
NO;
(Stasch et al., 2001)
• Possui propriedade heme-dependente por não ativar enzima desprovida deste grupamento;
(Stasch et al., 2001)
Introdução
BAY 41-2272
• BAY 41-2272 induz vasodilatação em anéis aórticos in vitro e reduz a pressão
arterial in ratos normotensos e hipertensos;
(Straub et al., 2002)
• Vasodilatação induzida por este composto não causa tolerância como os
orgânicos.
(Stasch et al., 2002)
nitratos
• BAY 41-2272 aumenta o débito cardíaco e preserva a função renal em
cardíaca congestiva experimental;
insuficiência
(Boerrigter et al., 2003)
• BAY 41-2272 relaxa anéis aórticos de coelho tanto na presença quanto na
endotélio;
ausência de
(Priviero et al., 2005)
• BAY 41-2272 interage com NO endógeno e exógeno causando um potente
do músculo anococcígeo de rato;
(Teixeira et al., 2006)
relaxamento
Objetivo Geral
Objetivo Geral
Avaliar a capacidade do BAY 41-2272 de induzir o relaxamento de corpo
cavernoso e investigar os mecanismos envolvidos neste fenômeno, em
coelho, rato e humano.
Objetivos Específicos
Objetivos específicos
•
Investigar a participação das vias guanilil ciclase-GMPc e adenilil
ciclase-AMPc, bem como dos canais de potássio, nas respostas
relaxantes de corpo cavernoso induzidas pelo BAY 41-2272;
• Investigar a capacidade do BAY 41-2272 em produzir ereção peniana em
modelo experimental in vivo
Materiais e Métodos
Experimentos in vitro
Materiais e Métodos
Preparação de corpo cavernoso
• humano: segmentos proximais de corpo cavernoso de 11 pacientes entre 16 e 55
anos submetidos a doação múltipla de órgãos após consentimento apropriado. (protocolo
CEP - HC UNICAMP 281/2001)
• coelho: New Zealand ( 2 -3 kg) anestesiados com pentobarbital sódico
(Hypnol®, 40 mg/kg, i.v) submetidos à penectomia.
(protocolo CEEA_IB UNICAMP 547-1)
• rato: Wistar (250-350 g) anestesiados com halotano exsangüinados pela carótida e
submetidos à penectomia. (protocolo CEEA_IB UNICAMP 547-1)
• tecidos colocados em solução de Krebs seguido da remoção da túnica albugínea e
tecidos conectivos
Materiais e Métodos
Registro da tensão isométrica
• Strips submetidos a tensão de 5, 10 e 20 mN
para
rato,
coelho
e
humano
respectivamente, em banho para órgão
isolado
• Tensão
isométrica (Sistema de Aquisição de
Dados PowerLab 400 ™; software Chart,
version 4.0, AD Instruments,
MA, USA);
• Tempo de estabilização: 60 minutos;
O2`- CO2
Materiais e Métodos
Protocolos Experimentais
• Adição de fenilefrina (10 µM) : viabilidade tecidual
• Curvas concentração-efeito cumulativa ao BAY 41-2272 ( 0.01 – 10 µM) na ausência e
na presença das seguintes drogas:
ODQ (10 µM) – inibidor da GCs
L-NAME (100 µM) – inibidor da NOS
rolipram (10 µM) – inibidor da PDE4
Materiais e Métodos
• Inibidores de canais de potássio : glibenclamida ( 10 µ M, ATP-dependentes)
apamina (100 nM, Ca2+-dependentes)
charibdotoxina (100 nM, Ca2+-dependentes)
4-aminopiridina (1mM, voltagem-dependentes)
tetraetilamônio (3 mM, voltagem-dependentes e ativados por Ca2+);
• Curva ao estímulo elétrico (EFS) e ao NO exógeno na presença e na
ausência do BAY 41-2272 (100 nM);
- EFS ( 2- 32 Hz, 1ms, 50V) - Estimulador Grass S8
- NO ( 1, 3, 10 uM) – solução de NaNO3
Materiais e Métodos
Determinação do conteúdo de GMPc tecidual
• Corpo cavernoso de rato dissecado e posto em período de estabilização de 30 minutos
em solução de Krebs aquecida e oxigenada;
• Estimulação durante 5 minutos;
• BAY 41-2272 (1 µM) ,
• SNP (1µM)
• na ausência e na presença de ODQ (10 µM);
• Dosagem do nucleotídeo cíclico de acordo com a técnica de imunoensaio
enzimático através de kit comercial (Cayman Chemical Cyclic GMP EIA kit,
Ann Arbor, MI, U.S.A.);
Análise Estatística
ANOVA duas vias e Student’s T-teste pareado bi caudal com
P < 0,05 considerado estatisticamente significativo.
Resultados : corpo cavernoso de coelho – in vitro
Resultados - Coelho
Curva concentração- efeito em resposta ao BAY 41-2272
Relaxamento (%)
0
Coelho
pEC50:
40
E max (%):
80
120
-8
-7
-6
log [BAY 41-2272] (M)
-5
6,82 + 0,06
116 + 2
Resultados - Coelho
Efeito do ODQ e do L-NAME
BAY 41-2272
BAY 41-2272 + ODQ
GTN
GTN + ODQ
40
(10 M)
80
120
-8
-7
-6
-5
log [Droga] (M)
BAY 41-2272
BAY + L-NAME (100 M)
BAY + L-NAME + ODQ
ACh
ACh + L-NAME
0
Relaxamento (%)
Relaxamento (%)
0
40
80
120
-8
-7
-6
log [BAY 41-2272] (M)
-5
Resultados - Coelho
Duração do relaxamento
(s)
Efeito do BAY 41-2272 na resposta ao estímulo elétrico
**
Controle
BAY 41-2272 (100 nM)
90
*
50
*
*
2.0
4.0
10
8.0
16.0
EFS (Hz)
* P < 0,05; ** P < 0,01 comparado ao controle
32.0
Resultados - Coelho
Duração do relaxamento
(s)
Efeito do BAY 41-2272 na resposta ao NO exógeno
250
200
**
**
**
**
30.0
100.0
**
150
100
50
1.0
3.0
10.0
NO (M)
** P < 0,01 comparado ao controle
Controle
BAY 41-2272 (100 nM)
Resultados - Coelho
Efeito do rolipram
Relaxamento (%)
0
40
80
controle
rolipram (10 M)
120
-8
-7
-6
-5
log [BAY 41-2272] (M)
Potência (pEC50)
forskolin
Controle
rolipram
a
7,12  0,02
7,58  0,05a
P < 0,01 comparado ao controle
Resultados - Coelho
Efeito dos inibidores dos canais de potássio
Em áx
relaxamento (%)
150
controle
glibenclamida
apamina
ChTx
TEA
4-AP
100
50
0
pEC50
7.5
5.0
2.5
0.0
controle
glibenclamida (10 M)
apamina (100 nM)
ChTx (100 nM)
TEA (1 mM)
4-AP 3 mM)
Resultados : corpo cavernoso humano – in vitro
Resultados - Humano
Curva concentração- efeito em resposta ao BAY 41-2272
% Relaxamento
0
Humano
pEC50:
E max (%):
50
100
-8
-7
-6
log [BAY 41-2272] (M)
-5
6,12 + 0,12
100 + 3
Resultados - Humano
Efeito da ODQ e do L-NAME
BAY 41-2272
BAY 412271 + ODQ
GTN
GTN + ODQ
40
(10 M)
80
120
-8
-7
-6
-5
log [Droga] (M)
0
Relaxamento(%)
Relaxamento (%)
0
BAY 41-2272
+ L-NAME (100 M)
+ L-NAME + ODQ
ACh
ACh + L-NAME
50
100
-8
-7
-6
log [BAY 41-2272] (M)
-5
Resultados - Humano
Efeito do rolipram
Relaxamento(%)
0
40
80
controle
rolipram (10 M)
120
-8
-7
-6
-5
log [BAY 41-2272] (M)
Potência (pEC50)
forskolin
Controle
rolipram
a
6,17  0,05
6,51  0,02a
P < 0,01 comparado ao controle
Resultados - Humano
Efeito dos inibidores dos canais de potássio
Em áx
relaxamento (%)
150
controle
glibenclamida
apamina
ChTx
TEA
4-AP
100
50
0
pEC50
7.5
5.0
2.5
0.0
controle
glibenclamida (10 M)
apamina (100 nM)
ChTx (100 nM)
TEA (1 mM)
4-AP (3 mM)
Resultados : corpo cavernoso de rato – in vitro
Resultados –Rato – in vitro
Curva concentração efeito em resposta ao BAY 41-2272
Rato
% Relaxamento
0
pEC50:
50
E max (%):
100
150
-9
-8
-7
-6
log [BAY 41-2272] (M)
-5
6,44 + 0,10
124 + 6
Resultados –Rato – in vitro
Efeito do ODQ
50
100
controle
ODQ (10 M)
150
-9
-8
-7
-6
-5
Efeito do L-NAME
-4
log [BAY 41-2272] (M)
0
% Relaxamento
% Relaxamento
0
50
100
controle
L-NAME (100 M)
150
-9
-8
-7
-6
-5
log [BAY 41-2272] (M)
-4
Resultados –Rato – in vitro
**
Níveis de nucleotídeo cíclico em corpo
cavernoso isolado de rato
(pmol GMPc/mg de proteína)
AMPc
(pmol/mg protein)
40
30
20
10
0
#
**
20
GMPc
(pmol/mg protein)
CTL
BAY
(1 µM)
SNP
(1 µM)
BAY
+ SNP
ODQ
(10 µM)
BAY
+ ODQ
18
16
**
4
3
**
2
1
0
CTL
BAY
(1 µM)
SNP
(1 µM)
** P < 0,01 comparado ao controle
BAY
+ SNP
ODQ
(10 µM)
BAY
+ ODQ
SNP
+ ODQ
FK
(1 µM)
# P< 0,01 comparado aos grupos BAY e SNP sozinhos
SNP
+ ODQ
FK
(1 µM)
Resultados : corpo cavernoso de rato – in vivo
Materiais e Métodos
Técnica de medida de pressão intracavernosa (ICP)
Ratos Wistar machos (250 -350g);
Anestesia: uretana (1,2g / kg)
intraperitonial;
Artéria carótida direita
(MAP; transdutores Grass, Astro-Med
Industrial Park, U.S.A.).
corpo cavernoso esquerdo
(ICP; transdutores Grass, Astro-Med
Industrial Park, U.S.A.).
corpo cavernoso direito
para administração de drogas.
Abertura da cavidade abdominal
Resultados –Rato – in vivo
Efeito do BAY 41-2272 no relaxamento induzido pelo EFS
controle
BAY 41-2272
( 0,6 mol/kg)
Resultados –Rato – in vivo
Efeito do BAY 41-2272 no relaxamento induzido pelo EFS do nervo
cavernoso
A
Controle
BAY 41-2272 (0,6 mol/Kg)
ICP/MAP
0.6
*
**
0.4
0.2
0.0
1.0
3.0
5.0
2000
Freqüência (Hz)
Controle
BAY 41-2272
AUC
*
1000
0
1.0
3.0
Freqüência (Hz)
* P < 0,05; ** P < 0,01 comparado ao controle
5.0
Sumário
Sumário
• BAY 41-2272 promoveu um relaxamento duradouro e concentraçãodependente
do músculo liso de corpo cavernoso em coelho, rato e
humano in vitro;
• O BAY 41-2272 potencializou a resposta erétil de rato induzida pelo
elétrico in vivo;
estímulo
• O relaxamento promovido pelo BAY 41-2272 em coelho, rato e humano in
foi dependente da via GCs - GMPc, mas não da via ACs – AMPc;
vitro
• O NO endógeno liberado tanto de fibras nitrérgicas quanto de endotélio sinusoidal
participa do relaxamento induzido pelo BAY 41-2272
aumentando sua resposta
relaxante;
• Os canais de potássio não interferiram no relaxamento de corpo cavernoso
coelho e humano promovido pelo BAY
41-2272, in vitro .
de
Conclusão Final
Conclusões finais
TABAGISMO
DIABETES
ENVELHECIMENTO
HIPERTENSÃO
DISLIPIDEMIAS
Fibras NANC
Endotélio cavernoso
nNOS
eNOS
GCs
GMPc
GTP
BAY 41-2272
Músculo liso
relaxamento