Transport of L -arginine and nitric oxide formation in human platelets Maria G.. The other component of L-arginine transport identified with the system y+L approximately 60–70% of the tot
Trang 1Transport 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+ System y+, that accounts for 30–40% of the total
transport, is characterized by low affinity forL-arginine, is
unaffected byL-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
forL-arginine, is insensitive to N-ethylmaleimide treatment,
unaffected by changes in membrane potential, sensitive to trans-stimulation and inhibited byL-leucine in the presence
of Na+ Moreover a strict correlation betweenL-arginine transport and nitric oxide (NO) production in whole cells was found N-ethylmaleimide andL-leucine decreased NO production as well as cGMP elevation, and the effect on NO and cGMP were closely related It is likely that theL-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 acidL-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 ofL-arginine still far exceed the Kmfor
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 ofL-arginine Thus
the L-arginine transport through the membrane exerts a
regulatory role in the pathwayL-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 plateletsL-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 (Km¼ 10–30 lM), 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 upL-arginine by two transport systems which are compatible with the systems y+L and y+ The two 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: leoncini@unige.it
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)
Trang 2AG Gabexate mesylate was a gift from Lepetit.
L-[2,3,4-3H]arginine was from NEN-Perkin Elmer
Titer-tekTMfilters were from Flow Laboratories cGMP-[3H] 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 130mM 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 mMtrisodium citrate,
42 mMcitric acid and 136 mMglucose), was resuspended in
pH 7.4 Hepes buffer (145 mM NaCl, 5 mM KCl, 1 mM
MgSO4, 10 mM glucose, 10mM Hepes) Centrifugations
were carried out at 4C
Flux measurements
Influx experiments were performed as described previously
with some light modifications [25] Washed platelets
(2.0· 108platelets), prewarmed at 37C for 10 min with
NaCl/Pior N-ethylmaleimide when required, were
incuba-ted for 1 min at 37C in a Dubnoff water bath under gentle
shaking in the presence of 1 lCiÆmL)1L-[2,3,4-3H]arginine,
unlabelled L-arginine and L-leucine when required (final
volume 1.2 mL) At the end of the incubation, aliquots of
1.0mL were withdrawn, immediately filtered through a
TitertekTMfilter and washed twice with large volumes of
cold NaCl/Picontaining 10mM L-arginine The
radioacti-vity corresponding to the incorporatedL-[2,3,4-3H]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, unlabelledL-arginine and
L-[2,3,4–3H]arginine, immediately filtered, were subtracted
from the experimental values In Na+-free incubation
buffer NaCl and Na2HPO4were replaced by choline salts
In some experiments washed platelets were resuspended in
pH 7.4 Hepes buffer containing 2 lM prostaglandin E1
In these conditions the platelet L-arginine influx was
unchanged
For efflux experiments washed platelets, resuspended at
1.0· 109platelets in pH 7.4 Hepes buffer containing 2 lM
prostaglandin E1 were loaded at 37C for 15 min with
1 lCiÆmL)1 L-[2,3,4-3H]arginine and unlabelledL-arginine
(5 lM), in the presence of N-ethylmaleimide when required
Incubation was stopped by centrifuging samples at 4C
Pellet was washed once with ice-cold pH 7.4 Hepes buffer
The total incorporatedL-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 22C 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
37C 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 ofL-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 theL-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· 109 platelets in
pH 7.4 Hepes buffer containing 2 mM CaCl2, were pre-warmed at 37C for 10 min with N-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)1glycine-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 0.1M H2SO4, bidistillated water and assay buffer The nitrite + nitrate (NOx) concentration, deter-mined by the Griess reagent (1% sulphanilamide in 2.5%
H3PO4, 0.1% naphtylenediamine dihydrochloride), was measured at 540nm using a sodium nitrite calibration curve
Measurement of platelet cGMP formation cGMP intracellular level of human platelets incubated in the presence of N-ethylmaleimide orL-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 2Mperchloric 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 0 5)
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 onL-arginine influx, platelets were preincubated with the sulphydryl reagent for 10min at
37C In these experimental conditions N-ethylmaleimide inhibited L-arginine uptake in a dose-dependent manner and at 200 l produced the maximal inhibition, that
Trang 3generally ranged from 30to 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 lM Moreover L-leucine inhibited dose-dependently
the rate of entry of theL-arginine, reaching the maximum
effect, in the range of 60–70% of the total flux, at 300 lM
L-leucine (Fig 1B) As it was reported that y+L mediates
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+L and y+ These two transport systems can be distinguished for theirL-arginine affinity The kinetic parameters of the system y+L, deter-mined experimentally in N-ethylmaleimide-treated platelets, were Km¼ 29 ± 5 lM and Vmax¼ 85 ± 4 pmol per 2.0· 108platelets 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: Km¼ 63 ± 8 lM and Vmax¼
51 ± 6 pmol per 2.0· 108platelets per min The kinetic parameters of the total influx were Km¼ 30± 2 lMand
Vmax¼ 127 ± 3 pmol per 2.0 · 108 platelets per min (Fig 3).L-Arginine total influx was competitively inhibited
by L-leucine In these conditions Km value increased to
103 ± 18 lM while Vmax did not change In agreement with previous data [16],L-arginine uptake was also competi-tively inhibited byL-glutamine,L-methionine andL-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 depolarizahyperpolariza-tion 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+]out¼ 5 mM) The system y+L measured in N-ethylmaleimide-treated plate-lets was unaffected, while totalL-arginine uptake and the system y+were significantly stimulated by the addition of valinomycin (Fig 4) The dependence ofL-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 , 20 0 l M
N-ethylmaleimide or 500 l M L -leucine were added as detailed above.
Each bar represents the mean ± SD of four experiments performed in
duplicate wP < 0 0 0 0 5 vs 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 10min at 37 C with NaCl/P i or N-ethylmaleimide as indicated (A), were incubated with 5 l M L -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.
Trang 4L-Arginine efflux from human platelets
Some preliminary experiments indicated that the efflux rate
was too rapid at 37C Thus efflux studies were carried out
at 22C 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 ofL-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 thatL-arginine produced a dose-dependent acceleration that reached saturation The half-saturation constant (Km) for external L-arginine was found to be
15 ± 4 lM for the total efflux, 16 ± 3 lM for the y+L component and 25 ± 3 lM for the y+ component The
Vmaxvalues were 55 ± 8 pmol per 2.0· 108 platelets per min for the total efflux, 32 ± 2 pmol per 2.0· 108platelets min for the system y+L and 18 ± 2 pmol per 2.0· 108 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 10min 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 10min 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 0 1; dP < 0 0 25 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 10min at
37 C with NaCl/P i , 20 0 l M N-ethylmaleimide or 50 l M tetrapen-thylammonium chloride Incubation was started by adding 5 l M
L -arginine and 500 l M L -leucine when required Each bar represents the mean ± SD of four determinations carried out in duplicate.
wP < 0 0 1 NEM, N-ethylmaleimide.
Trang 5Effect ofN-ethylmaleimide andL-leucine on NO
formation and cGMP levels
To evaluate the effect of N-ethylmaleimide orL-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 onL-arginine
uptake Moreover the addition to platelet ofL-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 (y¼ 0.284404x) 0.862385;
r2¼ 0.99)
Gabexate mesylate, a known inhibitor of cNOS [7,27], affected cGMP formation in a dose-dependent manner, producing at 100 lMan 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 orL-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% andL-leucine reduced cGMP production
by 60% The effects of N-ethylmaleimide orL-leucine on NOx formation and on cGMP levels were strictly correlated (y¼ 0.008165x) 0.01734; r2
¼ 0.99) Moreover the addi-tion to platelets of N-ethylmaleimide or L-leucine in the absence ofL-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] studied theL-arginine trans-port on small samples (50 lL) of very concentrated platelets (2.5· 109 platelets), after a prolonged preincubation (90min at 37C) 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 C with 1 lCiÆmL)1L -[2,3,4- 3 H]arginine
and unlabelled L -arginine (5 l M ) in the presence of saline (j Total
efflux: y+and y+L systems) or 20 0 l 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 1.0m M L -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 0 0 0 5; §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 10min 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 M L -leucine and 100 l M L -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.
Trang 6platelets for 30min in the presence of very high
concentra-tions of the sulphydryl reagent (2.0mM) 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 inL-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 plateletL-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 depolari(hyperpolari-zation) 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 ofL-arginine influx byL-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 ofL-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+L that is sensitive to
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 inL-arginine transport, but also to clarify
whetherL-arginine transport can modulate NO formation
Data show a close relationship betweenL-arginine uptake
and NO formation as determined directly by the detection
of NOx and indirectly by the assay of cGMP level,
suggesting that theL-arginine transport systems y+L and
y+are both implicated in NO production Thus
extracel-lularL-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] extracellularL-arginine concentration
is the most determinant ofL-arginine availability for cNOS,
despite the fact that intracellular arginine concentrations
greatly exceed the Km of endothelial NOS [37] The compartmentalization ofL-arginine within cells may explain the dependence of NO synthesis on extracellularL-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 thatL-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, theL-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+L amino acid 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 afterL-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 ofL-arginine transport might
be a therapeutic target to regulate intracellular NO production
In conclusion this study demonstrates that human platelets take upL-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 Coordinated regulation of NO production and arginine transport.
References
1 Moncada, S., Palmer, R.M & Higgs, E.A (1989) Biosynthesis of nitric oxide from L -arginine A pathway for the regulation of cell function and communication Biochem Pharmacol 38, 1709– 1715.
Trang 72 Mayer, B & Hemmens, B (1997) Biosynthesis and action of nitric
oxide in mammalian cells Trends Biochem Sci 22, 477–481.
3 Radomski, M.W., Palmer, R.M & Moncada, S (1990 )
Char-acterization of the L -arginine/nitric oxide pathway in human
platelets Br J Pharmacol 101, 325–328.
4 Radomski, M.W., Palmer, R.M & Moncada, S (1990) An
L -arginine/nitric oxide pathway present in human platelets
regu-lates aggregation Proc Natl Acad Sci USA 87, 5193–5197.
5 Palmer, R.M & Moncada, S (1989) A novel citrulline-forming
enzyme implicated in the formation of nitric oxide by vascular
endothelial cells Biochem Biophys Res Commun 158, 348–352.
6 Muruganandam, A & Mutus, B (1994) Isolation of nitric oxide
synthase from human platelets Biochim Biophys Acta 1200, 1–6.
7 Leoncini, G., Pascale, R & Signorello, M.G (2002) Modulation
of L -arginine transport and nitric oxide production by gabexate
mesylate Biochem Pharmacol 64, 277–283.
8 Closs, E.I., Scheld, J.S., Sharafi, M & Forstermann, U (2000)
Substrate supply for nitric-oxide synthase in macrophages and
endothelial cells: role of cationic amino acid transporters Mol.
Pharmacol 57, 68–74.
9 Tsao, P.S., Theilmeier, G., Singer, A.H., Leung, L.L & Cooke,
J.P (1994) L -arginine attenuates platelet reactivity in
hyper-cholesterolemic rabbits Arterioscler Thromb 14, 1529–1533.
10 Boger, R.H., Bode-Boger, S.M., Mugge, A., Kienke, S., Brandes,
R., Dwenger, A & Frolich, J.C (1995) Supplementation of
hypercholesterolaemic rabbits with L -arginine reduces the vascular
release of superoxide anion and restores NO production
Athero-sclerosis 117, 273–284.
11 Wang, B.Y., Candipan, R.C., Arjomandi, M., Hsiun, P.T., Tsao,
P.S & Cooke, J.P (1996) Arginine restores nitric oxide activity
and inhibits monocyte accumulation after vascular injury in
hypercholesterolemic rabbits J Am Coll Cardiol 28, 1573–1579.
12 Clarkson, P., Adams, M.R., Powe, A.J., Donald, A.E., McCredie,
R., Robinson, J., McCarthy, S.N., Keech, A., Celermajer, D.S &
Deanfield, J.E (1996) Oral L -arginine improves
endothelium-dependent dilation in hypercholesterolemic young adults J Clin.
Invest 97, 1989–1994.
13 Creager, M.A., Gallagher, S.J., Girerd, X.J., Coleman, S.M.,
Dzau, V.J & Cooke, J.P (1992) L -arginine improves
endothelium-dependent vasodilation in hypercholesterolemic humans J Clin.
Invest 90, 1248–1253.
14 Wolf, A., Zalpour, C., Theilmeier, G., Wang, B.Y., Ma, A.,
Anderson, B., Tsao, P.S & Cooke, J.P (1997) Dietary L -arginine
supplementation normalizes platelet aggregation in
hypercholes-terolemic humans J Am Coll Cardiol 29, 479–485.
15 Tangphao, O., Grossmann, M., Chalon, S., Hoffman, B.B &
Blaschke, T.F (1999) Pharmacokinetics of intravenous and
oral L -arginine in normal volunteers Br J Clin Pharmacol 47,
261–266.
16 Deves, R & Boyd, C.A (1998) Transporters for cationic amino
acids in animal cells: discovery, structure, and function Physiol.
Rev 78, 487–545.
17 Vasta, V., Meacci, E., Farnararo, M & Bruni, P (1995)
Identifi-cation of a specific transport system for L -arginine in human
platelets Biochem Biophys Res Commun 206, 878–884.
18 Howard, C.M., Sexton, D.J & Mutus, B (1998)
S-Nitroso-glutathione/glutathione disulphide/Cu2+-dependent stimulation of
L -arginine transport in human platelets Thromb Res 91, 113–120.
19 Mendes Ribeiro, A.C., Brunini, T.M., Yaqoob, M., Aronson,
J.K., Mann, G.E & Ellory, J.C (1999) Identification of system
y + L as the high-affinity transporter for L -arginine in human
platelets: up-regulation of L -arginine influx in uraemia Pflugers
Arch 438, 573–575.
20 Deves, R., Angelo, S & Chavez, P (1993) N-ethylmaleimide
discriminates between two lysine transport systems in human
erythrocytes J Physiol 468, 753–766.
21 Closs, E.I., Graf, P., Habermeier, A., Cunningham, J.M & For-stermann, U (1997) Human cationic amino acid transporters hCAT-1, hCAT-2A, and hCAT-2B: three related carriers with distinct transport properties Biochemistry 36, 6462–6468.
22 Deves, R., Chavez, P & Boyd, C.A (1992) Identification of a new transport system (y + L) in human erythrocytes that recognizes lysine and leucine with high affinity J Physiol 454, 491–501.
23 Verrey, F., Jack, D.L., Paulsen, I.T., Saier, M.H Jr & Pfeiffer, R (1999) New glycoprotein-associated amino acid transporters.
J Membr Biol 172, 181–192,.
24 Leoncini, G., Maresca, M., Buzzi, E., Piana, A & Armani, U (1990) Platelets of patients affected with essential thrombocythe-mia are abnormal in plasma membrane and adenine nucleotide content Eur J Haematol 44, 116–120.
25 Giovine, M., Signorello, M.G., Pozzolini, M & Leoncini, G (1999) Regulation of L -arginine uptake by Ca2+in human plate-lets FEBS Lett 461, 43–46.
26 Leoncini, G., Signorello, M.G., Roma, G & Di Braccio, M (1997) Effect of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro Biochem Pharmacol 53, 1667–1672.
27 Colasanti, M., Persichini, T., Venturini, G., Menegatti, E., Lauro, G.M & Ascenzi, P (1998) Effect of gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, on the nitric oxide pathway Biochem Biophys Res Commun 246, 453–456.
28 Kavanaugh, M.P (1993) Voltage dependence of facilitated arginine flux mediated by the system y + basic amino acid trans-porter Biochemistry 32, 5781–5785.
29 Negendank, W & Shaller, C (1982) Effects of valinomycin on lymphocytes independent of potassium permeability Biochim Biophys Acta 688, 316–322.
30 Zharikov, S.I & Block, E.R (1998) Characterization of L -arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells Biochim Biophys Acta 1369, 173–183.
31 Deves, R & Angelo, S (1996) Changes in membrane and surface potential explain the opposite effects of low ionic strength on the two lysine transporters of human erythrocytes J Biol Chem 271, 32034–32039.
32 Eleno, N., Deves, R & Boyd, C.A (1994) Membrane potential dependence of the kinetics of cationic amino acid transport sys-tems in human placenta J Physiol 479, 291–300.
33 Dall’Asta, V., Bussolati, O., Sala, R., Rotoli, B.M., Sebastio, G., Sperandeo, M.P., Andria, G & Gazzola, G.C (2000) Arginine transport through system y + L in cultured human fibroblasts: normal phenotype of cells from LPI subjects Am J Physiol Cell Physiol 279, C1829–C1837.
34 Bro¨er, A., Wagner, C.A., Lang, F & Bro¨er, S (2000) The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine Biochem J 349, 787–795.
35 Bogle, R.G., Baydoun, A.R., Pearson, J.D., Moncada, S & Mann, G.E (1992) L -arginine transport is increased in macro-phages generating nitric oxide Biochem J 284, 15–18.
36 Palmer, R.M., Rees, D.D., Ashton, D.S & Moncada, S (1988)
L -arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation Biochem Biophys Res Commun 153, 1251–1256.
37 Pollock, J.S., Forstermann, U., Mitchell, J.A., Warner, T.D., Schmidt, H.H., Nakane, M & Murad, F (1991) Purification and characterization of particulate endothelium-derived relaxing fac-tor synthase from cultured and native bovine aortic endothelial cells Proc Natl Acad Sci USA 88, 10480–10484.
38 McDonald, K.K., Zharikov, S., Block, E.R & Kilberg, M.S (1997) A caveolar complex between the cationic amino acid
Trang 8transporter 1 and endothelial nitric-oxide synthase may explain
the arginine paradox J Biol Chem 272, 31213–31216.
39 Gross, S.S., Stuehr, D.J., Aisaka, K., Jaffe, E.A., Levi, R &
Griffith, O.W (1990) Macrophage and endothelial cell nitric oxide
synthesis: cell-type selective inhibition by NG-aminoarginine,
NG-nitroarginine and NG-methylarginine Biochem Biophys.
Res Commun 170, 96–103.
40 Kaye, D.M., Ahlers, B.A., Autelitano, D.J & Chin-Dusting, J.P.
(2000) In vivo and in vitro evidence for impaired arginine transport
in human heart failure Circulation 102, 2707–2712.
41 Mykkanen, J., Torrents, D., Pineda, M., Camps, M., Yoldi, M.E.,
Horelli-Kuitunen, N., Huoponen, K., Heinonen, M., Oksanen, J.,
Simell, O., Savontaus, M.L., Zorzano, A., Palacin, M & Aula, P.
(2000) Functional analysis of novel mutations in y (+) LAT-1
amino acid transporter gene causing lysinuric protein intolerance
(LPI) Hum Mol Gen 9, 431–438.
42 Kamada, Y., Nagaretani, H., Tamura, S., Ohama, T., Maruyama, T., Hiraoka, H., Yamashita, S., Yamada, A., Kiso, S., Inui, Y., Ito, N., Kayanoki, Y., Kawata, S & Matsuzawa, Y (2001) Vas-cular endothelial dysfunction resulting from L -arginine deficiency
in a patient with lysinuric protein intolerance J Clin Invest 108, 717–724.
43 Kayanoki, Y., Kawata, S., Yamasaki, E., Kiso, S., Inoue, S., Tamura, S & Taniguchi, N (1999) Reduced nitric oxide production by L -arginine deficiency in lysinuric protein intolerance exacerbates intravascular coagulation Metabolism
48, 1136–1140.
44 Reade, M.C., Clark, M.F., Young, J.D & Boyd, C.A (2002) Increased cationic amino acid flux through a newly expressed transporter in cells overproducing nitric oxide from patients with septic shock Clin Sci 102, 645–650.