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

Effects of indomethacin on the metabolism of glucose by isolated rat kidney tubules.

Biochemxal Pharmacology, Vol. 26. Pelgamon pp. 2463 -2468. Press, 1977. Printed tn Great !\r~tarn. EFFECTS @F INDOMETHACIN ON ?HE METABOLISM...
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