Kinetics of post-exercise phosphate transport in human skeletal muscle: An in vivo 31P-MR spectroscopy study

Vol. 176, No. 3, 1991 May 15, 1991

KINETICS

BIOPHYSICALRESEARCH

BIOCHEMICALAND

OF POST-EXERCISE

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PHOSPHATE TRANSPORT IN HUMAN SKELETAL

MUSCLE:

AN IN VIVO 31 P-MR SPECTROSCOPY STUDY

S. IOTTI,

R. FUNICELLO,

P. ZANIOL*,

Cattedra di Biologia Molecolare, Istituto Universita di Bologna, via U. Foscolo, *Istituto

April

di Clinica Neurologica, 7 - 40123 Bologna, ITALY

di Radiologia, Universita de1 Pozzo, 41100 Modena,

via Received

and B. BARB IROL

di Modena, ITALY

5, 1991

Summary. 31-Phosphorus magnetic resonance spectroscopy was used to investigate in vivo the kinetics of inorganic phosphate transport and intracellular Intracellular pH further decrepH after exercise in human skeletal muscle. ased from the value reached at the end of work showing a minimum between 25 and 45 set and then increased back to the resting value. Inorganic phosphate showed an initial fast rate of recovery corresponding to the decreasing phase corresponded to of PH, and a second phase in which a slow rate of recovery increasing pH. The biphasic patterns of both phosphate and pH recoveries are in agreement with and support in vitro evidence that Pi transport into mitochondria is modulated by pH. 0 1991 Academic Press, mc.

Many aspects dated

by 31-phosphorus

vivo

both

work

has focussed

in

(2)

cellular

acidosis

and

However, recovery

kinetics (9)

the

This of

inorganic

rate study

0006-291X/91

aimed

phosphate

information

correlation

(Pi)

in

linking the

and the

investigate relation

the to

available

the

1204

extent

(3)

during

exercise.

kinetics recovery

of

intra-

(3,

9).

on the

kinetics

exercise

end-of-exercise authors

after

and

Even in a recent

do not

intracellular

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

the

Much

recovery

resynthesis

generated

after

recovery,

to

linking

is

(10-18).

(PCr)

of PCr post-exercise

pH recovery

of Pi recovery

conditions

phosphocreatine

relationships

phosphate

a linear

inorganic

Copyright All rights

little

metabolism have been eluci31 spectroscopy ( P-MRS) performed -in

pathological

of

kinetics

of phosphocreatine

between

and

qualitative

and the

energy

resonance

regulation

intracellular

reporting

kinetics

on the the

muscle

(l-9)

relatively of

of

magnetic

physiological

exercise

of

of human skeletal

acidosis find

of

post-exercise cytosolic

paper to

the

any correlation

pH at the of

the

end of work. recovery pH. Results

Vol.

176, No. 3, 1991

show

that

the

creasing

and the

direct

influence

MATERIALS

rate low

linking

BIOCHEMICALAND

rate

of when

of

Pi

Pi

transport

cytosolic recovery

of pH on phosphate

BIOPHYSICALRESEARCH

is pH is and

high

when

increasing,

the

COMMUNICATIONS

intracellular showing

intracellular

pH is

de-

a relationship

pH and indicating

a

transport.

AND METHODS

NMR spectra were acquired on a G.E. 1.5 T Magnetic resonance spectroscopy: Signa System with a spectroscopy accessory as recently described (18). Briefly, radio frequency pulses at 25.866 MHz with a pulse width of 400 usec and a transmitter power of 0.5 kW (approximately 90 degrees flip angle at the center of the coil) were transmitted by a surface coil supplied by G.E. (20.5 cm diameter) and the resonance signals were collected by a 7.5 cm receiving coil. A data table of 1K complex points was collected for each FID. The band width was 4 kHz. The delay between transmission and reception was 0.5 msec sequence and the acquisition duration was 250 usec. The stimulation-response was repeated every 5 sec. All studies were performed on gastrocnemius muscle by placing the surAfter optimizing magnetic field homogeneity face coil directly on the skin. (FWMH 0.25-0.35 ppm) 120 transients were accumulated during rest (10 min), then exercise was begun and data collected for 2 min (24 FID's) following a 2 min "preparation" time for each level of work. During post-exercise recovery 2-FID data blocks (10 set) were recorded during the first 70 set, and longer time blocks thereafter (20 or 30 set blocks corresponding to 4 or 6 FIDs, respectively) were collected for another 9 min. The accumulated spectra were transferred to a Nicolet 1280 data station and processed using a 4 Hz line broadening and a manual phasing. The signal-to-noise ratio for l3-ATP was Peak areas were used to calculate the phospho35-40 at rest (120 FID's). creatine to inorganic phosphate (PCr/Pi) and the inorganic phosphate to R-ATP (Pi/O-ATP) ratios. Intracellular pH: The pH calculation using a chemical shift of dibasic, 3.29, 5.68, and 6.77, respectively determined from the center of the PCr

from the chemical shift of Pi was made monobasic phosphoric acid and a pKa of The chemical shift was carefully (19). peak to the center of the Pi peak.

Six male and four female (cases 2, 3, 7, Volunteers and Exercise Protocol: and 9) normal volunteers aged 20 to 30 accustomed to moderate physical activity were selected. No athletes were included in our study. Informed consent was obtained for all studies. All subjects performed isokinetic exercise by pressing a pedal (plantar flexion) connected with a pneumatic ergometer (20). One contraction was performed every 5 set immediately after acquisition Typically 6-8 levels of steady-state work for 4 min at every level of work. were reached by all subjects.

RESULTS AND DISCUSSION Figure lunteer

no.

1A reports 5 at

the

the

spectrum

last

level

of working of

steady-state 1205

gastrocnemius work.

muscle All

volunteers

from

vowere

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICALRESEARCH

COMMUNICATIONS

A

Pi

*

I3

yyyyJyJ&-f$(-+;, 10

Figure 1. Z-min normal volunteer (4 FIDs) spectrum

asked to

to have

pH

1 2 3 4 5 6 7 a 9 10

-20

they

reached

level

at

the

of

end

of

a PCr/Pi

metabolic work.

In

ratio activation,

our

experiments

"'P-MRS data of human gastrocnemius exercise. Values of time of minimum curve "a" are given in set from the

Table 1. steady-state

Case no.

until

comparable

intracellular

-10

PpM

(24 FIDs) spectrum of working gastrocnemius (no. 5) at the last level of steady-state from the same subject at 85 set of recovery

exercise a

0

Last level steady-state PCr/Pi

pH

0.70 1.20 0.90 0.90 1.05 1.20 1.10 1.00 0.90 0.75

7.00 6.98 6.99 6.94 7.00 6.93 6.89 6.86 7.00 6.89

of work Pi/B-ATP 4.70 4.10 4.90 4.90 4.30 4.60 4.50 4.60 4.50 4.20

Time of minimum PH (set) 25 45 45 45 45 45 45 45 45 35

1204

close

muscle work (A). (B).

from a 20-set

to

(Table

Pi

35 35 45 45 45 45 45 45 45 35

accumulation

none

muscle during pH and of last end of exercise

Time of last point of curve "a" (set)

unity

of

recovery point

Pi/R-ATP at rest

1.10 1.00 1.20 1.60 1.50 1.30 1.30 1.20 1.51 1.10

the

1) and

subjects

from fitting

Pi/R-ATP at 85 set of recovery 0.60 0.90 0.90 0.60 0.70 0.90 0.60 0.60 1.19 0.70

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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0.5 i 0

30 RECOVERY

60 90 TIME (set)

120

Figure 2. Post-exercise recovery of intracellular pH (A) and inorganic phosphate (B) in human gastrocnemius muscle (case 10). Zero time values correspond to the last steady-state level of work. In graph 6 experimental points best fitted separately part (al and part (b) of the same monoexponential function. Curve in part (a): R = 0.9917; TC = 29.9 sec. Curve in part (b): R = 0.9927; TC = 58.1 sec.

examined Park

showed

either

during

et al.

(21):

Pi lines

Figure

1B reports

the

Zero

time

the

level

of

steady-state

riments

3 min

of

exercise

brium,

steady-state 5%. During

data

blocks

(2 FIDs

the

signal

at rest

to noise 85 set

Figure the

each),

first

first

part

value

reached

of

in at back

data

post-exercise

which the to

subjects

is

of

recovery. characterized

intracellular

last

level

the

resting

were

value.

1207

This

line

intensilo-set

collected

to

improve

was found

lower

than

11.

These

and

equili-

seven

pH changes

data

are

by two markedly

work,

expe-

first

intracellular

pH shows of

the

to be

In our

a steady-state

Pi signal

(Table

pattern

4 min.

of PCr and Pi

blocks

by

of post-

was assumed

for

recorded

the

the exercise

a typical

pH recovery (a)

20-set

at 85 set

recovery

reach

a variation having

In all

stopping

2A reports

and show that

pH increases

ratio.

after

2 min

two

to

after

recovery,

same subject

performed

sufficient

as reported

and symmetrical.

pH and Pi

being

considered

Pi peak

unique

the

for

work

a split

were

from

point

were

being

within

cases

spectrum

recovery.

ties

or recovery

in all

exercise last

work

a further

a second pattern

part

from

case

during no.

10,

different

parts.

decrease

from

the

in which

the

(b)

was found

in

all

A

sub-

Vol.

176,

No.

jects

examined,

the

25 and 45 set Figure

minimum

in all

from ratio.

inorganic

The

B-ATP

for

The

pattern

low

of

exponential

equation.

points

were

best

ratios

plotted

exponential

was

concentration

relatively Pi

levels

On the

in

by the

nature

used

as an

based

on the

scale Pi

being

between

as inorganic internal

separate

against

of

the

that

ATP remains

All

by a single groups

mono-

of experimental

function,

and the

clearly

demonstrate

time

recovery.

to

15).

be described

two

phosphate

calibration

assumption

(13,

not

2 min of post-exercise

same monoexponential

of

COMMUNICATIONS

recovery

first

reported

hand,

a logarithmic

bi-phasic

are

could

other

during

the

of V/Vmax

recovery

fitted

during

Data

signal

RESEARCH

1).

Pi pattern

same subject.

BIOPHYSICAL

of pH reached

(Table

the

phosphate

constant

the

AND

value

subjects

2B reports

recovery B-ATP

BIOCHEMICAL

3, 1991

subjects

studied

Pi/ATP the

showed

the

same pattern. Interestingly value

during

recovery

10 subjects, found.

enough,

while

The rate

(b)

of

was

31 set

of

recovery:

much.

A high

ing,

while

resting

rate it

the

other

recovery average

all

cases

low

last

2 (cases was very

time

point

curve

(b)

was found

(a)

a lo-set in

(TC)

intracellular

of curve

different

constant

for

minimum

1 and 2)

the

(10

two

subjects)

TC was twice

intracellular

pH was

must

be transported

in

pH 8 out

discrepancy

to

when intracellular

when

phosphate

Present vivo

control

pathologies

are

transport

by a membrane

Our in

of ADP to occur. pH recovery

stimulated

muscle

Pi

phosphate

intracellular

the

the

the

parts for

of was

(a)

and

curve

(a)

three

times

as

pH was decreas-

increasing

back

to

the

value.

phosphorylation

that

in

that

to the

of Pi recovery was

Inorganic for

in

1 shows

corresponds

the

while

Table

results of that

form

The peculiar

consistent catalyzed

pH gradient the

Pi transport may involve

into

with by

(22, basis

the

mitochondrial

biphasic

patterns

and support a specific

in

matrix of Pi and

vitro

transport

evidence protein

is

23). for

further

by cytosolic Pi transport

experiments pH,

and to

to elucidate better

evaluate

(24 1.

ACKNOWLEDGMENTS This work was supported by grants from MURST (quota 40% and 60%). The Magnetic Resonance System is a generous gift of the late Mr. Enzo Ferrari (Maranello, Modena) who made this study possible. R.F. is recipient of a UILDM - Sezione di Modena fellowship. 1208

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES

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24.

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