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: 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) 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. 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(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. 2012 M. G. Signorello et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . production and regulation in human platelets. Keywords: L -arginine; nitric oxide; platelets; transport. The cationic amino acid L -arginine is the main source. Transport of L -arginine and nitric oxide formation in human platelets Maria G. Signorello, Raffaele Pascale and Giuliana Leoncini Dipartimento