Vol. 64, No. 1,1975
CORRELATION AND
BIOCHEMICAL
Cl*-
Department
Received
MITOCHONDRIAL TO OXYGEN
ACTIVITY Leena
Mela,
Cleon
of Surgery, University
March
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CYTOCHROME AVAILABILITY W.
Goodwin,
and
CONCENTRATION THE NEWBORN
IN Leonard
Harrison Department of Pennsylvania School Philadelphia, Pennsylvania
D.
Miller
of Surgical of Medicine 19174
Research
lo,1975
Effect of in vivo oxygenation on mitochondrial respiratory chain Summary: capacity was studied in newborn puppies. Heart mitochondria were isolated from 0 to 7 days. As the arterial PO2 rises sharply during the first 3-4 activity decrease days, State 3 respiratory capacity and Ca ++ transport linearly. Cytochrome c and a t a3 concentrations increase rapidly. Thus the enzymatic turnover of the respiratory chain decreases to one-fifth ofthe These data are in agreement with earlier results fetal term value in 4 days. indicating a strong controlling effect of in vivo oxygenation on mitochondrial respiratory capacity.
INTRODUCTION During
the
enzyme
first
proteins
chromes
is
levels
in
still
increasing
activity
the
of extrauterine
occurs.
quite
low
rat
liver
At (1). and
after
earlier
two
respiratory
of the
mitochondrial
affected
by
should
occur
rises is
the
to case
heart
(1).
weeks
(2).
adult (5).
In
week rat
It is
these
brain not
would
suggest
alterations
to first
levels.
adult
days, In an
Were
the
animals when earlier
oxidase
however, during
this
is
time
the period.
on mitochon-
of kinetic newborn
adult
whether
oxygenation
and the
reach
cytochrome
changes
cyto-
enzymes
clear,
of tissue
similarly the
first
of many
of mitochondrial
effects
(3,4).
development
concentration
systems
enzymes
during
rapidly
the
rapid
of direct
activity
oxygen
life the
enzyme
studies
drial
birth
During
of mitochondrial Our
this
days
activities
mitochondrial
humans
(3)
a large
very
low
fetal
arterial
report
we
have
indicated
enzymes change PO2 that
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMAMJNICATIONS
METHODS Puppies were used for these studies. Fetal animals at term before Newborns were studied at different ages breathing air served as controls. up to two weeks. Fetal animals at term were delivered by Caesarean section. The mothers were anesthetized with Nembutal. Before removal of the ascending aortic blood samples, and the hearts for mitochondrial preparations, the fetuses were not allowed to breathe. Newborn animals were anesthetized with Nembutal. The chest was opened and ascending aortic blood was collected for determination of blood Immediately after the withdrawal of blood samples, the heart was PO2. removed, placed in 100 ml of ice-cold medium, and cut into small pieces with scissors. Mitochondria were isolated in 0. 225 M mannitol and 0. 075 M sucrose supplemented with 100 PM EDTA at 0’ C according to conventional methods as described earlier (3). The homogenization of fetal and newborn hearts, up to the age of ten days, was performed without Nagarse. In older heart preparations 5 mg Nagarse per 2 grams of tissue was used. Isolated mitochondria were assayed for respiratory activity with a Clark oxygen electrode using 10 mM glutamate and malate or 10 mM succinate in the presence of 1OpM Rotenone as substrates, and ADP as the phosphate acceptor. The concentrations and steady state changes of mitochondrial cytochromes were determined in a Chance dual-wavelength spectrophotometer. Cytochrome oxidase was measured at 445-460 nm and cytochrome c at 550-540 nm. Kinetics of the Cat+ transport 7 in the dual wavelength spectrophotometer using indicator at 540-507 nm according to the method Protein concentrations of the mitochondrial the Biuret method.
activity Murexide of Mela samples
were also measured as the Ca tt and Chance (6). were determined by
RESULTS Arterial seen
in
oxygen Figure
breathing
the
of
arterial
prepared
with
varies
rise
theoretical
fetal
coupled,
glutamate
(RCR
to
The
ascending between
8 and
occurs.
This
normal
adult
and
= 8) were (3 for
malate
discarded. glutamate
PO2 PO2
15 mm phase
newborn
is
puppy
followed
is
very
at term
During
from
hearts
the by
low.
As
before
first
four
a slower
newborn
using
gentle
control
substrates. The
of the
fetus
days
increase
of
level.
respiratory as
of the
Hg.
preparations
exhibiting and
arterial aortic
of - mitochondrial from
well
PO2
PO2
-Quality
are
1, the
air
a rapid
tension.
Two
ADP/O
ratios
t malate,
2 for
385
hearts. short-term
ratios
(RCR)
samples
with
gave succinate).
Mitochondria
values
homogenization of 10 or looser close
above coupling
to
BIOCHEMICAL
Vol. 64, No. 1,1975
-0
--State respiring
Changes of arterial Each point indicates
3 respiratory
activity.
in
the
presence
respiratory
activities
indicator
of mitochondrial
When
State
puppies, 2.
respiratory
Figure
3,
also
the
three
protein
is
The
biphasically. to
a 100%
of the of adult
found
plotted
days
by
to
by
the
first,
increase.
A
week
of life
first
days
faster second cytochrome
3,
puppies.
while
almost
state,
was
during
the
was against
maximal
thus,
is
first
a good
arterial
200’5’0,
slower
from
phase oxidase
value.
386
is
starts reaches
3 newborns.
seen
in
5 increases a fetal
term
level
days after
in
change during value
of
Cytochrome between
seen
of the
As
Adult
week.
occurs
State
PO2
newborn
as
cytochromes
level.
first
in
days,
when
Cytochrome
adult
rise
4-5
concentration.
almost
of the
examined
obtained
the
of life.
almost end
State
reach
of mitochondrial
first
in
capacity.
concentrations the
state,
respiratory
cytochrome
protein
reached
active
correlation
were
newborn
0. 21 nmoles/mg
as a function of age individual animal.
respiration
were
linear
during
first
This
-of mitochondrial
dramatically the
inverse
20
mitochondria
vitro.
rates
12 16 Days
their
mitochondrial
activities
Changes
In
respiratory
3 heart
An
6 Age,
PO2 an
of ADP, in
decreasing
Figure
4
1:
Figure
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0. 68
of
nmoles/mg
oxidase 0 and day
2.
a concentration
increase
2 and By
amounts
the of
end 50%
BIOCHEMKAL
Vol. 64, No. 1, 1975
Figure
State 3 respiratory activities of isolated heart mitochondria as a function of age. Glutamate and malate were used substrates. Each point indicates an individual animal.
2:
Utilizing
the
respiratory The
calculated
age
up
data
to
7 days.
of newborn
one
the
alterations
molecule
indicate
turnover
that
occurs
mitochondrial It
3,
in
be
in
the
noted
of
of cytochrome an
extremely
State
3 with
capacity
should
as
to that
absence
oxidase. large increasing
accumulate Cat’
transport
of added
phosphate
7.
transport During
adult
2 and
4,
mitochondria
day
ion
transient.
reach
Figure
the
heart
decreasing are
in oxidase
at
Figures per
dramatically.
completely
meters
shown
Similarly
capacity
The
in
calculated
of cytochrome
decreases
above
were
data,
calcium
ceases
presented
activity
decrease
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
and
respiratory
next
few
capacities
weeks
all
described
mitochondrial
para-
levels. DISCUSSION
The are (3-5).
data
sensitively Under
presented
here
affected acute
by hypoxia
support changes a 60%
our
earlier
of tissue decrease
387
contention oxygen of liver
that
mitochondria
concentration tissue
PO2
in in
the
vivo rat
Vol. 64, No. I,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r
0.75
d z e Q 0 f
0. I 5 c
\
t \o h 2 3
cytochrome
(0 t $I
0
s B c
o-
0
2
4
6
-I 6 Aqe
Figure
induced 20-40
twice
normal
minutes
a-- t a3, induced
(4).
of dog c and an
heart
hypoxia
mitochondria
mitochondrial each
oxygenation
induces
respiratory
chain, well
(3).
other increased and
as
of liver
also
induced
parallel
with
increased
human,
oxygenation
induces
decreased
animals
these
decreased
respiratory
in
of cytochrome
of hypoxic All
in
data
are
vivo
tissue
capacity
in
c
respiratory
a decrease
that
mitochondrial
that
increased
change.
demonstrating
c and for coefficients.
19 rnGml
mitochondria
Reoxygenation activity
in
I 6
, Days
capacities
-b concentrations
with
as
respiratory Chronic
opposite
agreement
animals,
I 6
Concentrations of heart mitochondrial cytochrome a t a3 as a function of age. For calculations and 160 mM-’ for a t a3 were used as extinction Each point indicates an individual animal.
3:
capacity
I 4
I 2
O0
chronically
in
per hypoxic
mitochondrial
respiratory
capacity. The in
vivo
site is
of interaction presently
not
of clear.
oxygen
with
However,
388
mitochondrial our
data
respiratory suggest
that
chain possibly
BIOCHEMICAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL
l
accumulation utilization
cat+
0 02
0
Figure
not
4:
only
one,
various
in
but
several
sites
this
and/
or
subst
Our who
and rate
no
respiratory Since
are effect
transport
is
rates
are
of State
are
transport,
as
3 respiration,
of
point
is
of changing
oxygen
in not
vitro
capable
unless
seems
those
in
of
Chance
The the
most
indicated
possibly
(7)
concentrations critically
of affecting
directly
mechanism
ADP
low
Liibbers
(8),
on mitochondrial tissue
mitochondrial
vitro,
and
PO2 respiratory
a cytoplasmically
mediated
were
reached. or
ion
in
vivo
necessary. ACKNOWLEDGMENTS
These 1 ROl
GM
studies 19867,
U. S. P. H. S.
CDA
a
involved.
mechanisms Cat+
mole
Each
functions
include
conditions
per
of age.
transport
to
capacities
control
accumulation
mitochondrial
contradictory
activity oxygen
6
transport.
findings
found
6
as a function animals.
These
under
4
Catf
membrane
of interaction.
report,
2
oxidase of 3-4
of the
carrier-mediated
probable
and
02 utilization cytochrome mean value
RESEARCH COMMUNICATIONS
were and
supported
by
U.
1 TOl-GM-01540.
lK04-GM
S. Leena
50318.
389
Public Mela
Health is
Service a recipient
Grants of
Vol. 64, No. 1, 1975
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8.
Hallman, Wilson, Park, Aberdeen, Mela, R. R. Goodwin, Surg.
M.
(1971) Biochim. Biophys. E. (1972) J. Neurochem. C. D., Mela, L., Wharton, R., E. (1973) J. Surg. Res. 14, L., Miller, L. D., Bacalzo, L. (1973) Ann. Surg. 178, 727-735. C. W. , Mela, L., Park, C. Forum 25, 185-187. J.
Acta 253, 19, 223-227. Reilly, J., 139-146. V., Olofsson, D.,
and
360-372. Fishbein, K.,
Campbell,
Mela, L. and Chance, B. (1968) Biochemistry 7, 4059-4063. Chance, B. (1965) J. Gen. Physiol. 49, 163-188. Liibbers, D. W., and Kessler, M. (1968) in: Liibbers, D. u. c., Thews, G., and Witzleb, E., Eds., Oxygen transport and tissue, pp. 90-99, George Thieme Verlag, Stuttgart.
390
P.,
and
and
White,
J.
(1974)
W., in
Luft, blood
CORRELATION AND
BIOCHEMICAL
Cl*-
Department
Received
MITOCHONDRIAL TO OXYGEN
ACTIVITY Leena
Mela,
Cleon
of Surgery, University
March
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CYTOCHROME AVAILABILITY W.
Goodwin,
and
CONCENTRATION THE NEWBORN
IN Leonard
Harrison Department of Pennsylvania School Philadelphia, Pennsylvania
D.
Miller
of Surgical of Medicine 19174
Research
lo,1975
Effect of in vivo oxygenation on mitochondrial respiratory chain Summary: capacity was studied in newborn puppies. Heart mitochondria were isolated from 0 to 7 days. As the arterial PO2 rises sharply during the first 3-4 activity decrease days, State 3 respiratory capacity and Ca ++ transport linearly. Cytochrome c and a t a3 concentrations increase rapidly. Thus the enzymatic turnover of the respiratory chain decreases to one-fifth ofthe These data are in agreement with earlier results fetal term value in 4 days. indicating a strong controlling effect of in vivo oxygenation on mitochondrial respiratory capacity.
INTRODUCTION During
the
enzyme
first
proteins
chromes
is
levels
in
still
increasing
activity
the
of extrauterine
occurs.
quite
low
rat
liver
At (1). and
after
earlier
two
respiratory
of the
mitochondrial
affected
by
should
occur
rises is
the
to case
heart
(1).
weeks
(2).
adult (5).
In
week rat
It is
these
brain not
would
suggest
alterations
to first
levels.
adult
days, In an
Were
the
animals when earlier
oxidase
however, during
this
is
time
the period.
on mitochon-
of kinetic newborn
adult
whether
oxygenation
and the
reach
cytochrome
changes
cyto-
enzymes
clear,
of tissue
similarly the
first
of many
of mitochondrial
effects
(3,4).
development
concentration
systems
enzymes
during
rapidly
the
rapid
of direct
activity
oxygen
life the
enzyme
studies
drial
birth
During
of mitochondrial Our
this
days
activities
mitochondrial
humans
(3)
a large
very
low
fetal
arterial
report
we
have
indicated
enzymes change PO2 that
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMAMJNICATIONS
METHODS Puppies were used for these studies. Fetal animals at term before Newborns were studied at different ages breathing air served as controls. up to two weeks. Fetal animals at term were delivered by Caesarean section. The mothers were anesthetized with Nembutal. Before removal of the ascending aortic blood samples, and the hearts for mitochondrial preparations, the fetuses were not allowed to breathe. Newborn animals were anesthetized with Nembutal. The chest was opened and ascending aortic blood was collected for determination of blood Immediately after the withdrawal of blood samples, the heart was PO2. removed, placed in 100 ml of ice-cold medium, and cut into small pieces with scissors. Mitochondria were isolated in 0. 225 M mannitol and 0. 075 M sucrose supplemented with 100 PM EDTA at 0’ C according to conventional methods as described earlier (3). The homogenization of fetal and newborn hearts, up to the age of ten days, was performed without Nagarse. In older heart preparations 5 mg Nagarse per 2 grams of tissue was used. Isolated mitochondria were assayed for respiratory activity with a Clark oxygen electrode using 10 mM glutamate and malate or 10 mM succinate in the presence of 1OpM Rotenone as substrates, and ADP as the phosphate acceptor. The concentrations and steady state changes of mitochondrial cytochromes were determined in a Chance dual-wavelength spectrophotometer. Cytochrome oxidase was measured at 445-460 nm and cytochrome c at 550-540 nm. Kinetics of the Cat+ transport 7 in the dual wavelength spectrophotometer using indicator at 540-507 nm according to the method Protein concentrations of the mitochondrial the Biuret method.
activity Murexide of Mela samples
were also measured as the Ca tt and Chance (6). were determined by
RESULTS Arterial seen
in
oxygen Figure
breathing
the
of
arterial
prepared
with
varies
rise
theoretical
fetal
coupled,
glutamate
(RCR
to
The
ascending between
8 and
occurs.
This
normal
adult
and
= 8) were (3 for
malate
discarded. glutamate
PO2 PO2
15 mm phase
newborn
is
puppy
followed
is
very
at term
During
from
hearts
the by
low.
As
before
first
four
a slower
newborn
using
gentle
control
substrates. The
of the
fetus
days
increase
of
level.
respiratory as
of the
Hg.
preparations
exhibiting and
arterial aortic
of - mitochondrial from
well
PO2
PO2
-Quality
are
1, the
air
a rapid
tension.
Two
ADP/O
ratios
t malate,
2 for
385
hearts. short-term
ratios
(RCR)
samples
with
gave succinate).
Mitochondria
values
homogenization of 10 or looser close
above coupling
to
BIOCHEMICAL
Vol. 64, No. 1,1975
-0
--State respiring
Changes of arterial Each point indicates
3 respiratory
activity.
in
the
presence
respiratory
activities
indicator
of mitochondrial
When
State
puppies, 2.
respiratory
Figure
3,
also
the
three
protein
is
The
biphasically. to
a 100%
of the of adult
found
plotted
days
by
to
by
the
first,
increase.
A
week
of life
first
days
faster second cytochrome
3,
puppies.
while
almost
state,
was
during
the
was against
maximal
thus,
is
first
a good
arterial
200’5’0,
slower
from
phase oxidase
value.
386
is
starts reaches
3 newborns.
seen
in
5 increases a fetal
term
level
days after
in
change during value
of
Cytochrome between
seen
of the
As
Adult
week.
occurs
State
PO2
newborn
as
cytochromes
level.
first
in
days,
when
Cytochrome
adult
rise
4-5
concentration.
almost
of the
examined
obtained
the
of life.
almost end
State
reach
of mitochondrial
first
in
capacity.
concentrations the
state,
respiratory
cytochrome
protein
reached
active
correlation
were
newborn
0. 21 nmoles/mg
as a function of age individual animal.
respiration
were
linear
during
first
This
-of mitochondrial
dramatically the
inverse
20
mitochondria
vitro.
rates
12 16 Days
their
mitochondrial
activities
Changes
In
respiratory
3 heart
An
6 Age,
PO2 an
of ADP, in
decreasing
Figure
4
1:
Figure
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0. 68
of
nmoles/mg
oxidase 0 and day
2.
a concentration
increase
2 and By
amounts
the of
end 50%
BIOCHEMKAL
Vol. 64, No. 1, 1975
Figure
State 3 respiratory activities of isolated heart mitochondria as a function of age. Glutamate and malate were used substrates. Each point indicates an individual animal.
2:
Utilizing
the
respiratory The
calculated
age
up
data
to
7 days.
of newborn
one
the
alterations
molecule
indicate
turnover
that
occurs
mitochondrial It
3,
in
be
in
the
noted
of
of cytochrome an
extremely
State
3 with
capacity
should
as
to that
absence
oxidase. large increasing
accumulate Cat’
transport
of added
phosphate
7.
transport During
adult
2 and
4,
mitochondria
day
ion
transient.
reach
Figure
the
heart
decreasing are
in oxidase
at
Figures per
dramatically.
completely
meters
shown
Similarly
capacity
The
in
calculated
of cytochrome
decreases
above
were
data,
calcium
ceases
presented
activity
decrease
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
and
respiratory
next
few
capacities
weeks
all
described
mitochondrial
para-
levels. DISCUSSION
The are (3-5).
data
sensitively Under
presented
here
affected acute
by hypoxia
support changes a 60%
our
earlier
of tissue decrease
387
contention oxygen of liver
that
mitochondria
concentration tissue
PO2
in in
the
vivo rat
Vol. 64, No. I,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r
0.75
d z e Q 0 f
0. I 5 c
\
t \o h 2 3
cytochrome
(0 t $I
0
s B c
o-
0
2
4
6
-I 6 Aqe
Figure
induced 20-40
twice
normal
minutes
a-- t a3, induced
(4).
of dog c and an
heart
hypoxia
mitochondria
mitochondrial each
oxygenation
induces
respiratory
chain, well
(3).
other increased and
as
of liver
also
induced
parallel
with
increased
human,
oxygenation
induces
decreased
animals
these
decreased
respiratory
in
of cytochrome
of hypoxic All
in
data
are
vivo
tissue
capacity
in
c
respiratory
a decrease
that
mitochondrial
that
increased
change.
demonstrating
c and for coefficients.
19 rnGml
mitochondria
Reoxygenation activity
in
I 6
, Days
capacities
-b concentrations
with
as
respiratory Chronic
opposite
agreement
animals,
I 6
Concentrations of heart mitochondrial cytochrome a t a3 as a function of age. For calculations and 160 mM-’ for a t a3 were used as extinction Each point indicates an individual animal.
3:
capacity
I 4
I 2
O0
chronically
in
per hypoxic
mitochondrial
respiratory
capacity. The in
vivo
site is
of interaction presently
not
of clear.
oxygen
with
However,
388
mitochondrial our
data
respiratory suggest
that
chain possibly
BIOCHEMICAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL
l
accumulation utilization
cat+
0 02
0
Figure
not
4:
only
one,
various
in
but
several
sites
this
and/
or
subst
Our who
and rate
no
respiratory Since
are effect
transport
is
rates
are
of State
are
transport,
as
3 respiration,
of
point
is
of changing
oxygen
in not
vitro
capable
unless
seems
those
in
of
Chance
The the
most
indicated
possibly
(7)
concentrations critically
of affecting
directly
mechanism
ADP
low
Liibbers
(8),
on mitochondrial tissue
mitochondrial
vitro,
and
PO2 respiratory
a cytoplasmically
mediated
were
reached. or
ion
in
vivo
necessary. ACKNOWLEDGMENTS
These 1 ROl
GM
studies 19867,
U. S. P. H. S.
CDA
a
involved.
mechanisms Cat+
mole
Each
functions
include
conditions
per
of age.
transport
to
capacities
control
accumulation
mitochondrial
contradictory
activity oxygen
6
transport.
findings
found
6
as a function animals.
These
under
4
Catf
membrane
of interaction.
report,
2
oxidase of 3-4
of the
carrier-mediated
probable
and
02 utilization cytochrome mean value
RESEARCH COMMUNICATIONS
were and
supported
by
U.
1 TOl-GM-01540.
lK04-GM
S. Leena
50318.
389
Public Mela
Health is
Service a recipient
Grants of
Vol. 64, No. 1, 1975
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8.
Hallman, Wilson, Park, Aberdeen, Mela, R. R. Goodwin, Surg.
M.
(1971) Biochim. Biophys. E. (1972) J. Neurochem. C. D., Mela, L., Wharton, R., E. (1973) J. Surg. Res. 14, L., Miller, L. D., Bacalzo, L. (1973) Ann. Surg. 178, 727-735. C. W. , Mela, L., Park, C. Forum 25, 185-187. J.
Acta 253, 19, 223-227. Reilly, J., 139-146. V., Olofsson, D.,
and
360-372. Fishbein, K.,
Campbell,
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