Platelets Affect the Activity of Amino Acid Transporter SNAT4 in HuH-7 Human Hepatoma Cells

Abstract

Platelets have been reported to exert diverse actions besides hemostasis and thrombus formation in the body. However, whether platelets affect transporter activity remains to be determined. In this study, we examined the effects of platelets on the activity of amino acid transporter system A, which is known to be changed by various factors, and we clarified the mechanism by which platelets affect system A activity. Among system A subtypes, we found that sodium-coupled neutral amino acid transporter (SNAT) 4 played a central role in the transport activity of system A in HuH-7 human hepatoma cells. Interestingly, platelets showed a biphasic effect on system A activity: activated platelet supernatants (APS) including the granule contents released from platelets downregulated system A activity at lower concentrations and the downregulation was suppressed at higher concentrations. The downregulation was due to a decrease in the affinity of SNAT4 for its substrate and not a decrease in the SNAT4 abundance on the plasma membrane. In addition, APS did not decrease the expression level of SNAT4 mRNA. On the other hand, platelets did not affect system A activity when the platelet suspension was added to HuH-7 cells. These results indicate that platelets indirectly affect the transport activity of system A by releasing bioactive substances but do not directly affect it by binding to HuH-7 cells.

INTRODUCTION

Amino acids are precursors of many biomolecules such as proteins, nucleic acids, hormones and neurotransmitters, and they play important and diverse biological roles in the body. Amino acid transporters are involved in the pharmacokinetics of amino acids, and amino acid transport system A mediates the transfer of short-chain neutral amino acids such as glycine and alanine in a sodium-dependent manner.^1,2) System A transporter is widely expressed in most mammalian tissues^3,4) and α-(Methylamino)isobutyric acid (Me-AIB) is known to be a specific substrate for system A.²⁾ Accordingly, the activity of this transport system has been evaluated by observing Me-AIB uptake. System A has been reported to be composed of three subtypes: sodium-coupled neutral amino acid transporter (SNAT) 1, 2, and 4. SNAT1, 2, and 4 are also known as amino acid transporter A (ATA) 1, 2, and 3, respectively. SNAT1 is known to transport neutral amino acids with high affinity and is expressed in the brain, heart and placenta in humans.^5,6) SNAT2 is a high-affinity transporter of neutral amino acids that is expressed ubiquitously in the body and is the subtype that best represents the previously reported characteristics of system A.^7–9) The Michaelis constant (K_m) of Me-AIB uptake mediated by human SNAT2 was reported to be 0.39 ± 0.05 mM.⁸⁾ SNAT4 is a transporter that is almost exclusively expressed in the liver and is known to transport neutral amino acids in a sodium-dependent manner as well as basic amino acids in a sodium-independent manner.^10,11) SNAT4 transports neutral amino acids with low affinity and basic amino acids with high affinity: the K_m values of Me-AIB and arginine uptake mediated by human SNAT4 were reported to be 6.7 ± 0.8 and 0.30 ± 0.04 mM, respectively.¹¹⁾ In addition to their affinity for neutral and basic amino acids, SNAT2 and SNAT4 differ in their transport activities for neutral amino acids in response to pH. The transport activities of human SNAT2 and SNAT4 were maximal at pH 8.0 and 8.5, respectively.^8,11)

Previous studies have shown that the transport activity of system A is upregulated by hyperosmotic shock^12,13) and amino acid deprivation.^14,15) This is thought to be a mechanism to protect tissues or cells from hyperosmotic pressure or starvation. It has also been reported that system A activity is upregulated by heat shock¹⁶⁾ and hormones such as insulin¹⁷⁾ and glucagon.¹⁸⁾ It has been shown that the upregulation of system A activity by insulin was related to an increase in SNAT2 abundance on the plasma membrane and that SNAT2 was already present in the intracellular compartment and was recruited to the plasma membrane after insulin stimulation.¹⁹⁾ Therefore, SNAT2 can rapidly increase its transport activity by recruitment after insulin stimulation. In contrast, osmotic shock induced new synthesis of SNAT2, and although this response was slower than transporter recruitment, the increase in transport activity by osmotic shock was higher than that by insulin stimulation.²⁰⁾

Platelets are known to regulate physiological hemostatic mechanisms. Furthermore, platelets also play a critical role in pathological thrombosis. Because activated platelets not only aggregate but also release bioactive substances such as growth factors^21,22) and chemokines^23,24) from intracellular granules, platelets have a great variety of actions in addition to hemostasis and thrombus formation.^25–29) However, whether platelets directly and/or indirectly affect transporter activity remains to be determined.

In the present study, we first investigated the indirect effects of platelets on transporter activity of system A in human liver carcinoma cell lines using activated platelet supernatants (APS). We also examined the effect of APS on the activity of organic cation/carnitine transporter 2 (OCTN2). We then investigated whether the values of K_m and maximal velocity (V_max) of Me-AIB uptake are changed by APS stimulation. Additionally, we investigated whether mRNA levels of system A are changed by APS stimulation. We finally examined the direct effect of platelets on the activity of system A.

MATERIALS AND METHODS

Materials

Adenosine 5′-diphosphate disodium salt (ADP) was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Dulbecco’s modified Eagle medium (DMEM) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Fetal bovine serum (FBS) was obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan). Penicillin G potassium salt and streptomycin sulfate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively. Me-AIB and L-arginine were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). L-Alanine and L-carnitine were purchased from FUJIFILM Wako Pure Chemical Corporation. [1-¹⁴C]-Me-AIB (55 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO, U.S.A.). L-[Methyl-³H]-carnitine hydrochloride (75.98 Ci/mmol) was purchased from PerkinElmer, Inc. (Boston, MA, U.S.A.).

Animals

Six-week-old male Wistar rats were obtained from CLEA Japan, Inc. (Tokyo, Japan). The breeding environment was controlled with appropriate temperature, humidity, and lighting conditions. All rats were provided with standard rat chow and water ad libitum. The experiments, which were approved by Hokkaido University Animal Care Committee (Approval Number: 20-0040), were conducted using 7- to 10-week-old rats.

Platelet Preparation

After each rat had been deeply anesthetized by isoflurane, blood was drawn by cardiac puncture with a syringe containing 3.8% trisodium citrate and the final concentration of trisodium citrate was adjusted to 0.38%. The blood was centrifuged at 200 × g for 10 min, and the upper phase was then centrifuged at 200 × g for 5 min. The upper phase was placed into a tube and a one-tenth volume of 77 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) (pH 7.4) was added. The mixture was centrifuged at 900 × g for 5 min and the platelet pellet was washed once with a washing buffer (135 mM NaCl, 5 mM KCl, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄, and 10 mM EDTA, pH 7.2). After centrifugation at 900 × g for 5 min, the washed platelet pellet was suspended in serum-free DMEM and the number of platelets was adjusted to 5 × 10⁵ platelets/µL, which is almost the same as the platelet concentration in rat whole blood.³⁰⁾ To activate platelets, 50 µM ADP was added to the platelet suspension and the platelets were incubated for 30 min at 37 °C. Finally, the platelets were centrifuged at 900 ×g for 5 min and APS, the concentration of which was defined as 100%, was obtained by collecting the upper phase. APS was prepared by mixing the blood of two or three rats at a time, and we prepared APS six times in this study. APS was stored at −20 °C after preparation until use and repeated freeze-thawing of APS was avoided. To investigate the direct effect of platelets, the platelet suspension was prepared when used.

Cell Culture

HuH-7 cells (JCRB0403) and HepG2 cells (HB-8065) were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Tokyo, Japan) and American Type Culture Collection (ATCC) (Manassas, VA, U.S.A.), respectively. Both of the cell lines are human hepatocellular carcinoma cell lines. HuH-7 cells and HepG2 cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C under an atmosphere of 5% CO₂. To investigate the effects of platelets indirectly or directly on transport activity, confluent cells were incubated in serum-free DMEM for 24 h and then incubated in APS or the suspension of washed platelets in serum-free DMEM, respectively.

Measurement of Transporter Activity

To determine the transport activity of system A, cells were washed with a transport buffer (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 5 mM glucose, pH 8.5), followed by incubation with 0.4, 2.1 or 6.7 mM Me-AIB in a transport buffer containing 18.2 µM [¹⁴C]-Me-AIB or with 18.2 µM [¹⁴C]-Me-AIB alone in a transport buffer for 30 min at 37 °C. When determining the Me-AIB uptake mediated by transporters other than system A, 10 mM alanine, a good substrate for system A,^8,11) was added to the transport buffer. To determine the transport activity of OCTN2, cells were washed with a transport buffer (25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 5 mM glucose, pH 7.5), followed by incubation with 5 µM carnitine in a transport buffer containing 13.2 nM [³H]-carnitine for 30 min at 37 °C. Under these conditions, the uptake of Me-AIB and that of carnitine were linear up to at least 1 h (data not shown). For pH-dependent studies, the pH of a transport buffer was adjusted with Tris, HEPES and 2-(N-morpholino)ethanesulfonic acid (MES). To terminate the uptake, the radioactive buffer was removed and then the cells were washed twice with ice-cold transport buffer. The cells were lysed in 0.2 N NaOH containing 1% sodium dodecyl sulfate, and the radioactivity in the cell lysate was determined by a liquid scintillation counter (AccuFLEX LSC-8000, Nippon RayTech Co., Ltd., Tokyo, Japan). For kinetic analysis of Me-AIB uptake, the concentration of Me-AIB was prepared in the range of 0.5–30 mM. The uptake mediated by SNAT4 was calculated by subtracting the uptake in the presence of 10 mM alanine and 10 mM arginine, which are good substrates for SNAT4.¹¹⁾ Transport data were fitted to the Michaelis–Menten equation and were analyzed.

Real-Time PCR Analysis

Total RNA was prepared from HuH-7 cells using RNAiso Plus (TaKaRa Bio Inc., Shiga, Japan) and was reverse-transcribed into cDNA using ReverTra Ace qPCR RT Kit (TOYOBO Co. Ltd., Osaka, Japan). Real-time PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio Inc.). The primer sets used to amplify human SNAT2, SNAT4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: 5′-AAGACCGCAGCCGTAGAAG-3′ (forward) and 5′-CAGCCATTAACACAGCCAGAC-3′ (reverse) for SNAT2 (Accession: NM_018976), 5′-TTGCCGCCCTCTTTGGTTAC-3′ (forward) and 5′-GAGGACAATGGGCACAGTTAGT-3′ (reverse) for SNAT4 (Accession: NM_ 018018) and 5′-AAGGTCATCCCTGAGCTGAA-3′ (forward) and 5′-TTCTAGACGGCAGGTCAGGT-3′ (reverse) for GAPDH (Accession: NM_002046). The PCR program was as follows: 30 s at 95 °C followed by 50 cycles of 5 s at 95 °C and 30 s at 60 °C. We selected GAPDH as an internal control and the mRNA expression level of SNAT4 was calculated by the ΔΔCt method.

Data Analysis

The results are expressed as means with standard error (S.E.). Statistical comparisons of values were made by Student’s t test after the F test for equal variance. For multiple comparisons, one-way ANOVA followed by the Dunnett test or the Tukey–Kramer test was used. A p-value of <0.05 was considered statistically significant.

RESULTS

Me-AIB Uptake in HuH-7 Cells Is Mediated by System A Subtype SNAT4

To determine whether SNAT2 and/or SNAT4 are involved in the uptake of amino acids in HuH-7 cells, we first examined the pH-dependence of Me-AIB uptake. If the Me-AIB uptake is mediated by SNAT2 or SNAT4, the uptake is expected to be maximal at pH 8 and 8.5, respectively. As stated above, the K_m values of Me-AIB uptake by human SNAT2 and SNAT4 were 0.39 ± 0.05 and 6.7 ± 0.8 mM, respectively.^8,11) Accordingly, SNAT2 is thought to play a central role in the uptake of 0.4 mM Me-AIB. When the concentration of Me-AIB was 6.7 mM, both SNAT4 and SNAT2 participated in the Me-AIB uptake. Unexpectedly, the uptake of 0.4 mM Me-AIB increased with increasing pH and reached a maximum at pH 8.5 (Fig. 1A). Similar to the 0.4 mM Me-AIB uptake, the 6.7 mM Me-AIB uptake was pH-dependent, with maximum uptake at pH 8.5 (Fig. 1B). Moreover, Me-AIB uptake was maximal at pH 8.5 at a lower concentration (18.2 µM) (Fig. 1C). These results indicate that Me-AIB uptake in HuH-7 cells is mediated by SNAT4 but not by SNAT2.

Fig. 1. pH Dependence of Me-AIB Uptake in HuH-7 Cells

HuH-7 cells were incubated with 0.4 mM (A) or 6.7 mM (B) Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB or with 18.2 µM [¹⁴C]-Me-AIB alone (C) for 30 min at 37 °C. The pH of a buffer was changed from 6.0 to 9.0 as indicated. Each value is the mean with standard error. (A) n = 4; (B) n = 4; (C) n = 3.

Transport Activity of SNAT4 Is Downregulated by Activated Platelet Supernatants

We then examined whether platelets affect Me-AIB uptake in HuH-7 cells using APS. After stimulation with APS for 24 h, the Me-AIB uptake decreased significantly in HuH-7 cells (Fig. 2A). This decrease in Me-AIB uptake after APS stimulation was also observed in HepG2 cells (Fig. 2B). When the Me-AIB uptake mediated by system A was inhibited by the addition of alanine, a good substrate for system A, the uptake was almost completely inhibited in HuH-7 cells (Fig. 2C), indicating that the Me-AIB uptake in HuH-7 cells is mediated by system A and not by other transporters and that the transport activity of system A is reduced by APS stimulation. On the other hand, the uptake of carnitine, a substrate for OCTN2, was hardly changed by stimulation with APS in HuH-7 cells (Fig. 2D), suggesting that the downregulation of transporter activity induced by APS is specific to SNAT4. We also investigated the time course of the effects of APS on Me-AIB uptake in HuH-7 cells. Me-AIB uptake decreased gradually with APS stimulation time and the decrease was significant when the stimulation time was 8 and 24 h (Fig. 2E). We next examined the effect of APS concentration on the reduction in Me-AIB uptake after stimulation for 24 h. Interestingly, APS showed a biphasic effect on Me-AIB uptake depending on the concentration: although Me-AIB uptake decreased in a concentration-dependent manner up to 100%, the decrease was suppressed from 300% (Fig. 2F). In this study, we further investigated the effect of 100% APS, which is close to the blood concentration, on system A activity.

Fig. 2. Effects of Activated Platelet Supernatants on Transporter Activity

HuH-7 cells (A) or HepG2 cells (B) were stimulated with 100% activated platelet supernatants (APS) for 24 h at 37 °C. After the stimulation, the cells were incubated with 6.7 mM Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). * p < 0.05 versus control (Student’s t test). (C) HuH-7 cells were stimulated with 100% APS for 24 h at 37 °C. After the stimulation, the cells were incubated with 18.2 µM [¹⁴C]-Me-AIB with or without 10 mM alanine for 30 min at 37 °C (pH 8.5). * p < 0.05 versus respective control (Student’s t test). (D) HuH-7 cells were stimulated with 100% APS for 24 h at 37 °C. After the stimulation, the cells were incubated with 5 µM carnitine containing 13.2 nM [³H]-carnitine for 30 min at 37 °C (pH 7.5). (E) Time course of the effect of APS (100%) on Me-AIB uptake in HuH-7 cells. After stimulation for the indicated time, the cells were incubated with 6.7 mM Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). * p < 0.05 versus no stimulation (Dunnett test). (F) Effects of APS concentration on Me-AIB uptake in HuH-7 cells. The cells were stimulated with APS at the indicated concentration for 24 h at 37 °C. After the stimulation, the cells were incubated with 6.7 mM Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). C on the horizontal axis represents the control. * p < 0.05 versus control (Dunnett test). Each value is the mean with standard error. (A) n = 4; (B) n = 3; (C) n = 3; (D) n = 4; (E) n = 4; (F) n = 8.

Affinity of SNAT4 for Me-AIB Is Decreased by Activated Platelet Supernatants

To clarify the mechanism by which APS downregulated system A activity, we analyzed the kinetic parameters of Me-AIB uptake after APS stimulation (Fig. 3A). The K_m value of Me-AIB uptake was increased by APS stimulation compared to that without stimulation: the values were 6.5 ± 1.7 and 2.1 ± 0.3 mM, respectively. The V_max value, however, hardly changed: the values of Me-AIB uptake with and without APS stimulation were 144.4 ± 23.6 and 152.2 ± 9.8 nmol/10⁶ cells/30 min, respectively. These results indicate that the reduction in Me-AIB uptake with APS stimulation is due to a decrease in affinity of SNAT4 for Me-AIB and not a decrease in SNAT4 abundance on the plasma membrane. Because the K_m value of Me-AIB uptake calculated in this study was lower than that reported previously,¹¹⁾ we investigated whether the uptake of 2.1 mM Me-AIB was decreased by APS stimulation in HuH-7 cells. Similar to the 6.7 mM Me-AIB uptake, the uptake of 2.1 mM Me-AIB decreased significantly after APS stimulation for 24 h (Fig. 3B). This result reinforces the notion that the transport activity of SNAT4 is reduced by APS stimulation.

Fig. 3. Saturation Kinetics of Me-AIB Uptake in HuH-7 Cells

(A) HuH-7 cells were stimulated with 100% activated platelet supernatants (APS) for 24 h at 37 °C. After the stimulation, the cells were incubated with Me-AIB (0.5–30 mM) containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). Transporter-mediated uptake was calculated by subtracting the uptake in the presence of 10 mM alanine and 10 mM arginine. Each value is the mean with standard error. n = 3. Inset, Eadie-Hofstee plot of Me-AIB uptake. (B) HuH-7 cells were stimulated with 100% APS for 24 h at 37 °C. After the stimulation, the cells were incubated with 2.1 mM Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). * p < 0.05 versus control (Student’s t test). Each value is the mean with standard error. n = 3.

Expression Level of SNAT4 mRNA Is Not Changed by Activated Platelet Supernatants

Analysis of kinetic parameters suggested that the amount of SNAT4 on the plasma membrane does not decrease, but it is unclear whether the amount of SNAT4 in the intracellular pool changes. To further confirm whether APS affects new synthesis of system A, we performed real-time PCR analysis of the expression levels of system A. We confirmed that the expression level of GAPDH, an internal control, did not change significantly after APS stimulation for 6 and 24 h in HuH-7 cells (Fig. 4A). The real-time PCR cycle threshold (Ct) values of SNAT2 and SNAT4 in HuH-7 cells under basal conditions were 35.3 ± 0.3 and 22.8 ± 0.2, respectively. Since the primer sets for SNAT2 and SNAT4 are different, we cannot accurately compare SNAT2 and SNAT4 expression levels of mRNA. However, the expression level of SNAT2 appears to be much lower than that of SNAT4 in HuH-7 cells. The expression level of SNAT2 was almost unchanged at 24 h after APS stimulation (data not shown). Similarly, the expression level of SNAT4 did not change significantly after stimulation with APS for 6 and 24 h (Fig. 4B), indicating that APS does not affect mRNA synthesis of system A.

Fig. 4. Real-Time PCR Analysis of the Expression of SNAT4 mRNA in HuH-7 Cells

HuH-7 cells were stimulated with 100% activated platelet supernatants for 6 or 24 h at 37 °C and then total RNA was prepared. (A) The amounts of amplification products for GAPDH mRNA are shown. The value at respective controls is presented as 1 and each value is the mean with standard error. 6 h, n = 4; 24 h, n = 3. (B) The amounts of amplification products for SNAT4 mRNA were normalized to that for GAPDH mRNA. The value at respective controls is presented as 1 and each value is the mean with standard error. 6 h, n = 4; 24 h, n = 3.

Platelets Do Not Directly Affect the Transport Activity of SNAT4

Platelets perform a variety of functions in the body through their actions directly by binding to other cells or indirectly by releasing bioactive substances from intracellular granules. We finally examined whether platelets directly affect Me-AIB uptake in HuH-7 cells using the platelet suspension. Unlike the results for APS, Me-AIB uptake did not decrease after stimulation with the platelet suspension for 24 h. Moreover, Me-AIB uptake did not decrease significantly when platelets plus ADP to activate platelets were added to cells (Fig. 5). This result indicates that platelets do not have an effect on the transport activity of system A by binding to cells.

Fig. 5. Effects of the Platelet Suspension on Me-AIB Uptake in HuH-7 Cells

HuH-7 cells were stimulated with the platelet suspension (5 × 10⁵ platelets/µL) or 100% activated platelet supernatants for 24 h at 37 °C. To activate platelets, 50 µM ADP was added to the platelet suspension just before the addition of the platelet suspension to the cells. After the stimulation, the cells were incubated with 6.7 mM Me-AIB containing 18.2 µM [¹⁴C]-Me-AIB for 30 min at 37 °C (pH 8.5). * p < 0.05 versus control (Tukey–Kramer test). Each value is the mean with standard error. n = 4.

DISCUSSION

Platelets have been reported to exhibit various actions, some of which are homeostatic and some of which are involved in the pathogenesis or exacerbation of disease. The aim of the present study was to determine whether platelets have effects on the activity of amino acid transporter system A in human liver carcinoma HuH-7 cells. Me-AIB uptake in HuH-7 cells was maximal at pH 8.5 when the concentration was 0.4 mM, which is almost the same as the K_m value of Me-AIB uptake by human SNAT2 and much lower than that by human SNAT4. Nevertheless, Me-AIB uptake in HuH-7 cells fitted to a feature of uptake mediated by SNAT4 (Fig. 1A). The K_m value of Me-AIB uptake in HuH-7 cells under basal conditions was also close to that of human SNAT4 (Fig. 3A). Furthermore, the Ct value of human SNAT2 was considerably larger than that of human SNAT4 in HuH-7 cells under basal conditions in the real-time PCR analysis. These results consistently suggest that SNAT4 plays a pivotal role in Me-AIB uptake in HuH-7 cells among system A subtypes. SNAT2 is known to be expressed ubiquitously, including in the rat liver.⁷⁾ In humans, however, SNAT2 is expressed less in the liver than in other organs.⁸⁾ These previous reports also support our results. The transport activity of system A was significantly reduced after APS stimulation for 24 h (Figs. 2A, B), indicating that platelets indirectly downregulate the activity of system A by releasing bioactive substances. Our results also indicated that the inhibitory effect of APS on Me-AIB uptake is brought about after a lag period (after 8 h) (Fig. 2E) and that the effect is responsible for a decrease in affinity of SNAT4 for Me-AIB (Fig. 3A). In contrast, the amount of SNAT4 on the plasma membrane and the mRNA expression of SNAT4 were not decreased significantly by APS stimulation (Figs. 3A, 4B). Because platelets store large amounts of serotonin in their granules and release it upon activation,³¹⁾ APS is also considered to contain serotonin, which is synthesized from tryptophan and its structure closely resembles that of tryptophan. SNAT4 can hardly transport tryptophan,^10,11) and serotonin is therefore also unlikely be a substrate of SNAT4. Thus, the downregulation of system A is probably not due to the desensitization induced by exposure to serotonin.

Notably, APS showed a biphasic effect on Me-AIB uptake in HuH-7 cells in a concentration-dependent manner (Fig. 2F). Activated platelets have been reported to release more than 300 bioactive substances from intracellular granules.³²⁾ Some molecules may contribute to the inhibitory effect on Me-AIB uptake at lower concentrations and other molecules may contribute to the potentiating of Me-AIB uptake at higher concentrations. In addition, platelet granules are known to contain insulin-like growth factor I (IGF-I)³³⁾ and transforming growth factor β (TGF-β).²¹⁾ Both IGF-I and TGF-β have been reported to modulate uptake of amino acids: IGF-I increased the mRNA expression of L-type amino acid transporter (LAT) 1 and decreased LAT3 and LAT4 mRNA levels in myocytes,³⁴⁾ and TGF-β increased amino acid uptake in lung fibroblasts.³⁵⁾ These molecules may contribute to the effect of APS on Me-AIB uptake.

Platelets bind to other cells including hepatocytes,³⁶⁾ neutrophils,³⁷⁾ plasmacytoid dendritic cells²⁸⁾ and regulatory T cells,³⁸⁾ and they play a critical role in liver regeneration,^26,36) trapping pathogenic bacteria²⁷⁾ and systemic lupus erythematosus.^28,38) Furthermore, platelets release growth factors,^21,22) chemokines^23,24) and serotonin,³¹⁾ and they are involved in tumor growth,³⁹⁾ metastasis,²⁵⁾ liver regeneration^26,40) and non-alcoholic steatohepatitis.²⁹⁾ Unlike APS, the platelet suspension did not induce downregulation of system A activity (Fig. 5), indicating that the effect of platelets on Me-AIB uptake is brought about by bioactive substances released from platelets and not by the binding of platelets to HuH-7 cells. Therefore, Me-AIB uptake is expected to decrease when secretion of bioactive substances from platelets is induced by ADP treatment. However, the platelet suspension plus ADP did not also decrease Me-AIB uptake significantly (Fig. 5), and this is thought to be due to the higher concentration of bioactive substances around cells than those in 100% APS.

HuH-7 cells and HepG2 cells have been used as human hepatocyte models for a variety of studies, but these are cancer cell lines. Since cancer cells differ from normal cells in many ways, the effect of platelets on the activity of system A should be elucidated in other models such as primary culture cells in a future study. However, our results showing that the transport activity of system A is reduced by APS in HuH-7 cells and HepG2 cells are very interesting. Amino acids as well as glucose are known to be important for cancer cell proliferation.^41,42) It has also been reported that platelets are activated by cancer cells⁴³⁾ and release growth factors.^21,22) These growth factors promote the proliferation of cancer cells,³⁹⁾ whereas platelets may attempt to suppress the proliferation by inhibition of the uptake of amino acids in cancer cells.

In conclusion, this study is the first study demonstrating that bioactive substances released from platelets decrease the transport activity of system A in HuH-7 cells. The inhibitory effect is due to a decrease in affinity of SNAT4 for its substrate. Our results indicate a novel role of platelets in altering the activity of an amino acid transporter.

Funding

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant Number: 21K06706) and by a Grant provided by The Ichiro Kanehara Foundation (Grant Number: 20KI371).

Conflict of Interest

The authors declare no conflict of interest.

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