Page 101 - 79_04

Reduction
in
mitochondrial
membrane
peroxidizability
index…

2.
MATERIALS
AND
METHODS

Animals
and
diets

Six
week-­-old
male
Wistar
rats
were
obtained
from
Harlan
Laboratories

(Unide,
Italy).
The
animals
were
caged
individually
and
maintained
in
a
12:12

(light-­-dark)
cycle,
22ºC
±
2ºC
and
50%
±
10%
relative
humidity
at
the
animal

house
of
the
Complutense
University.
Sixteen
male
rats
were
fed
ad
libitum
with
a

standard
rodent
diet
(Panlab,
Spain)
and
were
divided
into
two
groups
of
8

animals
each:
CONTROL
and
ATENOLOL.
The
animals
in
the
atenolol
group
had

free
access
to
a
1
g/L
atenolol
solution
(Sigma,
A7655)
in
the
drinking
water.

Previous
studies
have
used
that
dose
of
atenolol
to
cause
blockade
of
ß-­-adrenergic

receptors
in
rats
(30).
The
control
group
had
free
access
to
the
same
aliquot
of

drinking
water
without
atenolol.

The
mean
water
intake
per
animal
per
day
was

not
significantly
different
between
both
groups.
After
2
weeks
of
treatment,
the

animals
were
sacrificed
by
decapitation.
Hearts
were
immediately
processed
to

isolate
functional
mitochondria,
which
were
used
to
measure
mitochondrial

respiration
and
rates
ROS
generation
rates.
The
remaining
mitochondria
were

stored
at
-­-80ºC
for
posterior
analyses.
These
experiments
in
Wistar
rats
were

approved
by
the
Animal
Experimental
Committee
from
the
Complutense

University.
The
present
investigation
conforms
to
the
Directive
2010/63/EU
of
the

European
Parlament.

Isolation
of
functional
mitochondria,
oxygen
consumption
and
ROS
production

Mitochondria
were
obtained
from
fresh
tissue
by
the
procedure
of
Mela
and

Seitz
(31)
with
modifications.
After
checking
the
functionality
and
phosphorylation

capacity
of
the
mitochondria
(high
respiratory
control
ratios)
the
rate
of
mtROSp

was
measured
by
the
fluorometric
method
established
at
our
laboratory
(32).
The

oxygen
consumption
rate
of
heart
mitochondria
was
measured
at
37ºC
in
a
water-­-
thermostatized
incubation
chamber
with
a
computer-­-controlled
Clark-­-type
O2

electrode
(Oxygraph,
Hansatech,
UK).

Oxidative
damage
to
mtDNA
(8-­-oxodG)

Isolation
of
mtDNA
was
performed
by
the
method
of
Latorre
and
cols
(33)

adapted
to
mammals
(34).
The
isolated
mitochondrial
DNA
was
digested
to

deoxynucleoside
level
and
the
level
of
oxidative
damage
in
mtDNA
was
estimated

by
measuring
the
amount
of
8-­-oxo-­-7,8-­-dihydro-­-2’deoxyguanosine
(8-­-oxodG)

referred
to
that
of
the
non-­-oxidized
base
(deoxyguanosine,
dG)
by
HPLC-­-EC
as

previously
described
(35).

Measurement
of
mitochondrial
complexes
I,
II,
III
and
IV,
AIF,
MnSOD,
SIRT3

and
SIRT5

The
amounts
of
a)
the
mitochondrial
respiratory
chain
complexes
(I
to
IV),

the
complex
I
regulatory
factor
AIF,
the
mitochondrial
biogenesis
protein

617