incorporation of 15n-leucine amine into atp of fast-twitch muscle following stimulation

Vol. 128, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 16, 1985 Pages 1254-l 260

INCORPORATION OF 15N-LEUCINE AMINE INTO ATP OF FAST-TWITCH MUSCLE FOLLOWING STlMULATIflN

Jan Gorski, David A. Hood, Oliver M. Brown and Ronald L. Terjung

Departments of Physiology and Pharmacology Upstate Medical Center

State University of New York Syracuse, New York 13210

Received March 22, 1985

SUMMARY: During intense contraction conditions, ATP content in fast-twitch muscle rapidly decreases (approx. 50%) by the deamination of AMP to IMP and NH . During recovery, the ATP content returns to normal by the reamination of IM8 from aspartate. We evaluated whether the donor amine may be obtained from brgnched chain amino acid uptake by perfusjfg muscle in situ with 1.0 mM I: Nl-leucine during a 1 hr recovery. [ N]-enriched adenine nucle ide accounted for 14% to 24% of the IMP reaminated, depending on whether [ fit N]- leucine was provided only during the recovery period or, in addition, 30 min prior to stimulation. Thus, the uptake of leucine by fast-twitch muscle may provide an important source of amine for adenine nucleotide resynthesis following contractions. 0 1985 Academic Press, Inc.

During fairly intense contraction conditions, mammalian fast-twitch

muscle can suffer a large loss in ATP concentration. The decline in ATP of as

much as 50% is not found as an increase in ADP or AMP contents, but results in

a stoichiometric increase in IMP concentration (approx. 3-4 umole/g) within

the muscle (l-5). This 70 - 90 fold increase in IMP is produced by the action

of AMP deaminase, the first reaction of the purine nucleotide cycle (6). The

IMP remains within the muscle and is used for adenylate resynthesis during

recovery following muscle contractions (3,4). This occurs via the return leg

of the purine nucleotide cycle and involves the enzymes adenylosuccinate

synthetase and adenylosuccinase (6). In addition, the restoration of adeny-

late concentration to resting levels requires an amine donated by aspartate.

Since the aspartate content within the muscle (approx. 0.3 umole/g wet weight)

is a small fraction of that needed to supply the amine nitrogen for adenylate

resynthesis, the amine must come from another source.

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. 1254


Muscle is a major tissue for branched chain amino acid oxidation (7,8)

and the uptake of leucine by muscle is increased during contractions (9). In

addition, recent evidence indicates that branched chain amino acid oxidation

increases during exercise (10-13). Further, a greater rate of branched chain

amino acid transamination is evident following exercise (14). Although the

oxidation of leucine diminishes following exercise, an increased plasma con-

centration of branched chain keto acids, presumably released from the working

muscles, is found (11). Thus, it is possible that the glutamate generated via

branched chain amino acid transamination could represent a significant source

of amine for the resynthesis of adenylates from IMP and aspartate. We tested

this hypothesis by supplying [15N]-leucine to muscle during recovery using a

perfused rat hindlimb preparation.

METHODS

Adult male rats (355 + 12 g) obtained from Taconic Farms, Germantown, N.Y.) were anesthetized (50 mg pentobarbital per kg body weight, IP) and prepared for hindlimb perfusion as described by Ruder-man, et al. (15). The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing rejuvenated red blood cells (Hb approx. 12 g/100 ml), as described by Rennie and Holloszy (16), 4.0 g/l?! ml albumin, 100 uU/ml insulin, 5 t@i glucose, 0.15 mM pyruvate, and 1.0 mM [ Nl-leucine, pH 7.4. The gastrocnemius-plantaris- soleus muscle group was stimulated for 3 minutes via the sciatic nerve (60 tetanic contractions/min; each 100 duration at 100 Hz, 0.1 msec square wave at 6 V) as used previously (3). These stimulation conditions result in a 50% depletion of the ATP content in the fast-twitch muscle (3). In the first series of animals, stimulation.

hindlimb perfusion was begun within 5 minutes follqqng muscle In Series II, perfusion with the medium containing [ Nl-leucine

was begun 30 minutes qr$or to the stimulation period to permit time for equilibration of the [ N]-leucine within the muscle. In each case, the hindlimb effluent was discarded during the first 20 min post-contraction, since we did not want to recycle the metabolites (e.g., lactate, ammonia) that leave contracting muscle (5). Following a 60 minute recovery period, the stimulated/recovered plantaris muscle and then the contralateral non- stimulated plantaris muscle were clamp-frozen with aluminum tongs cooled to liquid N temperature.

Ass $y procedures. The muscles were extracted in cold alcoholic per- chloric acid, Eutralized and used for assay as previously reported (3). Evaluation of [ ‘NJ-amine incorporation into adenine nucleotides was performed on the derived adenine using a mass spectrometry technique (Finnigan 3100 mass spectrometer equipped with a 6100 data system and an electron ionization source). Adenine was prepared by acid hydrolysis (3.5% HC104 at 100° for 1 hour) of the adenine nucleotide isolated from the muscle extracts by activated charcoal (80% recovery), similar to that described by Vinkler, et al. (17). The resulting adenine originates primarily from ATP, since muscle ATP content is approximately 90% of the total adenine nucleotides (3,4). The mass spectrum of an adenine standard at 70 eV appears in Figure lA, and shows the characteristic fragments for adeninq (18), base peak and the molecular ion (M ),

including m/e 135 whic$ is both the m/e 136 which is (M + 1) and m/e 108

and 81. We monitored m/e 135 and 136 for all samples as they were released

1255


w from a enine The mass spectrum of an adefine standard (A), adenine prepared

nucleotides isolated from [ N&free muscle (B), and adenine prepared from a&nine nucleotides isolated from stimulated/recovered muscle ppgfused with [ N]-leucine (C). Note the shift in m/e 135/136 ratio with [ NJ-enrichment (C).

from the solid probe by controlled heating. The areas under the resulting curves (pseudo-maf5 fragmentograms) were used to calculate m/e 135/136 ratios.

The amount of [ N]-adenine was assessed from the shift in the 135/136 m/e ratio and related to the amount of IMP reaminated following stimulation. Although the adenine samples, prepared as described above, yielded more fragments than purified adenine, they were at lower m/e than those of interest and did not interfere with the adenine quantitation. This was verified by mass spectra of adenine obtained from control (Figure 1B) and experimental (Figure 1C) muscle samples that were further purified by paper chromatogrfghy.

All reagents were obtained from Sigma Chemical Co. except for I: Nl- leucine which was obtained from KOR Isotopes, Cambridge, MA.

RESULTS

The 135/136 ratio of 10.4 + 0.57 (n=6) for adenine prepared from [15N]-

free muscle was the same as that obtained with purified adenine. The 135/136

ratio of adenine prepared from the non-stimulated contralateral control

muscles of 8.71 t 0.38 represented an incorporation of 0.16 Pmole/g over the

60 -90 min perfusion period. Although this enrichment represents a low basal

rate of purine nucleotide cycle turnover (tI,2 approx. 34-50 hours), consis-

tent with the low rate of AMP deaminase activity expected for resting muscle

(19), our experiments were not carried out long enough to provide a meaningful

estimate of the basal rate of IMP reamination, independent of muscle stimula-

tion. Following 50% depletion of the adenine nucleotide content the incor-

poration of [15N]-amine was significant, representing approximately 14 - 24%

of the derived amine (Table l), depending on whether C15N]-leucine was

available during only the recovery period or, in addition, during the prior 30

min equilibration period.

1256

TABL

E 1.

IN

CORP

ORA

TIO

N OF

[1

5N]-L

EUCI

NE

AMIN

E IN

TO

ATP

DURI

NG

RECO

VERY

FO

LLOW

ING

STIM

ULAT

ION

ATP

RESY

NTHE

SIZE

DC

NETd

m

/e

ATP

4;

[15N

] um

ole/

g TA

Nb

[15N

l-TAN

FR

OM

IMP

%

FROM

%

FR

OM

1351

136

nmol

e/

g um

ol

e/g

umol

e/

g ['5

N]-L

EU

[15~

1-LE

U

Cont

rala

tera

l Co

ntro

l Le

ga

(N=lO

) 8.

71

2.04

6.

79

7.64

0.

16

+0.3

8 +a

.55

20.1

3 20

.13

20.0

42

[15N

]-Leu

cine

Durin

g 1

Hr

Reco

very

On

ly (N

=7)

5.52

8.

39

6.13

6.

98

0.59

2.

80

20.9

14

.3%

+0

.39

t1.2

1 kO

.22

LO.2

2 LO

.088

AO

.17

~2.7

7 23

.34

[15N

]-Leu

cine

30

Min

Befo

re

1 Hr

Re

cove

ry

(N=7

)

4.77

to

.28

-

10.7

0 Ll

.07

5.95

+0

.24

-

6.80

0.

73

~0.2

4 ~0

.078

2.

54

to.2

1 -

29.3

L3

.09

23.6

%

22.7

8

Valu

es

are

x+S.

E.

aCom

bina

tion

of

anim

als

perfu

sed

in

Serie

s I

and

Serie

s II.

bT

AN=T

otal

Ad

enin

e Nu

cleot

ides

. -O

btain

ed

by

incl

udin

g AD

P (0

.75

Umol

eJg)

an

d AM

P (0

.05

umol

eJg)

co

nten

ts

(3).

'Obt

ained

fro

m

the

mea

sure

d AT

P co

nten

t m

inus

on

e-ha

f

the

cont

rala

tera

l m

uscle

te

tani

Jmin

de

plet

es

ATP

by

50%

(3

). 6

Corre

cted

ATf5

co

nten

t, si

nce

stim

ulat

ed

at

60

for

the

amou

nt

of

[ N]

-TAN

m

easu

red

in

non-

stim

ulat

ed

cont

rol

mus

cle.


DISCUSSION

A large enrichment of [15N1-ATP was found in recovered muscle in Series

I, even though the C15N1-leucine was available to the muscle only during the 1

hour recovery period. This attests to the significant rate of leucine uptake

and transamination in skeletal muscle. The sequence of events for the [15N]-

leucine amine to appear in ATP must have included uptake of the extracellular

leucine, production of [15N]-glutamate via the branched chain amino acid

transaminase, and production of C15N]-aspartate via glutamate-oxaloacetate

transaminase. The [15N1-aspartate then donated the [15N]-amine to IMP via the

activity of adenylosuccinate synthetase. In each of the transamination

reactions the [15N]-amine would be diluted with the amine of intracellular

origin. Thus, the net incorporation of 14.3% of the IMP reamination was

probably an underestimate of the potential rate of amine originating from

extracellular leucine. This was verified in Series II where the perfusion

medium containing 1.0 t&i [15N]-leucine was initiated 30 min prior to muscle

stimulation. This additional time should have permitted equilibration of the

branched chain transamination process with extracellular [l'N]-leucine (8) and

a better saturation of the muscle's glutamate and aspartate pools with [15Nl-

amine. This procedure increased the enrichment of [15N]-adenine nucleotides

to approximately 25% of the IMP reaminated. Collectively, these results

indicate that extracellular leucine taken into the muscle can serve as an

important source of amine used to recover adenine nucleotide content following

intense contraction conditions.

It is probable that our results are physiologically relevant. Skeletal

muscle serves as a major tissue for the uptake and oxidation of branched chain

amino acids (7,8). While the uptake of leucine is enhanced during muscle

contractions (9) its oxidation contributes only a relatively small fraction of

the total energy supply (10-13). Further, muscle has an abundance of other

energy substrates (e.g., extracellular glucose, glycogen, fatty acids) avail-

able for oxidation. Therefore, the significance of branched chain amino acid

uptake by muscle may be coupled to another function, independent of its use as

1258


a carbon source for oxidation. This may be related to its amine supply as

identified by our data. For example, the uptake of leucine is increased post

exercise (14) and it is likely that the efflux of branched chain keto acids

from muscle accounts for the elevated plasma concentration during recovery

after exercise (20). This could yield a net deposition of amine within the

muscle for IMP reamination. Alternatively, the amine from leucine could be

transaminated to alanine (21) and released from the muscle. This latter

process may be dominant in resting muscle when the rate of IMP reamination is

not excessive. Although we used a high perfusion concentration of leucine in

these experiments, estimates of the rate of leucine uptake by skeletal muscle

at normal plasma concentration (7) indicate that the enrichment of adenine

nucleotides by the amine of leucine that we observed, could also occur in -

gvJL Thus, the present experiments suggest a potentially important role for

the branched chain amino acid uptake observed in muscle following intense

muscle contractions.

ACKNOWLEDGEMENTS

This work was supported by NIH Grant AM 21617 and NIH Research Career Development Award AM 00681 (to R.L.T.). The excellent technical assistance of Mrs. Judy Freshour is greatly appreciated. Jan Gorski was on leave from the Bialystok Medical School, Bialystok Poland, supported, in part, by the Francis Hendricks Endowment for Medical Research.

1. 2.

3. 4. 5.

6. 7. 8.

9.

10. 11.

12.

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incorporation of 15n-leucine amine into atp of fast-twitch muscle following stimulation

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