Impaired Corpus Cavernosum Relaxation

Is Accompanied by Increased Oxidative

Stress and Up-Regulation of the Rho-
Kinase Pathway in Diabetic (Db/Db) Mice

Fernanda B. M. Priviero, Haroldo A. F. Toque, Kenia Pedrosa Nunes, Denise G.

Priolli1, Cleber E. Teixeira, R. Clinton Webb

PLoS ONE 11(5), May 26, 2016

 Introdução

Ereção
peniana

NO

Principal mediador do relaxamento

do músculo liso do corpo cavernoso

MORELLI, Annamaria et al. Physiology of Erectile Function: An Update on Intracellular

Molecular Processes. Eau-ebu Update Series, [s.l.], v. 4, n. 3, p.96-108, jun. 2006.
Introdução
Introdução
Disfunção erétil

Incapacidade persistente ou
recorrente de obter ou
manter uma ereção que
permita um desempenho
sexual satisfatório
 Hipertensão Hipercolesterolemia

Aterosclerose Diabetes

Obesidade Síndrome metabólica

Envelhecimento Tabagismo

Apneia do sono
 Estresse oxidativo

Redução da
biodisponibilidade
de NO

Aumento das
espécies reativas de
oxigênio
Hipótese
• Via de sinalização RhoA/Rho-cinase deve estar hiper-regulada em
camundongos db/db.
Métodos
• Animais

Camundongos db/db (obesidade e diabetes do tipo II,

causada por uma mutação no receptor de leptina)

Camundongos heterozigóticos (db/+) (magros, não

diabéticos)
Métodos

 Temperatura: 37 °C
 Gaseificada
(O2 95%+CO2 5 %)
 Tensão: 2 mN
 CC (11x1x1 mm)
 Resultados
Via RhoA/Rho-cinase no relaxamento do corpo cavernoso nos
camundongos diabéticos
Fig 1.
A) Relaxation response induced by the Rho-
kinase inhibitor, Y-27632, in cavernosal tissue of
db/+ and db/db mice pre-contracted with
phenylephrine (PE 10 μmol/l); B) Inhibition of
the electrical field stimulation-induced
contraction in the absence (B) or in the presence
of increasing concentrations of Y-27632 (EFS 16
Hz; Y-27632 0.01–30 μmol/l). C) Protein
expression of RhoA in the corpora cavernosa of
db/+ and db/db mice. Increased levels of active
RhoA (membrane fraction) were observed in
db/db compared db/+ mice. Densitometry
values were normalized to β-actin and expressed
as the fold increase of control db/+ mice.
Resultados

RhoGDI (Rho-guanine dissociation inhibitor)

Rho-GEFs (guanine nucleotide exchange factors )
Subunidade regulatória MYPT-1
Resultados Fig 1.
D) Phenylephrine (PE; 10 μmol/L) induced
phosphorylation of MYPT-1. It was observed
a higher magnitude of phosphorylation in
db/db mice, compared to the db/+ mice. In
the presence of Y-27632, MYPT-1
phosporylation was quite inhibited in db/+
mice and only partially inhibited in db/db
mice. All data represents the mean ± S.E.M.
of 4–6 experiments per group. *P<0.05 and
**P<0.01, compared to db/+ mice; aP< 0.05
compared to the respective control; aa P<
0.01 compared to the respective control; bP<
0.05, compared to PE only.
Métodos - Western blot
Anticorpos

eNOS (1:1000),
nNOS (1:4000),
Cu/Zn SOD (1:1000),
Mn SOD (1:1000),
RhoA (1:200),
Rho-kinase α (1:500),
Rho-kinase β (1:200),
RhoGDI (1:2000),
PDZ-RhoGEF (1:500)
p115RhoGEF (1:200),
p190RhoGAP (1:200),
LARG (1:200),
MYPT-1 (1:1000),
pMYPT-1 (1:1000).
Resultados

Fig 2. Increased protein

expression of Rho-kinase α
and RhoGEFs subunits
p115RhoGEF, PDZ-RhoGEF
and LARG in the corpora
cavernosa of db/db mice.
Data represents the mean ± S.E.M.
of 4–6 experiments per group.
*P<0.05 and **P<0.01, compared
to db/+ mice.
Resultados

Fig 2. Increased protein

expression of Rho-kinase α
and RhoGEFs subunits
p115RhoGEF, PDZ-RhoGEF
and LARG in the corpora
cavernosa of db/db mice.
Data represents the mean ± S.E.M.
of 4–6 experiments per group.
*P<0.05 and **P<0.01, compared
to db/+ mice.
Métodos
Métodos - Nível de NOx

 Kit de ensaio
Nitrato/Nitrito
(Cayman Chem,
Ann Arbor, MI)
 Resultados
Via NO/sGC/cGMP no relaxamento do corpo cavernoso de camundongos
diabéticos

Fig 3. Protein expression of eNOS (Panel A) and nNOS (panel B) were similar in the CC of db/+ and
db/db mice.
Panel C shows decreased plasma and urine levels of nitrite/nitrate (NOx) in db/db mice compared to db/+. Data
represents the mean ± S.E.M. of 4–6 experiments. *P<0.05 and **P<0.01, compared to the db/+ mice.
Resultados

Fig 4. Protein expression of

sGCα (Panel A), sGCβ
(panel B), HuR (Panel C)
and PDE5 (Panel D) were
similar in the CC of db/+
and db/db mice.
Data represents the mean ±
S.E.M. of 4–6 experiments.

sGC (subunidades α e β)
Resultados

Fig 4. Protein expression of

sGCα (Panel A), sGCβ
(panel B), HuR (Panel C)
and PDE5 (Panel D) were
similar in the CC of db/+
and db/db mice.
Data represents the mean ±
S.E.M. of 4–6 experiments.

HuR (the sGC mRNA-stabilizing protein)

Proteína estabilizadora de mRNA sGC

PDE5 - fosfodiesterase tipo 5 

Métodos – Determinação dos níveis de 8-isoprostano

 Kit (Imunoensaio enzimático)

8-isoprostrano (Cayman
Chemical, Ann Arbor, MI)
8-iso-PGF2α é o principal isoprostano biologicamente ativo
produzido pela peroxidação não enzimática do ácido araquidônico
 Resultados
Avaliação da peroxidação lipídica

Fig 5. Increased lipid

peroxidation, measured by
formation of 8-isoprostane, in
CC (Panel A), plasma (Panel B)
and urine (Panel C) of db/db
mice compared to db/+.
Data represents the mean ± S.E.M.
of 4–6 experiments *P<0.05 and
**P<0.01, compared to db/+ mice.
Métodos – Determinação da atividade da enzima
superóxido dismutase (SOD)

 Kit de ensaio SOD (Cayman

Chemical, Ann Arbor, MI)
 Resultados
Avaliação de atividade e expressão de SOD
Fig 6. Decreased activity of
the antioxidant enzyme
SOD in the corpus
cavernosum (Panel A) and
serum (Panel B) of db/db
mice compared to db/+
mice.
Protein expression of CuZn
(Panel C) and Mn SOD (Panel
D) measured by western blot
were diminished in the CC of
db/db mice. Data represents
the mean ± S.E.M. of 4–6
experiments. *P<0.05 and
**P<0.01, compared to db/+
mice.
Ciclo catalítico da superóxido dismutase citosólica (SOD-Cu/Zn) e mitocondrial (SOD-Mn)
Resultados

Fig 6. Decreased activity of

the antioxidant enzyme
SOD in the corpus
cavernosum (Panel A) and
serum (Panel B) of db/db
mice compared to db/+
mice.
Protein expression of CuZn
(Panel C) and Mn SOD (Panel
D) measured by western blot
were diminished in the CC of
db/db mice. Data represents
the mean ± S.E.M. of 4–6
experiments. *P<0.05 and
**P<0.01, compared to db/+
mice.
Pearson Correlation Analysis
It was observed several associations among variables of the study in the RhoA/Rock signaling pathway and
oxidative status (Pearson correlation), as shown in S1 Fig, which suggests a cross-talk between these pathways.
There was a marked negative correlation between ROCKα expression and SOD activity on serum.
Evaluation of the target SOD expression in the corpus cavernosum (SODCC) according to the
predictors included in the model.
Data revealed that the strain db/db is the predictor of the reduced amount of SOD expressed in the corpus
cavernosum.
Conclusões

Os dados demonstram que em camundongos db/db, a hiper-

regulação da via de sinalização RhoA/Rho-cinase foi acompanhada
por diminuição da biodisponibilidade de NO e aumento do estresse
oxidativo que pode ser responsável pelo relaxamento prejudicado do
corpus cavernoso de camundongos db/db.