Nov 14, 1978 - shown by Brashear and Ross (I) for ADP clearance in the pulmonary circu- lation and by .... Marchesi, V.T., and Barnett, R.J. (1963) J. Cell. Biol.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 85, No. 1, 1978 November 14, 1978
A.M.
Pages
COMPARATIVE
DEGRADATION
OF A D E N Y L N U C L E O T I D E S
BY C U L T U R E D
ENDOTHELIAL
CELLS A N D FIBROBLASTS
DOSNE,
C. LEGRAND,
B. BAUVOIS,
E. B O D E V I N
and J.P.
183-189
CAEN
L a b o r a t o i r e d'H~mostase et de T h r o m b o s e exp4rimentale, ERA 335 CNRS, U 150 INSERM, H6pital Saint-Louis, 2, place du D o c t e u r A. Fournier, 75475 PARIS Cedex i0, France
Received
July
10,1978
SUMMARY The abilities of cultured human endothelial cells and skin fibroblasts to degrade adenylnucleotides were compared. The cells were incubated, either adherent in the culture dish or in suspension, for 5 m i n at 37 ° C w i t h 10-5M (14C) - ATP, -ADP and -AMP and the m e t a b o l i t e s in the supernatant were analyzed. E n d o t h e l i a l cells showed a much greater ability to degrade ATP and ADP while fibroblasts were m o r e efficient to degrade AMP. Due to the small amount of adenosine d e a m i n a s e activity of endothelial cells, there was an accumulation of adenosine in the medium, while fibroblast suspensions were able to convert a large p a r t of adenosine to inosine. N u c l e o t i d e p h o s p h o rylation o c c u r r e d m a i n l y in suspensions of fibroblasts w h i c h c o n v e r t e d ADP p r e f e r e n t i a l l y to ATP. These results suggest a p o s s i b l e c o n t r i b u t i o n of endothelial ADP d e g r a d a t i o n and of the subsequent adenosine a c c u m u l a t i o n in the endothelial cell inhibition of p l a t e l e t aggregation. Differences in the enzymatic activities e x h i b i t e d by adherent and scraped cells were apparent. INTRODUCTION There tides
is evidence
are degraded
shown by B r a s h e a r
by the vascular and Ross
lation and by Frick heart,
that about
been identified
vesicles
have shown that ADPase prepared
methods,
in the capillaries
in the p i n o c y t i c
from whole
Degradation role in the local
wall
nucleotide
activities
(5). F u r t h e r
in the m e m b r a n e
formation
circu-
in the p e r f u s e d
(3, 4) and more
(6) and in aortic
of ADP and subsequent of b l o o d
phosphatase
endothelium
was p r e s e n t
This was
in the p u l m o n a r y
of m y o c a r d i u m
aortic homogenate
adenylnucleo-
than they are by blood.
(2) for AMP d e g r a d a t i o n
of capillary
activity
control
more c i r c u l a t i n g
(I) for ADP clearance
and Lowenstein
with histochemical
I0 t i m e s
have
accurately studies
fractions
rings
of a d e n o s i n e
(7). may play a
flow and also in the inhibition
of
0006-291x/78/0851-0183501.00/0
183
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any ]orm reserved.
VoI. 85, No. 1, 1978
platelet
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
aggregation
by e n d o t h e l i a l
Mason
(8). Such nucleotide
kinds
of cells,
fibroblasts(10),
either
phosphatases
in membrane
platelet
also been
preparations
of m o n o l a y e r
with
and s u s p e n d e d
by Saba and
demonstrated
of hepatocytes
work was to emphasize
ATP and ADP in comparison
different b e h a v i o u r
MATERIAL
have
[11) or on intact g l i o m a
The aim of the p r e s e n t cells to degrade
cells w h i c h was d e s c r i b e d
cells
(9),
(12).
the ability
fibroblasts endothelial
in many
of endothelial
and to explore
the
cells.
AND METHODS
Cell cultures Human umbilical e n d o t h e l i a l cells were c u l t u r e d al (13). Primary s u b c o n f l u e n t cultures c o r r e s p o n d i n g were used for these experiments.
a c c o r d i n g to Jaffe et 2 to a m o n o l a y e r of 9 cm
Human skin fibroblasts were s u b c u l t u r e d from the 20th to the 26th p a s s a g e using the same conditions as for e n d o t h e l i a l cells. S u b c o n f l u e n t cultures representing a 9 cm area were used. At subeonfluency (5-10 days after plating) the cultures were w a s h e d four times w i t h serum free M e d i u m 199 in the culture dish. T h e y were e x p o s e d to the adenyl nucleotides e i t h e r as adherent plaques in the dish or in suspension. Suspensions were o b t a i n e d by gentle scraping of the cells with a p l a s t i c spatula. In the first case, incubations ~ere done in the presence of 1 ml of serum free medium, w h i c h covered a 9 cm e n d o t h e l i a l or fibroblast tissue surface. In the s e c o n d case, the cells were s c r a p e d and collected in one ml of serum free medium. The number of cells was 180 000 - 200 000 as d e t e r m i n e d after complete dissociation of the cells of replicate control cultures.
Nucleotide
degradation
investigations
Adherent c ~ l cultures ~r scraped cells were e x p o s e d to [14C] ATP, [14CJ ADP and [ e] AMP, i0- M final concentration, at 37 ° C in Medium 199 p H _ ~ . 4 for 5 rain. Then an excess of non l a b e l l e d adenylnucleotides, 1.6 x i0 M final concentration, was added. S u p e r n a t a n t was rapidly collected and deprived of cells by filtering on Millipore filter (0.2 ~). Nucleotides and metabolites p r e s e n t in the supernatant were analyzed by u n i d i m e n s i o n a l thin layer chromatography on P E I - C e l l u l o s e according to C h i v o t et al (.14). Nucleotide separation was achieved by developing the c h r o m a t o g r a m in three successive formate solutions at p H 3.5 : 0.5 M, 2 M and 4 M. Nucleosides and purine bases were w a s h e d off by methanol. N u c l e o s i d e s and bases were s e p a r a t e d on another c h r o m a t o g r a m d e v e l o p e d in d i s t i l l e d water. The spots were i d e n t i f i e d hy autoradiography and then cut out. T h e i r radioactivities were c o u n t e d in i ml d i s t i l l e d w a t e r + i0 ml Unisolve. F o r each component the results were e x p r e s s e d as the p e r c e n t a g e of the sum of all spot radioactivities, w h i c h represented their relative distribution in the s upernatant.
184
Vol. 85, No. 1, 1978
Labelled
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
compounds
8 8 8 8 8 8 8
~ 14 14CJ F 14C! ~ 14CJ [ 14C! [ 14 I [_ 14C! I. C[
were p u r c h a s e d
Radiochemical
from
adenosine 5' monophosphate adenosine 5' diphosphate adenosine 5' t r i p h o s p h a t e adenosine inosine hypoxanthine adenine
59 61 53 59 57 59 61
Centre
Amersham
:
mCi/mmol mCi/mmol mCi/mmol mel/mmol mCi/mmol mCi/mmol mCi/mmol
---LI4Cj
ATP was contamined with QA9 % ~IL14c] ADP. ~-ILI4Cj ADP was c o n t a m i n e d with 3.4 % AMP and 1.9 % adenine. I~'=CjAMP was c o n t a m i n e d with 2 % adenine. These values were d e t e r m i n e d by the same c h r o m a t o g r a p h i c and a u t o r a d i o g r a p h i c procedures as d e s c r i b e d above. RESULTS i) Comparison Comparing (table
of adherent
adherent
fibroblasts
I) that endothelial
in adenosine
which
cells
represented
condition
fibroblasts
degraded
adenosine
but a small
amount
When
labelled
and adenosine
Fibroblasts
transformed
When mainly
while
labelled
degraded
cells
72 % of the degradation
of inosine
cells
after exposition
degraded
and into a very small
in the various cells was n o t
was p r e s e n t cells,
amount
Using s c r a p e d
fibroblasts
representing
as ATP.
57 % was
transformed Fibroblasts
and some inosine.
used,
the formation
e x c e p t in very small
cells
or e n d o t h e l i a l
among the activities of endothelial
than h a l f
amounts
AMP.
o f s c r a p e d endothelial
the presence
compounds.
of inosine.
conditions
found,
2) c o m p a r i s o n
differences
in
40 % of the
ADP among which more
added to e n d o t h e l i a l
to labelled
ATP m a i n l y
67 % of the degraded
20 % of the metabolite
by endothelial
In the same
was also detected.
12 % of the exogenous
that,
ATP m a i n l y
products.
only 34 % of the exogenous
of inosine
it can be shown
58 % of the exogenous
73 % of the AMP to a large amount of adenosine
It can be n o t e d
adenosine
and fibroblasts
and e n d o t h e l i a l
represented
AMP was
into adenosine
degraded
cells
ADP was used e n d o t h e l i a l
nucleotide,
was as adenosine,
endothelial
and .fibroblasts
cells
of the two types
cells m o r e
than
If),
larger
of cells were
found.
70 % of ATP was
h a l f of the degraded products
185
(table
without
In
degraded, any inosine
VoI. 85, No. 1, 1978
Table I.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Distribution of radioactivities (in percent) of nucleotides and of their metabolites in the supernatant of adherent fibrobl~sts (F) and endothelial cells (E.C.) exposed to ATP, ADP or AMP i0- M for 5 min at 37 ° C. 2 experiments were done with fibroblasts and 6 with endothelial cells.
Supernatant
Labelled compound added
ATP
ADP
AMP
Adenosine
Inosine
2.8
F.
66.7
1.9
6.0
21.0
ATP I
E.C.
42.0
1.3
14.0
42.0
ADP
F.
2.3
86.5
3. i
6.7
60.2
12.7
27.0
F.
27.0
67.0
5.6
E.C.
43.0
54.5
I .5
{
E.C.
AMP
Table II.
I
Distribution of radioactivities (in percent) of nucleotides and of their metabolites in the supernatant of scraped fibrobla~ts (F) and endothelial cells (E.C.) exposed to ATP, ADP or AMP i0- M for 5 min at 37 ° C. 3 experiments were done with fibroblasts and 5 with endothelial cells
Labelled compound added
Supernatant
I F.
ATP
ADP
71.6
13.6
5.2
4.5
28.2
3.5
33.2
34.9
30.1
51.9
6.0
6.5
1.6
32.5
22.7
42.8
1.7
54.8
43.0
48.1
50.2
1.6
AMP
Adenosine
ATP E.C.
I F. ADP E.C.
I F. AMP E.C.
186
Inosine
4.7
5.0
Vol. 85, No. 1, 1978
formation.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In the same
30 % of ATP,
inosine
representing
When endothelial degraded
(2/3)
Using cells
Using
fibroblasts about
fibroblasts,
was
apparently
15 % of the
cells were e x p o s e d
and the main catabolite
into inosine. mainly
condition,
to ADP,
adenosine
converted
less than
catabolites.
68 % of the n u c l e o t i d e
without
was
any t r a n s f o r m a t i o n
h a l f of the e x o g e n o u s
ADP was t r a n s f o r m e d
into ATP.
labelled AMP the differences
d e g r a d e d h a l f of the nucleotide
catabolized
almost entirely
were
into
the nucleotide
also e v i d e n t
adenosine
since e n d o t h e l i a l
while
into similar
fibroblasts
amount of adenosine
and inosine. DISCUSS ION This
investigation
endothelial which
cells in the degradation
may be involved
endothelial
cells
inhibit p l a t e l e t
(8). A comparison aggregation
interpretative
in o r d e r to emphasize
of adenylnucleotides,
in the inhibition
The investigation some
was undertaken
the p h y s i c a l
state
ly o r g a n i z e d
in a tissue
was made w i t h
activities
related
of the cells.
Endothelial
structure.
The
surface,
the cells or by e x p o s i n g
cells.
For endothelial
scraping.
A minimal
fibroblasts well
of e n z y m a t i c
as enhancement
procedure
conversion
the scraping
some
activity
cells,
of cultured
cells
ADP,
by which
do not
activities
observed,
cells may raise
of the cultures
and fibroblasts of the
by e x p o s i n g
different
upon
of AMP and adenosine
may alter the balance
and m u s t be taken into account when i n v e s t i g a t i n g cells.
187
cellular
faces.
the nature
were i n c r e a s e d
transformation degradation.
mechanical-
greater
of ADP to ATP could be also observed. a marked
a n d to
are n o r m a l -
cells,
cellular
depended
ATP and ADP d e g r a d a t i o n s
induced
that is employed
fibroblasts,
dissociation
may modify
modifications
aggregation
to the m a t u r i t y
ly or enzymatically, by injuring
especially
(15) .
of enzymatic
problems,
of p l a t e l e t
the role of
The of the
after For
of ADP into ATP,
as
The cell d i s s o c i a t i o n
among e n z y m a t i c functions
activities
of c u l t u r e d
Vol. 85, No. 1, 1978
Whatever exhibited
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the physical
a greater
ability
leading
to an a c c u m u l a t i o n
convert
it into inosine.
activity amounts
in endothelial
of adenosine
cells.
of ATP after exposure synthetized
significant
in the presence
Adenosine
fibroblasts. capacity
ATP.
Endothelial
that the ADPase cyclocxygenase thrombotic
inhibitors,
events.
of adenosine
Thus,
P r e s e n t work
report
enzymatic
of endothelial
from p l a t e l e t
is p o t e n t i a t e d
natural m e c h a n i s m
of ADP associated
calcium
by increasing
release
(16).
cells has been
suggested
by two for limiting
w i t h the accumulation
m e d i u m may also contribute
aggregation
salvage
to their
(7), Lewis et al have
to endothelial
platelet
cyclic AMP
as it has been proposed
tion of ADP b y endothelial
cells
and the subsequent
could play a role in the inhibition inhibits
of p l a t e l e t
ADP-induced
We thank Pr. Morin of the M a t e r n i t y the u m b i l i c a l
fibroblasts.
for
that the degrada-
accumulation
aggregation
platelet
of adenosine
by endothelial
aggregation
(21).
ACKNOWLEDGEMENTS
providing
(19)
(20).
shows that we have to take into account
since adenosine
of scraped
activities
(15, 17, 18)
scraped
of endothelial
with the low purine
rings which
the d e g r a d a t i o n
the p l a t e l e t
smooth m u s c l e
activity
could be another
of p l a t e l e t
or by inhibiting
cells,
in the presence
of p r o s t a c y c l i n
in the surrounding
cell inhibition
vascular
than in presence
deaminase
of aortic
deaminase
was more
to AMP, which has been related
in a recent
activity
a low adenosine
of AMP to adenosine
was greatest
and adenosine
to the formation
were able to
in the same conditions
of fibroblasts
exposed
However,
cells
cells did not form appreciable
to ADP while
Until now the a n t i a g g r e g a t i n g
endoperoxides.
endothelial
fibroblasts
suggested
These data are in good agreement
h i g h 5'nucleotidase
attributed
while
Degradation
deamination
of fibroblasts
cultured
to d e g r a d e ATP and ADP than did fibroblasts,
These results
fibroblasts
cells.
state of the cells,
of L a r i b o i s i ~ r e
cords and C a r m e n B e r n a r d
This work was supported
by Grant
188
Hospital
for giving human
INSERM,
ATP 78-100.
for skin
Vol. 85, No. 1, 1978
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2. 3. 4.
5. 6. 7. 8.
9. I0. ii.
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189
