L-Proline uptake in human fibroblasts: Evidence for a high-affinity system in addition to system A

Vol.

142,

January

No. 15,

BIOCHEMICAL

1, 1987

BIOPHYSICAL

COMMUNICATIONS

RESEARCH

Pages

1987

L-PROLINE

UPTAKE

IN

HUMAN SYSTEM

Madeleine

des

IN

TO

ADDITION

Central

de l3iochimie I-e Kremlin-

20,

FOR

SYSTEM

Nicole , and Alain

CuI!PIAT*

221-227

A HICII-AI-FINITY

A

WATTI', LEMONNICR*

Juan

MAlX'AII

- Centre tHospitaller Bic&tre, France and

Pharm::ceutiques et 92296 Ch~tcnay-llalabry,

Scierlces

October

: EVIDFNCE

Audrey

?4275

$ Facult6

FIBROULASI-S

FCNIIANT-TIII[IB~JLT",

Laboratoire

Received

AND

de

Rioloqiques

de

IO”,

Uict'tre,

l'Univcrsit6

Paris-Sud,

rrnricr?

1986

in hurnan skin fibroblasts by ::i.multoneot:s Proline uptake was studied running of kinetic and inhibition experiments on the same cell lines. Two systems for proline uptake were shown : a high-affinity system not inhibited by a-(methylomino)isobutyric acid and a 30~ affinity system inhibited by this amino acid (i.e. system A). These results appear to be of interest, firstly because up till now, system A was considered preferable for prolinc uptake in human fibroblasts, and secondly because tilcy illustrate the need for combined inhibition and kinetic studies of amino acid uptake, especially .when the substrate concentration range used and the respective Km of the systems do not allow their dktection by kinetic analysis alone. Furthermore, this high-affinity system may have major physiological implications, a 1987 Academic Press, Inc.

We previously uptake Since

in

human

L-proline

activity by

cultured

by

Me-AIR,

may system and

studied

be

this

uptake

system.

experiments

on

same

MATERIALS

abdominal conditions 'Gazzola

AND

the

a

type

defined

(Z),

on uptake

amino we

acid

examined

results

1Je therefore skin

one

tracer

obtained

human

cycloleucine

fibroblast

in the

for

of

evaluating

the of

the

existence kinetic

amino

acid

only

(1).

proline

inhibition

simultaneous cell

kinetics

system

suggesting

conducted

ttle

transport proline

more than

of and

uptake

inhibition

lines.

METHODS

Human Cibroblasts were obtained from skin biopsies taken from children during surgery. Five different cell lines were grown under perfectly standardized as previously described (3). We adopted the cluster tray method described by et al. (4) with minor modifications.

sbreviations Phosphate-buffered System

of

and as

cell

surprise

one proline

effect

fibroblasts considered

A in

to our

the

A,

: Pie-BIB saline

alanine-preferring

solution, system,

isotctyric

: a-(methylamino) pl-i

7.45,

System

supplemented AX,

acid, with

alanine,

P.O.S-G

5.56

ser.tnr

ml.1 glk~ose,

2nd

c:ysteinc-ptcf~:rrjn7

system. 0006-291X/87 Copyrght 221

All

rights

0

1987

of reproduction

by Academic in any

$1.50 Press,

,form

Inc.

reserved.

Vol.

142,

No.

1, 1987

BlOCHEMlCALANDBlOPHYSlCAL

RESEARCH

COMMUNICATIONS

For measurement of amino acid transport, about 3 X 104 ceils between passages 3 and 6 were seeded in 2 cm2 wells of disposable mcrlLiwcl1 trays (Costar) and incubated for 72 or 96 h in 1 ml of growth medium, always replaced 24 I-I before each experiment., At this time the cells were just reaching confluency as estimated visually with an inverted phase microscope. The procedure for amino acid transport then began with PO min of preincubation with PBS-G, after which excess liquid was removed and the experimental cover immediately placed over the drained monolayers. The 24 tubes in the experimental covers each contained 0.3 ml of PBS-G, comprising 0.5 VCi of L-[u-"'Cl proline (260 mCi/ mmol, CEA-Gif sur Yvette France) plus the desired concentration of the non radioactive Lproline (Sigma Chemical Co) and when necessary, of the inhibitor. The contents of the 24 tubes were simultaneously transferred to the monolayers in 24 wells by vigorous horizontal shaking 01' the r/hole iray and cover, 3ric incubdtcd for 1 minutn at 37°C. Transport assays were terminated by dumping tlhe uptake riwdi.r.rrn in orle rrwti
KSULTS

AND

DISCUSSIOrj . Inhibition We chose

performed were

on

an

cell

uptake

ibitor-to-substrate

inh

lines

for

incubated

proline

of

l.arid

1 min,

in

As

2. the

ratio shown

presence

lri or

of 10 (6,

7;.

'ruble

I,

six

prollrte

absence

of

inhibitor.

These

were

experiments concentrations

proline

The

concen-

w Effect

of Me-AIB,

serrne

(5er)

or glycine

L-praline

NO"C?

A 2

Ser**

ME-RIB

uptake

in human flbroblasts

A Inhib

Uptake

% Inhib

20

14.8

21

la.1 26.1 35.5 48.0 65.0

79.2 61.7 56.9 47.3 a.0 8.5

29 31 31 31 44 44

9.6 11.9 17.1 26.8 34.0 46.4

86.9 74.8 71.7 60.2 35.0 34.0

16.7 29.5 31.3 37.5 49.3 57.8

la.1 24.0 24.0 14.4 1.6 0

Uptake

24 24 29 25 42 40

71.3 47.3 60.5 67.4 52.3 71.1

22 25 24 23 40 34

21.5 19.5 25.2 32.6 37.4 52.5

69.a

0.2 0.1 0.05 0.025 0.010

58.8

53.3 51.6 29.5 26.2

22 22 32 28

0.5 0.2 0.1 0.05 0.025 0.010

36 41 38 38 39 42

20.4 39.0 41.2 43.8 50.1 57.8

34 42 36 35 42 38

12.5 24.1 26.3 34.4 36.6 51.2

30.7 58.2 36.2 21.5 26.9 11.4

35 40 34 34 36 34

Protein*

(dpm/pg protein) YBS measured at 1 min incubation time in two different ranging from 0.010 to 0.5 ml1 in the presence of 10 [S] inhibitor, as described Each result is the average of 6 experimental values.

pg per well

+w cx*

Effect Effect

of Ser on cell of Gly on cell

line line

1 2

222

and Ser

Protein+

Protein*

l

Me-RIB % Inhlb

Uptake

Uptake

01‘ ay*+* Uptake

Protein' 0.5

N i ;:t

on L-praline

INHIEITOR

concentration (mW

E ;: 2 i

(Gly)

cell lines For proline concentrations under Materials and Methods.

([S])

Vol.

142,

tration

range

systems,

used

of

more

Even

0.010 amino

respectively.

30

10

[S]

did

not

clxceed

!;*;C

or

iciinoglycine

than

in

of

this

that

I

than

for

we

also

noticed

to

cell

line

density

cell.

line

that

of

of

cell

the

it

the

be

no

Scrine

may

to

known

ovary

cells,

the

D ne\u

system

contributjng

the of

iTn!y

obwrved

A: i

min

111 prollnr:

of

the

and

uptake

0.025

mM.

a system

other

thar1

tested

(results

also in

the

by

Ple-AIB

absence

of‘

density)

c~as

wit11

the

whose

A,

inhibitiol the

of tif

density

riot

of

A,

rr;uch

our

experimental rates

smaller

in

conof

these line

1

sensitivity

dwreascs

3s ceil

uptake

‘lint:

cell

for

cell

well-&.nown

this

than

larger

was

p:oline

shown)

sodium

growth

acti.vitv

inhibitiorl

the

proline

the

cliff?rc:nt

agreement

is

by

both

the

best

the

ASC

serine

and

al

the

fact

with (12)

(preincubation

foun?

in

the

fc;:,

shown

human

in

LIP MD-IIIB

is

lomr

than

the

DO 9; inhibition serum)

show

that

a

very

RX

A I'/),

hamster

al

(8)

In

humarl

tt131

at

i, cunci:r:t

19).

i!~!i;:~-:I]

UT‘ ~2.2

hc:rc,

p;Jrt.iclpEit.!

but to

to

define fit?rar;1-

et

al

i 1!

:~i[: :ti'tr:r

.I:) ;iSC , ii 'r's!: on

t-lowcver,

fle-AIB,

thougil

et

: o c;>;.,:

r?tcre known

in

P.

1 I: uptakr:

tl~r

that,

even

Chinese

systcrll

lildii,::te

is

characterized system,

In

cor!cerltr;!iio~,

by

OF

been

Moffett.

called

conceniration.

sub:;trate

fibro!,lasts

h&s

(6).

.jy!;tcm

J p1~01ln~

non-inilibitab

presence

[or

allowr:d they

substrhte

as

in sy stem

A systems

liti:?Lltui:c

tIllit,

tile

and

the

fur

this

AX

substrate

wl,i.ch

in

I.!+AID

attributed

uptake the

giutaminc

uptake

r!:pCrted by

by

proline for

.J prcfererntial

decreases et

of

considered

Me-AIB,

explained

mM Ga;zola

study substrate

to proline

and

at

their

of

sy:;!:e~:~

:i

a ccrnceni.ral.ior cxperimrnt:al

enhance

the

uptake

by

A. . Kinetic The

in

involved

particularly this

the

studic::

uptake

conditions system

use

pFuli~-ie

be

was

cell

greater that

specific

20 % inllibitiorl

may

to

system

fact

transported

incubation,

rc%lts

04‘ 0.1

be

1 mfl,

the

be

inhibition

t-ion

In

imino-glycine

added,

standarclizatiorl

(i.e

the

limiting

time,

is

!,lasts,

wells

are

that

glycine

uptake.

inhibition

owing

and

ASC inhibited

OF 0.010

stronger

the

and

2.

factor

cells.

the

results

to

bu

of

Me-AIB-intlibited

due

line

present

these

of

These

to

probably

A,

high-affinity

serine

simultaneously

uptake

I&spite

Consequently,

1 may

One >,t

2.

increases.

2.

that,

conient

prolinc

percentage

line

were

proline

found

the acids

indicates

in

was

of' amino

serine

of

(Me-AIB),

concentrations

finding

in

the

cell

pr0tei.11

of

intervenes

uptake

three

proline [S]

sodium-ion

We noticed

cell

density

This

Me-AID.

the

lines,

I).

of

line

ditions,

(Table

%

of

presence

cell

10

uptake

the

COMMUNICATIONS

investigation acid

proline of

lowest

and

BIOPHYSICALRESEARCH

isobutyric to

Me-AIB

role

inhibition the

two

systems

lhe

preferential

None

the

of 35

m?l allowed

contribution

for

%

AND

methylamino

acids,

the

than

BIOCHEMICAL

0.50

systems

when

in

to

to test

transport

and

1, 1987

Three

were

by

No.

probability

studie:;

present proline

inhibition uptake.

effective we

studies

It

at very conducted

is

probably

low

proline

further

high

affinity

concentrations

kinetic

studies 223

other

a system

(O.Olii on

three

than systcrn and

other

:;ystem as O.U25

cell

A is

it

was mbl).

lines,

IO 3,

check (4

Vol. 142, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

2

3

IL-Proline]

,mM

1

Figure

RESEARCH COMMUNICATIONS

4

5

in

a human

1.

Kinetics cell line.

of

L-proline

in the

uptake,

presence

of

Me-AIB,

skin

fibroblast

Initial rates of uptake (1 min) were measured for prolinc concentrations ([S]) ranging from 0.010 to 5 mM in the presence of 10 [S] Me-AIB as described under Materials and Methods. Each point is the average of 6 experimental results. Fitting is obtained with the model described under table II.

Xld

5 . l:r: used

.)rescnce 3t

of

the

10

end

- In The

of and

period,

of

an uptake

was

were

models

appropriate

process

system

This

model

of fair the

in

is

:

v

this

affinity

system estimates

existence

OF

in

affinity

of

Me-AIB,

unknown

estimates.

iriitial

inodified

five

obtained of

order

for

system (i.e

only

5 mM,

1 min

after

with

time.

linear analysis

(13)

systematically

in

the

absence

and

ensuring

that

Kinetic

transport

as

previously

(e.g.

in

descri-

tested.

:

Km = 0.0505

to

correct

which

constants,

the

the

not

inhibitable

to

Me-AIB

the

one

saturable take

, and

for

for to

+ KD [sl

our

system

system.

simultaneous

model

is

could

not

high-affinity

concentration

inhibitable

latter

cell

line

this

Me-/JIB

non-

0.124

mmoljl

(Table

4,

[S]

Therefore, and

the

t

the

system

striking

Cd

Vmax

=

a high

absence

includes

constants

affinity

has

the

initial

of

was particularly

Km

- rherefore,

still

Co

tie-AIB

model

inhibitable

0.013

time

regression

Tig.1). The

from

incubation

by non-linear

a saturable

of

ooncentratior,s

chose

mathematical

presence

existence

13

prolinc

determined

Uiffsrent

the

range

Me-AIB

this

were

(1).

3ed

[S]

of

3azarneters

wide

a

range,

based

on

224

of

calculated

MC-AIB.

in we

Owing

the

the

constants

that

the

and

of

high

the

low the

assuming

procedure with

absence

account

Therefore defined

mathematical

experiments

the

took to

WC postulated

the

Accordingly, account

be

was preponderant.

system) were

:

system, by

II)

without

the

was inhi-

Vol.

142,

No.

1, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

Table

Kinetic

parameters for

line

3

Cell

line

system

4

Cell

line

5

vmax

1.61

+ 0.24

0.866

.-+ 0.126

1.19

+_ 0.20

XUl

0.124

+ 0.026

0.0560

f 0.0115

0.0505

+ 0.0172

XD

1.75

+ 0.23

3.01

+ 0.29

1.75

+ 0.38

Approximate lines of

COMMUNICATIONS

II

of the Me-AIB non-inhibitable uptake, in human fibroblasts

L-proline Cell

RESEARCH

for lO[S]

model

initial

praline He-AIB,

fitting

rates

of

uptake

concentrations as the

described

under

experimental

are expressed

min)

were

0.010

and Methods.

is

to

in

three

5 I+!

in

As

shown

+

30-

E” . .E

20-

(Km as mol/l

Kinetics

_/

I the

; &ax

as nmol/min/mg

.I

protein)

J , ’ 83

,’

/

2

3

[L-Prolinel

2.

of L-proline

figure

,/”

1 Figure

in

cell

presence

XD [S]

2 ; .0 z z

different the

:

--ILL Km + [S]

f S.D.

measured

from

Materials

results

v * Vmax

kineticparamecers

(I

ranging

C(S])

uptake

in human skin

4

5

, mM

fibroblasts

in the presence

and absence

of

Me-AIB. Initial

([s])

rates from 0.010 under Materials

ranging

described

and the described

of uptake (1 to 5 m19, in and Methods.

min) the

were measured for presence or absence

0

expkrimental of 6 results.

points

in

the

presence

0

experimental of 6 results.

points

in

the

absence

Curves inhibitable under

1 and 2 respectively one (i.e system Table III.

represent A uptake)

225

of

of

Me-AIB

Me-AIB

of

; each

; each

the process plus

proline

a diffusion

not

10

concentrations (S] Me-AIB,

point

point

inhibitable process,

in

as

the

average

is the average by fitted

Me-AIB as

Vol.

142,

No.

1, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

Table Kinetic

Cell

Pie-AIB inhibitable non

for

human

line

COMMUNICATIONS

III

parameters

in

RESEARCH

L-proline

uptake

fibroblasts

3

Cell

line

4

Cell

line

5

0.26

vcnax ,

2.04

+

0.46

1.00

+

0.16

I .4.7

$

Kml

0.191

+

0.053

0.0697

+

0.0134

0.0809

+_ 0.0261

+, 13.69

7.51

+

3.38

3.74

+

1.36

I .4 I

+

0.75

1.49

l

0.38

2.78

2

0.35

system

I

Me-AIB inhibitable system

44.66

Vmax2 xm2

Approximate lines

for

initial

proline

experiments

were

Me-AIB.

Both

rates

results

were

hitor.

parameters

This

line

: cell

(e.g

and

non

enabled

inhibitable

to

may

this

each

be

due

more

results

than than

for

proline

ting

for

proline.

and

for

the

of

line,

10 (S]

PI

the

as

nmol/min/mg

in

for

(Table

to

the

each

ccl:

inhibitable

one cell

the

two

for

the

IJI).

from

addition

of

points

constants

observed

existence

protein).

experimental

process

density the

K~

kinetic

parameters

cell

of

cell cell

.

; Vmax

diffusion

kinetic

each

2.78

[S]

Fitting

well-defined

different

For

absence

+

line

variability

systems

to the inherent

is

obvious

for

line. The

geneous

correct

Nevertheless,

model

+ +

5 u&f.

and

+_ 23.5

4.70

three

in

to

presence

(Km as mol/I

and

of

measured 0.010

the

IsI Km2

2)

differences

material.

live cell

to

Vmax

f S.D.

of the

the by

2

obtain

systems

in

[SJ

Fig

variability

The

other

to 3.

uere from

account

+

+

expressed

us line

into

IsJ

are

uptake

ranging

conducted taken

Km]

of

([S])

simultaneously

v = Vmax]

kinetic

(1 min)

concentrations

49.7

those

of for

the

uptake

system

One

of

most

uptake such

uptake,

Nevertheless,

in

is may

of

it

account

is the

system

For much 226

the

literature

may

system

be

A is

exhibiting of

the

(1,2,9,10,11,14).

drawn

From the

these

only

thus

system

mask

of

results

affinity

highest and

more homopresence

the

alone

not the

uptake,

are

showed

studies

which that

in

investigators

the

kinetic

conclusions

fibroblasts

neither

none

performing

striking

human

it

as when

reported

studies

inhibition,

one

the

kinetic

accoun-

For the

existence

8lOCHEMlCAL

Vol. 142, No. 1, 1987

if

another

system.

:ombined

Our

careful able

to

1.07

to

0.19

rnrnol/l)

10th

the

A and

lroline

demonstrate not

ASC

it further.

has been

a second over

the

presence

considered

is

not

the

glycine,

but

a system for

with

as

was we

seems

uptake

by

levels

in

to

not

yet

A in

up

able

to

to

till

human

(0.060

=

from

been as

sy:;tem

(Krri

differ

important,

plasma

studies.

system

shown have

ue

inhibition

iI-igh-aff-inity

system

usual proline

the

k may have major physiological

such

i~~nsmuch

range

This

cclbstrate

best

in view or

by

of

i‘r~Itfu1,

Naf-dependent,

Me-AIB.

used

existence

proved

RESEARCH COMMUNICATIONS

concentration

of: an by

It The

system

a large

inhibitable

systems.

ILasts. Furthermore,

for

studies

kinetic

le, were

,haracterize

search

AND BIOPHYSICAL

now,

F'ibro0.260

mmol/l)

iemonnier,

A.

implications.

REFERENCES 1. . Fenkant,

M.,

Moatti,

N.,

Pluccsrio,

Gauticr,

J.,

II.,

Cucp?oui,

S.

i(ondall,

i7.3.

(1951)

and

(1984) Eiochem. J. 224, 309-315. '). . 3. 4. 5. Loury,

O.tl.,

1tosebrougF1,

N.J.,

l'orr‘,

A.L.

wu

Siol.

J.

C!lem.

193, 265-275. 6. Franchi-Gazzola,

If. , Gozzola, Chem. 257, 9582-3585.

-,, . uass, ii.,

Hedl;gaard, H.U., Uillehay, Chem. 256, 10259-10266.

J. Biol.

J., &net.

5. tloffett,

cell.

9. Revsin,

L.C. , Ual!‘AsLa,

Curri.den, S., 9, 183-213.

B. and

Morrow;

.o.

Tramacere, +I., Petronini, Res. 151, 70-79.

.l.

Russell,

S.B.,

Russell,

.2.

Gazzola,

G.C.,

Dall'Asta,

.3.

Metzler,

C.M.,

Elfring,

.4,

Longo, aqd

N., Gazzola,

Franchi-Gazzola,

L..,

Ertsey,

G.

(1979j

Exp.

P.G.,

Severini,

J.D.

and

V.,and G.L.

R.,

and R.,

G.C. (1985) Biocbim.

Mendiaz, Cell.

E. Res.

A.

and

J,S,

Guidotti,

G.S.

Ewen,

Russolati,

A.J, O.,

and

Englesberg,

119,

(1984) (!1980)

3. Uioi.

C. (i3Bl)

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