Báo cáo khoa học: Transport of L-arginine and nitric oxide formation in human platelets pdf

Transport of
L
-arginine and nitric oxide formation in human platelets
Maria G. Signorello, Raffaele Pascale and Giuliana Leoncini
Dipartimento di Medicina Sperimentale, sezione Biochimica, Universita
`
di Genova, Italy
The results of the present study show that human platelets
take up
L
-arginine by two transport systems which are
compatible with the systems y
+
and y
+
L. These Na
+
-
independent transporters have been distinguished by treat-
ing platelets with N-ethylmaleimide that blocks selectively
system y
+
.Systemy
+
, that accounts for 30–40% of the total
transport, is characterized by low affinity for
L
-arginine, is
unaffected by
L
-leucine, is sensitive to changes of membrane

potential and to trans-stimulation. The other component
of
L
-arginine transport identified with the system y
+
L
(approximately 60–70% of the total flux) shows high affinity
for
L
-arginine, is insensitive to N-ethylmaleimide treatment,
unaffected by changes in membrane potential, sensitive to
trans-stimulation and inhibited by
L
-leucine in the presence
of Na
+
.
Moreover a strict correlation between
L
-arginine transport
and nitric oxide (NO) production in whole cells was found.
N-ethylmaleimide and
L
-leucine decreased NO production as
well as cGMP elevation, and the effect on NO and cGMP
were closely related. It is likely that the
L
-arginine transport
systems y
+

and y
+
L are both involved in supplying sub-
strate for NO production and regulation in human platelets.
Keywords:
L
-arginine; nitric oxide; platelets; transport.
The cationic amino acid
L
-arginine is the main source for
the synthesis of nitric oxide (NO) in many cell types [1]. NO
exerts different functions in the regulation of vascular tone
and blood pressure and in neurotransmission in central
nervous system [2]. One of the most relevant functions
of NO is the inhibition of platelet aggregation [3,4]. The
regulation of platelet activation is crucial to prevent platelet
aggregation, thrombus formation and stroke. Human
platelets synthesize NO through the action of a soluble
calcium/calmodulin-dependent constitutive NO synthase
(cNOS) [5], that is active in the presence of the same
cofactors as other constitutive NOSs but it has a different
molecular weight [6]. As the plasma and assumed intracel-
lular concentrations of
L
-arginine still far exceed the K
m
for
cNOS [7], the enzyme should be saturated with the amino
acid under physiological conditions. Nevertheless different
studies have shown that the

L
-arginine extracellular con-
centration regulates NO formation in platelets [7], macro-
phages and endothelial cells [8]. Moreover experimental
[9–11] and clinical studies [12–15] demonstrated that the
decrease of vascular and platelet NO activity can be reversed
by oral and intravenous administration of
L
-arginine. Thus
the
L
-arginine transport through the membrane exerts a
regulatory role in the pathway
L
-arginine/NO.
In most mammalian cells arginine requirements are met
primarily by uptake of extracellular arginine via specific
transporters, such as systems y
+
,b
+
,B
+
,y
+
L [16]. Not all
transporters are found in every cell type and activities of
specific transporters can be regulated in response to specific
stimuli [16]. Previous studies demonstrated that in human
platelets

L
-arginine transport is mediated by y
+
transport
system [17,18] or by system y
+
L [19]. Both systems are
Na
+
-independent exchange mechanisms for cationic amino
acid, but they have different properties [16]. System y
+
is
membrane potential dependent, interacts with the neutral
amino acids with low affinity and is selectively inhibited
by N-ethylmaleimide [20]. The specificity of system y
+
is
restricted to cationic amino acids and the activity is due to
the cationic amino acid transporter (CAT), among which
CAT-1, CAT-2A and CAT-2B are the best characterized
members of the family [16,21]. System y
+
L recognizes
L
-arginine with higher affinity (K
m
¼ 10–30 l
M
), is not

sensitive to membrane potential and exhibits a high affinity,
Na
+
-dependent interaction with neutral amino acids such
as
L
-leucine [22]. System y
+
L is an heterodimeric amino
acid transporter formed by a light and a heavy subunit.
The latter is the glycoprotein 4F2hc, while two alter-
native light chains (y
+
LAT1 and y
+
LAT2) have been
characterized [23].
The results of the present study show that human
platelets take up
L
-arginine by two transport systems which
are compatible with the systems y
+
Landy
+
.Thetwo
transporters, distinguished by the use of the sulphydryl
reagent N-ethylmaleimide, have been characterized. Both
systems seem to be involved in substrate supply for NOS,
contributing to NO formation and regulation.

Experimental procedures
Materials
Amino acids, gramicidin D, dibutyl phthalate, N-ethyl-
maleimide, valinomycin and chemicals were from Sigma
Chemical Co. Tetrapentylammonium chloride from Fluka
Correspondence to G. Leoncini, Dipartimento di Medicina
Sperimentale, sezione Biochimica, Viale Benedetto XV,
1, 16132 Genova, Italy.
Fax: + 39010354415, Tel.: + 390103538154,
E-mail:
Abbreviations: CAT, cationic amino acid transporter; LPI, lysinuric
protein intolerance; cNOS, constitutive NO synthase; NO, nitric
oxide; NOx, nitrate + nitrite.
(Received 18 November 2002, revised 12 March 2003,
accepted 14 March 2003)
Eur. J. Biochem. 270, 2005–2012 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03572.x
AG. Gabexate mesylate was a gift from Lepetit.
L
-[2,3,4-
3
H]arginine was from NEN-Perkin Elmer. Titer-
tek
TM
filters were from Flow Laboratories. cGMP-[
3
H] RIA
kit was from Amersham Pharmacia Biotech.
Blood collection and preparative procedures
Human blood from normal healthy volunteers, who have
not taken drugs known to affect the platelet function, was

collected in 130 m
M
aqueous trisodium citrate anticoagu-
lant solution (9 : 1). Washed platelets were prepared as
previously described [24]. Briefly platelet-rich plasma,
obtained by centrifugation of the whole blood at 100 g for
25 min, was centrifuged at 1000 g for 15 min. Pellet, washed
once with pH 4.8 ACD solution (75 m
M
trisodium citrate,
42 m
M
citric acid and 136 m
M
glucose), was resuspended in
pH 7.4 Hepes buffer (145 m
M
NaCl, 5 m
M
KCl, 1 m
M
MgSO
4
,10m
M
glucose, 10 m
M
Hepes). Centrifugations
were carried out at 4 °C.
Flux measurements

Influx experiments were performed as described previously
with some light modifications [25]. Washed platelets
(2.0 · 10
8
platelets), prewarmed at 37 °Cfor10minwith
NaCl/P
i
or N-ethylmaleimide when required, were incuba-
ted for 1 min at 37 °C in a Dubnoff water bath under gentle
shaking in the presence of 1 lCiÆmL
)1
L
-[2,3,4-
3
H]arginine,
unlabelled
L
-arginine and
L
-leucine when required (final
volume 1.2 mL). At the end of the incubation, aliquots of
1.0 mL were withdrawn, immediately filtered through a
Titertek
TM
filter and washed twice with large volumes of
cold NaCl/P
i
containing 10 m
ML
-arginine. The radioacti-

vity corresponding to the incorporated
L
-[2,3,4-
3
H]arginine
was directly measured by liquid scintillation counting of
the filter in a Packard model TRI-CARB 1600 TR liquid
scintillation analyzer. Blank values, obtained by measuring
an iced-cold mixture of platelets, unlabelled
L
-arginine and
L
-[2,3,4–
3
H]arginine, immediately filtered, were subtracted
from the experimental values. In Na
+
-free incubation
buffer NaCl and Na
2
HPO
4
were replaced by choline salts.
In some experiments washed platelets were resuspended in
pH 7.4 Hepes buffer containing 2 l
M
prostaglandin E
1
.
In these conditions the platelet

L
-arginine influx was
unchanged.
For efflux experiments washed platelets, resuspended at
1.0 · 10
9
platelets in pH 7.4 Hepes buffer containing 2 l
M
prostaglandin E
1
were loaded at 37 °Cfor15minwith
1 lCiÆmL
)1
L
-[2,3,4-
3
H]arginine and unlabelled
L
-arginine
(5 l
M
), in the presence of N-ethylmaleimide when required.
Incubation was stopped by centrifuging samples at 4 °C.
Pellet was washed once with ice-cold pH 7.4 Hepes buffer.
The total incorporated
L
-arginine was immediately meas-
ured. To initiate efflux the washing buffer was aspirated and
replaced by Hepes buffer at room temperature. The efflux
was followed at 22 °C. Suitable aliquots of platelets were

withdrawn in tubes containing dibutyl phthalate and rapidly
centrifuged. The supernatant radioactivity was assayed by
liquid scintillation counting. To eliminate effects of trans-
stimulation due to variation in intracellular substrate levels,
in several experiments washed platelets were incubated at
37 °C for 1 h in the absence of any substrate. In these
conditions the kinetic behaviour of
L
-arginine flux was
unchanged. These parameters, assayed before loading
washed platelets with labelled arginine and at the end of
the efflux experiments, were not different. The kinetic
parameters of
L
-arginine influx and efflux were calculated
by Lineweaver–Burk plot. The
L
-arginine flux through
system y
+
L was measured in N-ethylmaleimide-treated
platelets and the
L
-arginine flux via system y
+
was measured
by subtracting the flux via system y
+
L from total flux.
Measurement of platelet NOx formation

Washed platelets, resuspended at 1.0 · 10
9
platelets in
pH 7.4 Hepes buffer containing 2 m
M
CaCl
2
,werepre-
warmed at 37 °Cfor10minwithN-ethylmaleimide and
incubated with
L
-arginine and
L
-leucine when required.
Incubation was stopped by sonicating samples in ice.
Suitable aliquots of supernatant, added to equal volumes
of pH 9.7 assay buffer (15 gÆL
)1
glycine-NaOH) containing
cadmium beds, were incubated overnight at room tempera-
ture under horizontal shaking. Cadmium beds were activa-
ted immediately before each experiment by subsequent
washings with 0.1
M
H
2
SO
4
, bidistillated water and assay
buffer. The nitrite + nitrate (NOx) concentration, deter-

mined by the Griess reagent (1% sulphanilamide in 2.5%
H
3
PO
4
, 0.1% naphtylenediamine dihydrochloride), was
measured at 540 nm using a sodium nitrite calibration
curve.
Measurement of platelet cGMP formation
cGMP intracellular level of human platelets incubated in the
presence of N-ethylmaleimide or
L
-leucine was assayed as
previously described [26]. Some experiments have been
carried out in the presence of gabexate mesylate, known
inhibitor of cNOS [7,27]. The reaction was stopped by the
addition of cold 2
M
perchloric acid. Extracts were neutral-
ized and analyzed for the cGMP content by RIA kit.
Data analysis
Data are the mean ± SD of at least four independent
determinations, each performed in duplicate. Reported
drawings are also representative of four experiments.
Statistical analysis was performed using the unpaired
Student’s t-test considering significant the difference
between control and each treatment at least at 5% level
(P <0.05).
Results
L

-Arginine influx in human platelets
N-ethylmaleimide inhibits selectively system y
+
, leaving
system y
+
L functionally intact [20]. Thus it can be
employed to discriminate the transport systems involved
in the uptake of cationic amino acids. To evaluate the
N-ethylmaleimide effect on
L
-arginine influx, platelets were
preincubated with the sulphydryl reagent for 10 min at
37 °C. In these experimental conditions N-ethylmaleimide
inhibited
L
-arginine uptake in a dose-dependent manner
and at 200 l
M
produced the maximal inhibition, that
2006 M. G. Signorello et al.(Eur. J. Biochem. 270) Ó FEBS 2003
generally ranged from 30 to 40% of the total flux (Fig. 1A).
The N-ethylmaleimide-inhibited component of
L
-arginine
flux was identified as the system y
+
. In all subsequent
experiments N-ethylmaleimide was used at the concentration
200 l

M
.Moreover
L
-leucine inhibited dose-dependently
therateofentryofthe
L
-arginine, reaching the maximum
effect, in the range of 60–70% of the total flux, at 300 l
M
L
-leucine (Fig. 1B). As it was reported that y
+
Lmediates
Na
+
-independent cationic and Na
+
-dependent neutral
amino acid transport [16], several experiments in the absence
of Na
+
were performed.
L
-Leucine was ineffective on
L
-arginine influx in the absence of Na
+
, confirming the
presence of the system y
+

L in human platelets (Fig. 2).
Data indicate that platelet
L
-arginine transport mainly
occurs by the action of the systems y
+
Landy
+
.Thesetwo
transport systems can be distinguished for their
L
-arginine
affinity. The kinetic parameters of the system y
+
L, deter-
mined experimentally in N-ethylmaleimide-treated platelets,
were K
m
¼ 29 ± 5 l
M
and V
max
¼ 85 ± 4 pmol per
2.0 · 10
8
platelets per min.
L
-Arginine influx via system y
+
,

which was evaluated by subtracting the influx via
system y
+
L from total influx, was characterized by the
following parameters: K
m
¼ 63 ± 8 l
M
and V
max
¼
51 ± 6 pmol per 2.0 · 10
8
platelets per min. The kinetic
parameters of the total influx were K
m
¼ 30 ± 2 l
M
and
V
max
¼ 127±3pmol per 2.0· 10
8
platelets per min
(Fig. 3).
L
-Arginine total influx was competitively inhibited
by
L
-leucine. In these conditions K

m
value increased to
103 ± 18 l
M
while V
max
did not change. In agreement
with previous data [16],
L
-arginine uptake was also competi-
tively inhibited by
L
-glutamine,
L
-methionine and
L
-lysine
(data not shown).
It was clearly established that system y
+
is electrogenic
and amino acid accumulation is driven by the cell plasma
membrane potential [28], but no clear data are available
concerning the effects of voltage changes on the activity of
system y
+
L. Thus the effect of membrane hyperpolariza-
tion or depolarization on these two transport systems was
studied. Hyperpolarization was induced by the addition of
K

+
ionophore, valinomycin [29] in the presence of an
outwardly directed K
+
gradient ([K
+
]
out
¼ 5m
M
). The
system y
+
LmeasuredinN-ethylmaleimide-treated plate-
lets was unaffected, while total
L
-arginine uptake and the
system y
+
were significantly stimulated by the addition of
valinomycin (Fig. 4). The dependence of
L
-arginine uptake
on membrane potential was further investigated by
inducing membrane depolarization with gramicidin D,
which dissipates both Na
+
and K
+
gradients [30]. The

y
+
L system was not modified by gramicidin, whereas the
y
+
component was significantly reduced (Fig. 4). In
addition, total
L
-arginine uptake and the flux via system
y
+
were significantly inhibited by tetrapenthylammonium
chloride (Fig. 5), while other K
+
channel blockers like
4-aminopyridine and glibenclamide were ineffective (data
not shown).
Fig. 2.
L
-Arginine uptake in the presence or absence of Na
+
in the
external medium.
L
-Arginine uptake was measured in washed platelets
(2.0 · 10
8
platelets) resuspended in pH 7.4 Na
+
-present or Na

+
-
free Hepes buffer (see Experimental procedures). NaCl/P
i
,200l
M
N-ethylmaleimide or 500 l
ML
-leucine were added as detailed above.
Each bar represents the mean ± SD of four experiments performed in
duplicate. wP <0.0005vs.Na
+
-present. NEM, N-ethylmaleimide.
Fig. 1.
L
-Arginine uptake in human platelets: sensitivity to N-ethylmaleimide and effect of
L
-leucine. Washed platelets (2.0 · 10
8
platelets), pretreated
for 10 min at 37 °C with NaCl/P
i
or N-ethylmaleimide as indicated (A), were incubated with 5 l
ML
-arginine. In the experiments in which the
L
-leucine effect was tested (B),
L
-leucine and
L

-arginine were added simultaneously. After 1 min, incubation was stopped and
L
-arginine uptake
measured as described in Experimental procedures. Data are the mean ± SD of four determinations carried out in duplicate. NEM, N-ethyl-
maleimide.
Ó FEBS 2003 Arginine transport and NO formation in platelets (Eur. J. Biochem. 270) 2007
L
-Arginine efflux from human platelets
Some preliminary experiments indicated that the efflux rate
was too rapid at 37 °C. Thus efflux studies were carried out
at 22 °C in the presence or in the absence of N-ethyl-
maleimide. In these experimental conditions we were able to
measure
L
-arginine total efflux and the y
+
L transport
component, that was not inhibited by N-ethylmaleimide.
Results of Fig. 6 show that y
+
L system is 60% of the total
L
-arginine efflux, while the system y
+
, N-ethylmaleimide-
inhibited, represents the minor fraction. The addition of
L
-arginine to the external medium was found to produce
marked acceleration of
L

-arginine efflux stimulating the rate
of labelled arginine exit by 2.8 ± 0.2-fold (Fig. 6, dotted
lines). The trans-stimulation involves both the systems y
+
L
and y
+
. Moreover the results of four independent experi-
ments indicated that
L
-arginine produced a dose-dependent
acceleration that reached saturation. The half-saturation
constant (K
m
)forexternal
L
-arginine was found to be
15 ± 4 l
M
for the total efflux, 16 ± 3 l
M
for the y
+
L
component and 25 ± 3 l
M
for the y
+
component. The
V

max
values were 55 ± 8 pmol per 2.0 · 10
8
platelets per
min for the total efflux, 32 ± 2 pmol per 2.0 · 10
8
platelets
min for the system y
+
L and 18 ± 2 pmol per 2.0 · 10
8
platelets per min for the system y
+
.
Fig. 3. Kinetic analysis of
L
-arginine uptake in human platelets. Washed platelets (2.0 · 10
8
platelets), preincubated for 10 min at 37 °C in presence
of NaCl/P
i
or N-ethylmaleimide, were incubated for 1 min with various
L
-arginine concentrations.
L
-Arginine uptake was measured as detailed in
Experimental procedures. j, total influx; m, influx via system y
+
L, determined experimentally by treating platelets with 200 l
M

N-ethylmaleimide.
d, Influx via system y
+
which was obtained by subtracting the influx via system y
+
L from total influx. Each point represents the mean ± SD of
seven experiments carried out in duplicate. In (B) data have been plotted according to Lineweaver–Burk.
Fig. 4. Effect of valinomycin and gramicidin D on
L
-arginine uptake.
Washed platelets (2.0 · 10
8
platelets) were preincubated with saline or
200 l
M
N-ethylmaleimide for 10 min at 37 °C when required. Uptake
was evaluated after 1 min incubation in the presence of 5 l
M
L
-arginine as described in Experimental procedures.
L
-leucine (500 l
M
)
was added simultaneously to
L
-arginine. Valinomycin (10 l
M
)or
gramicidin D (1 l

M
) were added 5 s before starting the assay. Data are
the mean ± SD of four determinations carried out in duplicate.
§P < 0.0005; wP <0.01; dP <0.025 vs. none. NEM, N-ethyl-
maleimide.
Fig. 5. Effect of tetrapenthylammonium chloride on
L
-arginine uptake.
Washed platelets (2.0 · 10
8
platelets) were preincubated for 10 min at
37 °C with NaCl/P
i
,200l
M
N-ethylmaleimide or 50 l
M
tetrapen-
thylammonium chloride. Incubation was started by adding 5 l
M
L
-arginine and 500 l
ML
-leucine when required. Each bar represents
the mean ± SD of four determinations carried out in duplicate.
wP <0.01.NEM,N-ethylmaleimide.
2008 M. G. Signorello et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Effect of
N
-ethylmaleimide and

L
-leucine on NO
formation and cGMP levels
To evaluate the effect of N-ethylmaleimide or
L
-leucine on
NO formation the level of nitrite + nitrate was measured.
It was shown that in N-ethylmaleimide-treated platelets the
NO formation was reduced by 35% of the total in close
correlation with the N-ethylmaleimide effect on
L
-arginine
uptake. Moreover the addition to platelet of
L
-leucine, able
to competitively inhibit
L
-arginine transport through the
y
+
L system in the presence of Na
+
, reduced NO synthesis
by 60% of the total (Fig. 7A). These data support a close
correlation between the
L
-arginine transport systems y
+
L
and y

+
and NO formation. The effects of N-ethylmaleimide
and
L
-leucine on
L
-arginine uptake and on NOx forma-
tion were closely correlated (y ¼ 0.284404x ) 0.862385;
r
2
¼ 0.99).
Gabexate mesylate, a known inhibitor of cNOS [7,27],
affected cGMP formation in a dose-dependent manner,
producing at 100 l
M
an inhibition by approximately 40%
(data not shown). Thus intracellular cGMP levels are
dependent on NO formation. NO increases intracellular
cGMP levels through the activation of the soluble guanylyl
cyclase. As additional evidence for the inhibition of NO
formation by N-ethylmaleimide or
L
-leucine, the effect of
these compounds on cGMP was measured in platelets
incubated in the presence of
L
-arginine. As shown in
Fig. 7B, N-ethylmaleimide treatment decreased cGMP
formation by 35% and
L

-leucine reduced cGMP production
by 60%. The effects of N-ethylmaleimide or
L
-leucine on
NOx formation and on cGMP levels were strictly correlated
(y ¼ 0.008165x ) 0.01734; r
2
¼ 0.99). Moreover the addi-
tion to platelets of N-ethylmaleimide or
L
-leucine in the
absence of
L
-arginine did not produce any effect on NO
basal formation or on the cGMP levels.
Discussion
L
-Arginine transport was previously studied in human
platelets and was identified as the system y
+
[17,18] or as the
system y
+
L [19], respectively. Data from those authors were
obtained under experimental conditions different from ours.
In particular Vasta et al.[17]studiedthe
L
-arginine trans-
port on small samples (50 lL) of very concentrated platelets
(2.5 · 10

9
platelets), after a prolonged preincubation
(90 min at 37 °C). Moreover Mendes Ribeiro et al.[19],
who identified in the system y
+
L the only transporter for
L
-arginine in human platelets and described a stimulatory
effect of N-ethylmaleimide on this system, incubated
Fig. 6.
L
-Arginine efflux in human platelets. Platelets (1.0 · 10
9
plate-
lets), loaded for 15 min at 37 °Cwith1lCiÆmL
)1
L
-[2,3,4-
3
H]arginine
and unlabelled
L
-arginine (5 l
M
) in the presence of saline (j Total
efflux: y
+
and y
+
Lsystems)or200l

M
N-ethylmaleimide (m system
y
+
L), were washed once with ice-cold buffer and resuspended in
pH 7.4 Hepes buffer in the absence (continuous lines) or in the pre-
sence (dotted lines) of 1.0 m
ML
-arginine. The
L
-arginine efflux via
system y
+
(d) was determined as difference between total and system
y
+
L efflux. Data are the mean ± SD of four determinations carried
out in duplicate. wP <0.0005;§P < 0.0025 vs. total efflux. NEM,
N-ethylmaleimide.
Fig. 7. Effect of N-ethylmaleimide and
L
-leucine on NO formation and cGMP levels in platelets. Washed platelets, resuspended in pH 7.4 Hepes
buffer containing 2 m
M
CaCl
2
(1.0 · 10
9
platelets), were pretreated for 10 min at 37 °C with NaCl/P
i

or 200 l
M
N-ethylmaleimide then 100 l
M
L
-arginine was added. In the experiments in which the effect of
L
-leucine was tested, 500 l
ML
-leucine and 100 l
ML
-arginine were added
simultaneously. After 5 min at 37 °C incubation was stopped by sonicating samples in ice (A) or by adding of ice cold 2
M
perchloric acid (B).
Nitrite + nitrate and cGMP levels of supernatants were measured as reported in Experimental procedures. Each bar represents the mean ± SD of
four experiments carried out in duplicate. wP < 0.0005 vs. none.
Ó FEBS 2003 Arginine transport and NO formation in platelets (Eur. J. Biochem. 270) 2009
platelets for 30 min in the presence of very high concentra-
tions of the sulphydryl reagent (2.0 m
M
). Moreover in both
cases [17,19] the technique used to isolate labelled platelets
was different from ours, which consisted of a rapid filtration
of platelets.
The present report demonstrates that two Na
+
-inde-
pendent main systems are involved in
L

-arginine transport
in human platelets. The properties of one of these systems,
responsible for 40% of the total carrier mediated transport,
are consistent with the properties of the system y
+
[16].
In human platelets this system shows low affinity for
L
-arginine, is inhibited by N-ethylmaleimide, not affected by
L
-leucine and sensitive to trans-stimulation. Moreover the
activity of y
+
is affected by changes of membrane potential
as described previously in other cell types such as human
erythrocytes [31], human placenta [32] and cultured human
fibroblasts [33]. The other component, which represents
approximately 60% of the platelet
L
-arginine transport, can
be identified with the system y
+
L [16]. Kinetic experiments,
performed over a wide range of substrate concentrations,
revealed that this system (y
+
L) has a high affinity for
L
-arginine, is insensitive to N-ethylmaleimide treatment,
unaffected by changes in membrane potential (hyperpolari-

zation or depolarization) and stimulated when cationic
amino acids are present on the trans-side of the membrane.
Moreover system y
+
L is inhibited by
L
-leucine in the
presence, but not in the absence of Na
+
.
The small inhibition of
L
-arginine influx by
L
-leucine in
the absence of Na
+
could be due probably to the presence
of the system b
+
[16] but this component accounts for
5–7% of the total arginine influx. Thus its contribution to
arginine influx seems to be minor.
To clarify the actual contributions of system y
+
and y
+
L
to the overall rate of
L

-arginine transport under physiologi-
cal conditions it would be suitable to measure the transport
in the presence of plasma concentrations of competing
amino acids. In addition both systems would be exposed to
many substrates at different concentrations on both sides of
the membrane in vivo. However it is likely that system y
+
,
which has a more restricted substrate specificity than y
+
L
[16], should make a more important contribution to
L
-arginine flux and to intracellular NO formation in human
platelets. On the other hand, system y
+
Lthatissensitiveto
trans-stimulation mechanisms could provide an effective
route of efflux for cationic amino acids in exchange for
neutral amino acids as recently shown [34].
The present study was addressed not only to revalue the
systems involved in
L
-arginine transport, but also to clarify
whether
L
-arginine transport can modulate NO formation.
Data show a close relationship between
L
-arginine uptake

and NO formation as determined directly by the detection
of NOx and indirectly by the assay of cGMP level,
suggesting that the
L
-arginine transport systems y
+
Land
y
+
are both implicated in NO production. Thus extracel-
lular
L
-arginine may modulate intracellular NO synthesis
by providing the substrate for cNOS. The crucial role of
L
-arginine transport in regulating NO production has been
recently demonstrated in human platelets [7] and in
endothelial cells and macrophages [8,35]. Moreover in
endothelial cells [36] extracellular
L
-arginine concentration
is the most determinant of
L
-arginine availability for cNOS,
despite the fact that intracellular arginine concentrations
greatly exceed the K
m
of endothelial NOS [37]. The
compartmentalization of
L

-arginine within cells may explain
the dependence of NO synthesis on extracellular
L
-arginine
despite saturating intracellular substrate levels. Immuno-
histochemical studies have shown that cationic arginine
transport system colocalizes in caveolae with membrane-
bound eNOS [38], suggesting a preferential channelling or
directed delivery of extracellular arginine to eNOS. Several
other observations support the evidence that extracellular
arginine is determinant for NOS activity. NO synthesis is
decreased by several
L
-arginine analogues [39] such as
gabexate mesylate [7,27] which are able to also inhibit
L
-arginine influx. Moreover several clinical studies indicate
that
L
-arginine is essential for endothelial NO synthesis and
demonstrate that a deficiency of endothelial NO production
generates an abnormal vasomotor tone and a prothrom-
botic state. In a group of patients affected with congestive
heart failure, a disease characterized by reduced ventricular
function, neurohormonal activation and impaired endo-
thelial function, the
L
-arginine transport was reduced during
arterial infusion and in mononuclear cells of peripheral
blood [40]. Lysinuric protein intolerance (LPI) is an

autosomal recessive disorder characterized by defective
transport of the cationic amino acids lysine, arginine and
ornithine at the basolateral membrane of the polar epithelial
cells in the intestine and renal tubules. LPI is caused by
mutations in the SLC7A7 gene encoding y
+
Laminoacid
transporters [41]. Kamada et al. [42] examined vascular
endothelial function in an LPI patient. The authors found
that endothelium-dependent vasodilation (EDV) and serum
levels of NO were markedly reduced in the patient
compared with controls. Endothelium-dependent vasodila-
tion and NO became normal after
L
-arginine infusion. In
addition to these abnormalities in vasomotor function, an
earlier report showed that the above mentioned patient had
a reduced circulating platelet count, increased plasma level
of the thrombin-antithrombin III complex and elevated
plasma fibrin(ogen) degradation products [43]. Intravenous
L
-arginine infusion normalized all these parameters. More-
over in other pathological states such as septic shock
[44] increased NO production due to increased activity of
L
-arginine transport in peripheral blood mononuclear cells
was shown. Thus the control of
L
-arginine transport might
be a therapeutic target to regulate intracellular NO

production.
In conclusion this study demonstrates that human
platelets take up
L
-arginine by two transporters compatible
with the systems y
+
and y
+
L. Both could provide adequate
amounts of substrate to cNOS for endogenous NO
production and regulation.
Acknowledgements
This study was supported by MURST Prin 2000 ÔCoordinated
regulation of NO production and arginine transportÕ.
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2012 M. G. Signorello et al.(Eur. J. Biochem. 270) Ó FEBS 2003