Arquivo

1

• Arquivo
• Mecanismo envolvido no potencial terapêutico da
  via óxido nítrico-guanilato ciclase solúvel GMP
  cíclico
• Dúvidas
• denucci_at_gdenucci.com
• Site
• www.gdenucci.com

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The NOsGCcGMP 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
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NOsGCcGMP 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
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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
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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
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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
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Homology model of the haem-binding domain of the
human soluble guanylate cyclase (sGC) ß-subunit.
NO-independent stimulators and activators of
soluble guanylate cyclase discovery and
therapeutic potential NATURE REVIEWS DRUG
DISCOVERY VOLUME 5 SEPTEMBER 2006 755
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Soluble guanylate cyclase (sGC) redox equilibrium.
NO-independent stimulators and activators of
soluble guanylate cyclase discovery and
therapeutic potential NATURE REVIEWS DRUG
DISCOVERY VOLUME 5 SEPTEMBER 2006 755
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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.
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Nitric oxide and overactive bladder
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.

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BAY 41-2272 potently stimulates soluble guanylate
cyclase (sGC) through cysteine 238 and cysteine
243 region in the a1-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.
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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.

14

• 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 400TM 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),
  L-NAME (100 µM) or sildenafil (100 nM).

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BAY 41-2272, but not SNP, GTN or acidified NaNO2,
potently relaxes the rabbit bladder
BAY 41-2272, but not SNP, GTN or acidified NaNO2,
potently relaxes the rabbit bladder
Figure 1. Relaxant effects of BAY 41-2272, sodium
nitroprusside (SNP), glyceryl trinitrate (GTN)
and acidified NaNO2 in the rabbit isolated
detrusor smooth muscle (n5). Data are the mean
S.E.M. plt0,001.
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BAY 41-2272-induced responses are not affected by
L-NAME, ODQ and 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 (n5).
Data are the Mean S.E.M.
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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.
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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 cet_at_fcm.unicamp.br
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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-1-benzyl 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-bpyridine-3-ylpyrimidin-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).
21
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.
Structure of BAY 41-2272
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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).
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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
37C in a total volume of 100 µl containing the
following 50 mM Tris-HCl (pH 7.4), 1 mM
3-isobutyl-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 95C 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.
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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-GCATGCATCTGGAGAAGG
G-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 (1100), sGC?1 (1200), gp91phox
(1500), p47phox (1500), p22phox (1500) or
p67phox (1500) 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 (12000).
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Cyclic GMP content of mouse corpus cavernosum
from wild-type (WT), nNOS-/- and eNOS-/-
plt0.05 and plt0.01 compared to respective
vehicles plt0.05 compared to the sum of BAY
41-2272 plus SNP values alone.
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Relaxing activity of BAY 41-2272 in corpus
cavernosum from WT, nNOS-/- and eNOS-/- mice
b
a
Concentration-response curves to BAY 41-2272
(n16) 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. plt0.01 compared to curves elicited
in WT and nNOS-/- strips.
27
Effects of ODQ and L-NAME on BAY 41-2272-induced
relaxations
a
b
c
d
e
f
Experimental values were obtained in the absence
(closed circles) and in the presence (open
circles) of ODQ (10 µM) or L-NAME (100 µM).
plt0.05 and plt0.01 compared to values in the
absence of inhibitor.
28
BAY 41-2272 enhances magnitude and duration of
nitrergic responses
a
b
Effects of increasing concentrations (0.01-0.1
?M n6) 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. plt0.05 and
plt0.01 compared to the respective control (CTL)
values.
c
d
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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).
30
Stimulation of cavernosal sGC activity by BAY
41-2272
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 n3) 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.
31
sGC expression in corpus cavernosum from WT,
nNOS-/- and eNOS-/- mice
a
b
c
d
Messenger RNA (panels a and b n6) and Western
blot (panels c and d n6) 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.
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Inhibition of superoxide production by BAY 41-2272
Inhibitory effect of BAY 41-2272 (0.1-1 ?M n4)
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. plt0.05 and plt0.01 compared to CTL
values in the absence of U46619 plt0.05 and
plt0.01 compared to CTL values in the presence
of U46619 plt0.05 and plt0.01 compared to BAY 1
?M.
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BAY 41-2272 inhibits NADPH oxidase expression in
the corpus cavernosum
Inhibitory effect of BAY 41-2272 (0.1-1 ?M n4)
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). plt0.05 and plt0.01
compared to CTL values plt0.05 and plt0.01
compared to BAY values alone.
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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)
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36
Introdução
37
Introdução
incapacidade persistente de alcançar e manter a
ereção peniana adequada para uma atividade sexual
normal.
Disfunção Erétil (DE)
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
38
Introdução
Classificação da DE masculina (Comitê da
Sociedade Internacional de Pesquisa em Impotência)
Orgânica
Psicogênica

• Vasculogênica
• Arteriogênica
• Cavernosa
• Associada

• Generalizada
• dessensibilização
• Inibição
• Situacional
• Relacionada ao parceiro
• Relacionada à performance
• Desajuste psicológico
• (p.e. depressão)

2. Neurogênica 3. Anatômica 4. Endocrinológica
Lisa Rosen, 1999 Dean Lue, 2006
39
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
40
Introdução
CONTROLE PERIFÉRICO
Grau de contração do músculo liso cavernoso
FLACIDEZ
EREÇÃO
Equilíbrio
Plexo Pélvico
Fatores Contráteis
Fatores Relaxantes
Controle Central e Períférico
Burnett, 1992 Andersson, 2001 Rampin, 2004 Dean
Lue, 2006
41
Introdução
Ereto
Plexo venoso subtunical
HEMODINÂMICA E MECANISMO DA EREÇÃO PENIANA
Veias emissárias
Espaços sinusoidais
Flácido
Artérias helicinais
Veia dorsal
Túnica albugínea
Músculo liso trabecular
Espaços sinusoidais
Plexo venoso subtunical
Corpos cavernosos
Artéria cavernosa
Saenz de Tejada et al., 1985 Lue Tanagho,
1987
Corpo cavernoso humano
42
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

Lue et al., 1983 Saenz de Tejada et al.,
1988 Hedlund et al.,1999 2000a,b Andersson,
2003.
Dean Lue, 2006
43
Introdução
PRINCIPAL VIA PARA A EREÇÃO
ESTÍMULO SEXUAL
Fibras NANC
Endotélio cavernoso
nNOS
eNOS
sildenafil tadalafil vardenafil
NO
GCs
PDE5
Músculo liso vascular/ cavernoso
GMPc
5GMP
GTP
relaxamento
Rapoport Murad, 1983 Lucas et al., 2000
44
Introdução
Biossíntese do NO
nNOS
eNOS
NOS
NADPH
1/2 NADPH
NO.

O2
O2
H2O
H2O
L-Arg
Nw-hydroxy-L-Arg
L-Cit
Marletta, 1988, 1993 Feldman et al., 1993
Fukuto Chaudhuri, 1995 Ignarro Murad,
1995 Kerwin et al., 1995 Korth et al., 1995.
45
Introdução
Guanilil Ciclase Solúvel
Guanilil Ciclase Solúvel (GCs)

• receptor mais importante da molécula de NO
• presente no citoplasma dos mamíferos
• inibição da agregação plaquetária
• relaxamento de músculo liso
• vasodilatação
• transdução de sinal neuronal
• imunomodulação

Collier and Vallance, 1989 Lucas et al., 2000
46
Introdução
Fe2 reduzida Fe3 oxidada HIS105 mutação
produz enzima não responsiva ao NO
Necessário para a ativação da enzima pelo NO
Craven DeRubertis, 1978
a
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
47
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)

48
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 nitratos orgânicos.
  (Stasch et al., 2002)

• BAY 41-2272 aumenta o débito cardíaco e
  preserva a função renal em insuficiência
  cardíaca congestiva experimental
• (Boerrigter et al., 2003)

• BAY 41-2272 relaxa anéis aórticos de coelho
  tanto na presença quanto na ausência de
  endotélio
• (Priviero et al., 2005)

• BAY 41-2272 interage com NO endógeno e exógeno
  causando um potente relaxamento do músculo
  anococcígeo de rato
• (Teixeira et al., 2006)

49
Objetivo Geral
50
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.
51
Objetivos Específicos
52
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

53
Materiais e Métodos
Experimentos in vitro
54
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

55
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

56
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

57
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

58
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.)

59
Análise Estatística
ANOVA duas vias e Students T-teste pareado bi
caudal com P lt 0,05 considerado
estatisticamente significativo.
60
Resultados corpo cavernoso de coelho in vitro
61
Resultados - Coelho
Curva concentração- efeito em resposta ao BAY
41-2272
Coelho
6,82 0,06
pEC50
116 2
E max ()
62
Resultados - Coelho
Efeito do ODQ e do L-NAME
(10 ?M)
(100 ?M)
63
Resultados - Coelho
Efeito do BAY 41-2272 na resposta ao estímulo
elétrico
P lt 0,05 P lt 0,01 comparado ao controle
64
Resultados - Coelho
Efeito do BAY 41-2272 na resposta ao NO exógeno
P lt 0,01 comparado ao controle
65
Resultados - Coelho
Efeito do rolipram
66
Resultados - Coelho
Efeito dos inibidores dos canais de potássio
67
Resultados corpo cavernoso humano in vitro
68
Resultados - Humano
Curva concentração- efeito em resposta ao BAY
41-2272
6,12 0,12
69
Resultados - Humano
Efeito da ODQ e do L-NAME
(10 ?M)
(100 ?M)
70
Resultados - Humano
Efeito do rolipram
71
Resultados - Humano
Efeito dos inibidores dos canais de potássio
72
Resultados corpo cavernoso de rato in vitro
73
Resultados Rato in vitro
Curva concentração efeito em resposta ao BAY
41-2272
6,44 0,10
74
Resultados Rato in vitro
Efeito do ODQ
Efeito do L-NAME
75
Resultados Rato in vitro
Níveis de nucleotídeo cíclico em corpo cavernoso
isolado de rato (pmol GMPc/mg de proteína)
P lt 0,01 comparado ao controle Plt 0,01
comparado aos grupos BAY e SNP sozinhos
76
Resultados corpo cavernoso de rato in vivo
77
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
78
Resultados Rato in vivo
Efeito do BAY 41-2272 no relaxamento induzido
pelo EFS
BAY 41-2272 ( 0,6 ?mol/kg)
controle
79
Resultados Rato in vivo
Efeito do BAY 41-2272 no relaxamento induzido
pelo EFS do nervo cavernoso
P lt 0,05 P lt 0,01 comparado ao controle
80
Sumário
81
Sumário

• BAY 41-2272 promoveu um relaxamento duradouro
  e concentração- dependente 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 estímulo elétrico in
  vivo
• O relaxamento promovido pelo BAY 41-2272 em
  coelho, rato e humano in vitro foi dependente
  da via GCs - GMPc, mas não da via ACs AMPc
• 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 de coelho e
  humano promovido pelo BAY 41-2272, in vitro .

82
Conclusão Final
83
Conclusões finais
TABAGISMO
DIABETES
ENVELHECIMENTO
HIPERTENSÃO
DISLIPIDEMIAS
Fibras NANC
Endotélio cavernoso
nNOS
eNOS
GCs
GMPc
GTP
BAY 41-2272
Músculo liso
relaxamento
84
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