Biochemxal
Pharmacology,
Vol. 26.
Pelgamon
pp. 2463 -2468.
Press,
1977.
Printed
tn Great
!\r~tarn.
EFFECTS @F INDOMETHACIN ON ?HE METABOLISM OF GLUCOSE BY ISOLATED PAT KIDNEY TUBULES
Gregory J. Cooney and Anthony G. Dawson Department of Biochemistry, University of Sydney, Sydney, N.S.W. 2006, Australia (Received I2Seprember I9 77)
The demonstration by Whitehouse
(1) that indomethacin, a non-
steroidal anti-inflammatory drug, uncoupled oxidative phosphorylation in isolated rat liver mitochondria has been amply confirmed (2 - 4) and the suggestion has been made that membrane sulphydryl groups are involved in this effect (2).
In addition indomethacin can induce
mitochondrial swelling through increasing mitochondrial permeability to alkali metal cations (3) and a recent report states that the drug also inhibits the electron transport chain at a site above cytochrome c (4). Such actions of indomethacin hint at the possibility that it might influence cell metabolism in ways which are unrelated to its role as an inhibitor of prostaglandin synthesis (5).
A previous report by one
of us (6) showed that
cylate, another anti-inflammatory drug,
affected m-
-at kidney tubules by uncouplinq oxidative -rest,therefore, to investigate whether lbule metabolism and, if so, whether ts uncoupling action. ules and their use in metabolic previously
(6 - 8).
Tubules (9mg
. phosphate-buffered medium
(6)
.on vessels shaken continuously td 2,4-dinitrophenol
(2,4-DNP)
Ise of its low solubility in 1 ethanolic solution to give systems containing ethanol few experiments were also
2464
Prellmmary Commumcatmns performed Oxygen were
consumption
used
glucose even
in which
indomethacin
was
was measured
manometrically
for the determination
(12).
though
Glucose
incubation glucose,
by liquid
on glass
CO2
Fnzymatic
not affected
by indomethacin
(13).
peroxidase, Possibly
acid deproteinization
during
the metabolism was
Protein
trapped after
methods
(11) and
inhibits
procedure
spectrometry
(8).
solution.
(lo), pyruvate
or L-Y1-14 Cllactate
discs
(9).
indomethacin
durinq
evolved
in aqueous
of the of D-rU- 14C!
in 2N-NaOH
precipitation
was measured
most
and as
bv the method
et al.(14).
The effects
of 2,4-DNP
by isolated
sole exogenous
kidney
substrate
i
.-
Fig.1.
incubated
tubules
value
on the consumption ,14 Clqlucose D-_
with
2,4-DMP
in Fig.1.
sliqhtly
T
T
of indomethacin
by tubules
and 2,4-DNP
metabolizinq
the rate of oxyqen activity.
Each
for 6 - 8 separate
uptake,
an effect
In contrast,
point
indome+
was
were
contain;
indomethac
represent-
experiments.
which
Tubules
medium
(1mM) and either
(0) as indicated.
+ S.E.M.
on oxvaen
qlucose.
for lh at 37O in 2ml of buffered
0.1 UCi of D-[U-l4 C:qlucose or 2,4-DNP
provided
are shown
I
Effects
consumption
and indomethacin
l.qr
a + :
uncoupling
that
scintillation
fibre
were
in the assay
removed 14
Cl-14Clpyruvate
of Lowry
oxygen
was
mixtures.
determined Ba14C03
reported
involved
of the indomethacin
of lactate
measurements
it has been
one of the enzymes
introduced
of as the increased
2465
Preliminary Communications
previously
found
either chain
did not
since
acid
respiration
was virtually
with
unaffected
consumption
The above
cycle
showed
to support
mechanisms
in the mitochondria.
2,4-DNP,
respiration
a study was made
on glucose
inhibitory
effects
2,4-DNP
on
respiratory substrate
again
stimulated
shown).
indomethacin without
strong
as the added
though
inhibited
directly
In order
the results
the ability
affecting
to determine
of the influence
metabolism:
This
or the mitochondrial
(data not
that
of glucose
was exerted
by direct
5mM 2-oxoglutarate
40%
in isolated
consumption.
by indomethacin
by about
results
phosphorylation
oxygen
seem to be caused
the tricarboxylic
oxygen
oxidative
(1 - 41, inhibited
mitochondria inhibition
to uncouple
oxidative
how this effect
of indomethacin, are presented
and of
in Fig.
2.
Cal
.fO. 15~
I_
~_.~
L _.__I___-
0.01
0
0.07
2,4-DNP
Fiq.2.
Effects
0
0.
I
0.2
lndom~ihacin L-
0
--.__L
c . ‘,
(mM)
_____1_d
3.01
on glucose
Tubules
were
0.07
incubated
Cal qlucose
as described used;
ICI lactate
formed.
point
represents
+ S.E.M.
for 6 - 8
mean
Each
value
and
metabolism.
Lb1 l4 CO2 produced;
the
0.03
separate 7,4-DNP
of indomethacin
2,4-DNP
in Fiq.1.
0.0:
(rnbt)
experiments.
(mM)
(0) or 2,4-DNP
Indomethacin
(0) present
as shown.
2466
Preliminary Communicatmns Both
indomethacin
kidney
tubules
metabolism while
the amount
its conversion whereas
2,4-DNP
indomethacin revealed
allowed
promoted
that
Cl-14C]pyruvate altered
its reduction
by indomethacin
occurred
when
in [
14
CO2 but
of 14co 2 indomethacin
stronqly
results
by
Clglucose
In contrast,
These
stimulated
indicated
that,
of glycolytically-produced Further
to lactate.
Neither
its conversion a slight
was present
pyruvate,
experiments
was not exerted
pyruvate.
though
indomethacin
14
to
of indomethacin
CO2 nor
Clglucose
in the liberation
small.
Clglucose
the oxidation
supplied 14
to
14
was
14
the increase
(Fig. 2b,c).
this effect
of exogenously
2,4-DNP
formed
of r
to lactate
the use of [
by an increase
of lactate
the oxidation
stimulated
With
(Fig. 2a).
was accompanied
inhibited
olism
and 2,4-DNP
on the metab-
the oxidation
to lactate
was
of added
siqnificantly
increase
in oxygen
consumption
(Fiq. 3).
From
it was
this
I i 0
L
1
L-_-.-l
0
0.
._2
0.2
I
Effect
Tubules
were
medium
formed
produced
Each
reduction
from glucose, involved
0.7
0.3
on pyruvate
,-’
CPM)
metabolism.
for 30 min at 37O in 2ml of buffered
(2.5mM). 0.1 1?Ci of rl- 14C]pyruvate 14 CO2 produced (0) and lactate [bl point
represents
indomethacin
the mean
+ S.E.M.
value
lowered
inhibited
the oxidation
it did not do so by interfer.ng in pyruvate
had to be looked
which
0. I
0
,”
1
___I
experiments.
that although
indomethacin
-_A
_____I.~_
consumed;
(0).
the mechanisms
L
lndomethacin
of indomethacin
incubated
for 8 separate concluded
L
(mM)
containinq
[a! oxygen
i!
ii . 3
irdomethacii
Fiq.3.
-
+
upon
the amount
as a positive available
directly
Instead
oxidation.
of pyruvate with
the effect
stimulation
for oxidation.
of
of pyruvate
2467
It may be speculated pyruvate
to lactate ratio
NADH/NAD+ position ratio
when
indomethacin
to any
increase
affect
glucose
or ethanolic
metabolism
Moreover,
account
for an increase
qlycolysis,
malate
"shuttles"
in Fig.
in this
the transfer
tissue
Results
(17).
was
it was
found
added
dehydrogenase
ratio
is that
to
produced
phosphate
this
the metabolism
carbo-
indomethacin
via the glycerol with
in the
hypothesis
equivalents,
consistent
to
in aqueous
activity
not to influence
reducing
it can be seen that
by this
can not be attributed
An attractive
in the NADH/NAD+
of cytoplasmic
the equilibrium
indomethacin
appears
(16).
in the cytoplasmic
is determined
ratio
alcohol
to the mitochondria
Here
4.
reaction
since
is low and ethanol
metabolism
during
oxidatron
since
in the same way whether
solution.
hydrate
inhibits
is present
in the NADH/NAD+
in ethanol
cortex
of an increase
dehydrogenase
An increase
(15).
of qlycolytically-produced
the diversion
is the consequence
of the lactate
rat kidney
that
and/or
idea are given
of L-cl-
14
Cllactate
: 0.4 .CT .a, K$ t 2 -a 1.5m e l.O\ -
;
\ d
0 E 2
z
72 g
3 m z 0 N 0
d
“\‘ 0.5-
\
0.2
;
, I
" ," 0.1
0-l
1 0.1
0
I 0.2
1 0.3
lndomethacin
Fig.
4.
Tubules medium
Effect were
consumed
incubated
(0).
affected
L.-i
Indomethacil
on lactate
Each
0.3
0.2
0.1
CrrlM!
metabolism.
for lh at 37O in 2ml of buffered
0.1 UCi of L-[l- 14C]lactate
consumed;
for 6 separate
0' N 0 q0 0 I I--I 0
(mM)
of indomethacin
containing
[al oxygen
was
K$
a g10.3 : E \ _ 0 t
[b] l4 CO2 produced point
represents
(2.5mM).
(0) and lactate the mean
value
+ S.F.M.
experiments.
by indomethacin
in the same way
as was
the metabolism
of
, glucose. inhibited
Oxygen
consumption
by indomethacin
lactate
and its conversion
earlier
that pyruvate
by tubules
metabolizing
(Fig. 2a) as were
the overall
to l4CO 2 (Fig. 2b).
oxidation
lactate
was unaffected
Since
was
consumption it was
by indomethacin
shown it
of
Prelmlmary