d ,L -homocysteine-regulated ileum motility via system L and B°,+ transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments

European Journal of Pharmacology 764 (2015) 471–479

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

L-cysteine/D,L-homocysteine-regulated ileum motility via system L and B°, þ transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments Satoshi Yamane a, Ryouya Nomura a, Madoka Yanagihara a, Hiroyuki Nakamura a, Hiromichi Fujino a, Kenjiro Matsumoto b, Syunji Horie b, Toshihiko Murayama a,n a b

Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, Chiba 283-8522, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 21 February 2015 Received in revised form 13 July 2015 Accepted 16 July 2015 Available online 19 July 2015

Previous studies including ours demonstrated that L-cysteine treatments decreased motility in gastrointestinal tissues including the ileum via hydrogen sulfide (H2S), which is formed from sulfur-containing amino acids such as L-cysteine and L-homocysteine. However, the amino acid transport systems involved in L-cysteine/L-homocysteine-induced responses have not yet been elucidated in detail; therefore, we investigated these systems pharmacologically by measuring electrical stimulation (ES)-induced contractions with amino acids in mouse ileum preparations. The treatments with L-cysteine and D,Lhomocysteine inhibited ES-induced contractions in ileum preparations from fasted mice, and these responses were decreased by the treatment with 2-aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), an inhibitor of systems L and B°, þ . The results obtained using ileum preparations and a model cell line (PC12 cells) with various amino acids and BCH showed that not only L-cysteine, but also aminooxyacetic acid and D,L-propargylglycine, which act as H2S synthesis inhibitors, appeared to be taken up by these preparations/cells in L and B°, þ system-dependent manners. The L-cysteine and D,L-homocysteine responses were delayed and abolished, respectively, in ileum preparations from fed mice. Our results suggested that the regulation of ileum motility by L-cysteine and D,L-homocysteine was dependent on BCH-sensitive systems, and varied depending on feeding in mice. Therefore, the effects of aminooxyacetic acid and D,Lpropargylglycine on transport systems need to be considered in pharmacological analyses. & 2015 Elsevier B.V. All rights reserved.

Keywords: L-Cysteine System L Contractility Mouse ileum Hydrogen sulfide

1. Introduction Several amino acids including L-cysteine and L-arginine have been shown to modulate signaling pathways in cells and in vivo as functional amino acids, in addition to their roles as the components of peptides/proteins (Chin-Dusting et al., 2007; Wu, 2009). For example, L-arginine is known to regulate enzyme activity, gene expression, and intracellular signaling and is also a precursor of the gas signaling molecule, nitrogen oxide. L-Cysteine has been shown to directly regulate protein function by modifying disulfide links and the transport of sulfur in proteins, and also indirectly regulate cellular responses by controlling glutathione levels and the formation of hydrogen sulfide (H2S), another gas signaling molecule (Wu, 2009; Ingenbleek and Kimura, 2013). Dysfunctions in nitrogen oxide- and H2S-mediated pathways have been n

Corresponding author. Fax: þ 81 43 226 2875. E-mail address: [email protected] (T. Murayama).

http://dx.doi.org/10.1016/j.ejphar.2015.07.042 0014-2999/& 2015 Elsevier B.V. All rights reserved.

implicated in various diseases including cardiovascular and neurodegenerative diseases (Chin-Dusting et al., 2007; Wu, 2009; Ingenbleek and Kimura, 2013). Homocysteine is a non-essential sulfur-containing amino acid. Hyperhomocysteinemia is a wellestablished risk factor for cardiovascular and chronic kidney diseases, and the possible involvement of H2S in these diseases has already been proposed (Ingenbleek and Kimura, 2013; Pushpakumar et al., 2014). In gastrointestinal tissues, a dysfunction in H2S signaling pathways (Givvimani et al., 2012; Chan and Wallace, 2013), hyperhomocysteinemia, and greater levels of homocysteine in the mucosa (Morgenstern et al., 2003; Danese et al., 2005; Oussalah et al., 2011) have been suggested to regulate pathophysiological processes including inflammatory bowel diseases. We previously reported that (1) L-cysteine and D,L-homocysteine treatments decreased electrical stimulation (ES)-induced contractions through the formation of H2S, and (2) a treatment with aminooxyacetic acid (AOAA), an inhibitor of H2S synthesis, enhanced ES-induced contractions in longitudinal ileum

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S. Yamane et al. / European Journal of Pharmacology 764 (2015) 471–479

preparations from fasted mice (Yamane et al., 2014). However, the transport systems involved in the L-cysteine-induced response have not yet been identified. The transport systems of amino acids have been examined extensively (Poncet and Taylor, 2013; Kanai et al., 2013; Fotiadis et al., 2013; Pramod et al., 2013). L-Cysteine is taken up by various systems including ASC (SLC1A4/5 gene family), B°, þ (ATB°, þ , SLC6A14), L (SLC7A5/8 and SLC43A1/2), and XC– (SLC7A11). System X–A,G (SLC1A1/2/3/6/7) takes up not only anionic amino acids, but also neutral amino acids including L-cysteine (Chen and Swanson, 2003; Hayes et al., 2005). L-Homocysteine is also taken up by  various systems: ASC, XA,G , B°, þ , B° (SLC6A18/19), y þ (SLC7A1-4), L, y þ L (SLC7A6/7), b°, þ (SLC7A9), XC–, and A (SLC38A1-6) (Hayes et al., 2005; Büdy et al., 2006; Jiang et al., 2007). The expression and activities of these transporters are known to be modified by various factors including diet, hormones, and neurotransmitters (Soriano-García et al., 1999; Howard et al., 2004; Kanai et al., 2013; Fotiadis et al., 2013; Pramod et al., 2013). Various inhibitors of amino acid transporters have been examined, and 2-aminobicyclo [2.2.1]heptane-2-carboxylate (BCH) has been established as a specific inhibitor of system L and B°, þ transporters (Palacín et al., 1998; Sloan and Mager, 1999; Fotiadis et al., 2013). In the present study, we investigated the effects of inhibitors of amino acid transporters and several amino acids including L-leucine, L- and Dserine, and D-cysteine on L-cysteine-induced contractile responses in ileum preparations. Our results demonstrated that BCH-sensitive amino acid transporters played critical roles in motility with Lcysteine/D,L-homocysteine, and that the fasting/feeding of mice modulated motility with these amino acids.

2. Materials and methods 2.1. Reagents AOAA and D,L-propargylglycine (PAG), inhibitors of H2S synthesis, were purchased from Sigma-Aldrich (St. Louis, MO, USA). BCH (an inhibitor of system L and B°, þ transporters), α-(methylamino) isobutyric acid (an inhibitor of system A transporters), and acetylcholine chloride were also obtained from Sigma-Aldrich. Amino acids including L- and D-cysteine and D,L-homocysteine were from Nacalai Tesque (Kyoto, Japan). The free base forms of amino acids were used in the present study. L-[3H]Leucine (5.66 TBq/mmol, 153 Ci/mmol) was from Amersham (Buckinghamshire, UK). 2.2. Animals, ileum preparations, and measurement of contractile responses Male ddY mice were purchased from SLC Co. (Shizuoka, Japan). Animals weighing 34–41 g were housed in groups of 5 or 6 under controlled environmental conditions (a temperature of 2472 °C and lights on between 7:00 a.m. and 7:00 p.m.) and fed commercial MF chow (Oriental Yeast Co. Ltd., Tokyo, Japan) for at least 1 week before the experiments. Mice were kept individually and fasted or fed for 18 h with free access to water. They were killed by cervical dislocation. The housing and handling of animals were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society, and experiments were approved by the Laboratory Animal Committee of Chiba University. The ileum was removed and placed in modified Krebs–Hanseleit buffer (112.0 mM NaCl; 5.9 mM KCl; 2.0 mM CaCl2; 1.2 mM MgCl2; 1.2 mM NaH2PO4; 11.5 mM glucose; 10 mM HEPES, and 25.0 mM NaHCO3; pH 7.4). Whole segments (10 mm in length) of the ileum were taken between 10 and 50 mm before the ileo–cecal junction, and these preparations were then suspended in a longitudinal

direction under a 0.5-g load in a 5-ml organ bath containing buffer. The bath was maintained at 37 °C and continuously bubbled with a mixture of 95% O2 and 5% CO2. The procedure used to measure contractile activity with ES (1-min intervals) and the conditions for ES were the same as those described in our previous study (Yamane et al., 2014). Briefly, one end of each preparation was attached to an isotonic transducer, and the other end was mounted on an anodal electrode placed at the bottom of the bath. After equilibration (20–30 min), the preparation was challenged with 3 μM acetylcholine at the beginning of each experiment to estimate the amplitudes of ES-induced contractions. The preparations were transmurally stimulated by platinum needle-ring electrodes. The conditions of ES were 10-V intensity, 0.2-ms duration, 200-ms interval between stimulations, 50 pulses, and a total stimulation for 10 s. The preparations were challenged with ES trials 3–5 times to obtained stable ES responses before drug administration. Stock solutions containing amino acids and the inhibitors of H2S synthesis were prepared with modified Krebs–Hanseleit buffer, and reagents were added to the organ bath after being diluted with buffer. The pH of the assay mixture was 7.4. AOAA, PAG, BCH, α-(methylamino)isobutyric acid, L- and D-cysteine, L-leucine, Lalanine, L- and D-serine, L-arginine, L-lysine, D,L-homocysteine, and L-cystine were added to the organ bath. The concentrations of the drugs and amino acids used were described in the text and in the legends to the Figures and Tables. The concentrations of the inhibitors of H2S synthesis and amino acid transporters were selected based on previous studies (Segawa et al., 1999; Teague et al., 2002; Nemoto et al., 2003; Nagao et al., 2011; Gil et al., 2011; Yamane et al., 2014), and the results obtained demonstrated the validity of the inhibitors at the concentrations tested. The amplitude of ES-induced contractions, namely, from the bottom of the trace (basal tone) to the peak, was defined as an ES response, and ES responses were approximately 30–50% of 3 μM acetylcholineinduced contractions depending on the preparations from fasted and fed mice. In the same preparation, ES-induced contractions showed similar amplitudes for 20 min with vehicle. At the beginning of the incubation, the responses observed were similar in all treatments such as amino acids and the inhibitors of amino acid transporters and H2S synthesis. Thus, the contraction before reagent administration was considered to be 100%, and the contractile response was expressed as a percentage of the control (% of control). In some cases, the area under the curve (AUC, dimension) of an ES-induced contraction was measured, and similar results were obtained. The T1/2 (min) was the time needed to obtain a response with an amplitude that was 50% of the first response. 2.3. Measurement of L-[3H]leucine uptake in PC12 cells The cultivation of PC12 cells, a neuroendocrine cell line derived from rat phaeochromocytoma, and an assay of L-[3H]leucine uptake were conducted as described previously (Nemoto et al., 2003) with minor modifications. Briefly, PC12 cells were re-suspended in modified Tyrode HEPES buffer (137 mM NaCl; 5 mM KCl; 5 mM glucose; 2 mM MgSO4; 2 mM CaCl2; 20 mM HEPES; pH 7.4). Cell suspensions (40–60 μg protein, 0.8–1.2  106 cells per tube, 200 μl) were incubated with 30 nM L-[3H]leucine in the presence of the indicated supplements for 8 min at 37 °C. The reaction was terminated by the addition of 2 ml of ice-cold buffer, and free and incorporated L-[3H]leucine was then separated by repeated washing with buffer. Cells were solubilized in a scintillation cocktail containing 20% Triton X-100, and radioactivity was determined using liquid scintillation spectrometry. 2.4. Statistical analysis Each experiment for the measurement of ileum contractions

S. Yamane et al. / European Journal of Pharmacology 764 (2015) 471–479

was conducted using different animals. Values were presented as means 7S.D. for the indicated numbers (n) of experiments. In the case of L-[3H]leucine uptake, values were the means 7S.E.M. for four independent experiments performed in duplicate. The significance of differences between two groups was assessed using the two-tailed Student's t-test. Multiple comparisons against a single control group were made by a one-way analysis of variance followed by Dunnett's test. Wilcoxon's test or the Mann–Whitney U-test was used for small data sets. P o0.05 was considered significant.

Table 1 L-Cysteine-induced inhibition of ES-induced contractions in the mouse ileum with various amino acids and BCH.

Vehicle L-Leucine L-Alanine L-Serine L-Arginine L-Lysine

(5 mM) (10 mM) D-Cysteine D-Serine BCH (1 mM) L-Lysine

3. Results 3.1. Effects of amino acid transporter inhibitors and amino acids on ES-induced contractions with L-cysteine and D,L-homocysteine in ileum preparations

ES-induced contractions (% of control)

The treatment with L-cysteine time-dependently decreased ESinduced contractions in the longitudinal preparations from fasted mice: the results obtained for 1 mM L-cysteine were shown in Fig. 1 and Table 1, while those for 3 mM L-cysteine were shown in Fig. 4A and Table 3. The amplitude of contractions with 1 mM Lcysteine 20 min after ES was 377 15% (n ¼8), which was significantly less than that of the control, 112 712% (n ¼6, Table 1). In preparations treated with 1 mM BCH, a specific inhibitor of system L and B°, þ transporters (Palacín et al., 1998; Sloan and Mager, 1999; Fotiadis et al., 2013), 1 mM L-cysteine did not inhibit ESinduced contractions. Similar results were obtained by measuring the area under the curve (AUC, dimension) of ES-induced contractions. When the AUC value of the control before the L-cysteine treatment was regarded as 100%, the values obtained with 1 mM Lcysteine at 20 min were 93 718% and 24713% with and without BCH, respectively (n ¼ 3). The treatment with 1 mM α-(methylamino)isobutyric acid, an inhibitor of system A (Palacín et al., 1998), did not change the L-cysteine response. The amplitude of ES-induced contractions with α-(methylamino)isobutyric acid at 20 min was 44 713% (n ¼3), which was almost the same as that without the reagent. The treatment with 5 mM D,L-homocysteine inhibited ES-induced contractions in a time-dependent manner (Fig. 4B), as previously reported (Yamane et al., 2014). In the presence of 1 mM BCH, ES-induced contractions with D,L-homocysteine were 93 75% and 73 710% at 10 and 20 min, respectively (n ¼3), which were greater than previously reported values without BCH (58.3 74.8% and 20.1 75.3% at 10 and 20 min, respectively, n ¼3, Yamane et al., 2014). The treatment with 1 mM α160 140 120

473

None Contraction (% of control)

1 mM L-Cysteine

112 712 (6) 101 72 (3) 104 711 (3) 106 77 (3) 1087 14 (3) 1027 9 (3) 94, 107 (2) 1157 14 (3) 1057 13 (3) 122 721 (3)

37 715a (8) 1027 11b (3) 1027 13b (3) 1117 15b (3) 75, 82 (2) 517 14a (3) 103 7 15b (3) 106 7 11b (3) 1107 11b (3) 1107 14b (3)

ES-induced contractions with and without 1 mM L-cysteine were measured for 20 min in ileum preparations from fasted mice. The indicated amino acids at 10 mM were further supplemented in the assay mixture. In some cases, 5 mM and 10 mM L-lysine and 1 mM BCH were used. The amplitudes of ES-induced contractions were expressed as percentages of the first ES-induced contractions in the respective preparations. Data showed the values obtained 20 min after ES, and are means 7S.D. of the indicated number (n) of independent experiments. Data for 10 mM L-lysine and 10 mM L-arginine/1 mM L-cysteine were from two experiments. a b

Po 0.05, significantly different from the control values without L-cysteine. P o 0.05, significantly different from the value with L-cysteine.

(methylamino)isobutyric acid did not affect the D,L-homocysteine response in a typical experiment (data not shown). We then investigated the effects of amino acids on ES-induced contractions with L-cysteine. System L has been shown to prefer neutral amino acids such as L-leucine, L-methionine, and L-alanine (Kanai et al., 1998; Poncet and Taylor, 2013; Fotiadis et al., 2013), while L-serine (Takarada et al., 2003) and L-homocysteine (Büdy et al., 2006; Tsitsiou et al., 2009) are both taken up via system L. The L-cysteine response was significantly inhibited by the cotreatment with the several amino acids tested; not only L-amino acids such as L-leucine, L-alanine, and L-serine, but also D-cysteine and D-serine were effective at 10 mM. Typical tracings for ES-induced contractions with 1 mM L-cysteine in the absence and presence of 10 mM L-alanine were shown in Supplementary Fig. 1. The co-treatment with 10 mM L-arginine slightly decreased the Lcysteine response 20 min after ES. The co-treatment with L-lysine at 5 mM did not change the L-cysteine response; however L-lysine at 10 mM canceled the L-cysteine response in preparations from fasted mice (Table 1). The amino acids tested at 1 mM had no effect on the L-cysteine response; the contractions observed at 20 min were approximately 31–55% (% of control). The results observed suggested that the inhibition of ES-induced contractions by L-cysteine and D,L-homocysteine appeared to be mediated by BCH-sensitive amino acid transporters. 3.2. Effects of AOAA and PAG on ES-induced contractions with L-cysteine and D,L-homocysteine in ileum preparations

100 80 60 40 0

0

2

4

6

8

10

12

14

16

18

20

22

Time (min) Fig. 1. Time-dependent enhancement in ES-induced contractions by AOAA and its delay with L-cysteine and PAG. ES-induced contractions with 1 mM AOAA were measured for 20 min in ileum preparations from fasted mice. L-Cysteine (■) and PAG (●) at 1 mM or vehicle (○) were further supplemented in the assay mixture. Contractions with 1 mM L-cysteine without AOAA were shown for comparison (□). Data are from typical examples in different preparations, and the amplitudes of ESinduced contractions were expressed as percentages of the first ES-induced contractions in respective preparations. Quantitative data were shown in Table 2.

AOAA has been shown to inhibit cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), while PAG is an irreversible and selective inhibitor of CSE (Asimakopoulou et al., 2013). The treatment with 1 mM AOAA alone enhanced ES-induced contractions from 3–5 min after ES, and this enhancement continued for 20 min (Fig. 1 and Supplementary Fig. 2A); however, PAG did not show a stimulatory effect (Yamane et al., 2014). The co-treatment with 1 mM L-cysteine delayed the appearance of the AOAA response (Fig. 1 and Supplementary Fig. 2B): the AOAA response with L-cysteine at 5 min was significantly less than that without Lcysteine, and the AOAA responses with L-cysteine observed after 10 min were the same as those without L-cysteine (Table 2). The co-treatment with 1 mM L-serine inhibited the appearance of the

S. Yamane et al. / European Journal of Pharmacology 764 (2015) 471–479

Time (min)

0 5 10 Contraction (% of control)

Control (7) AOAA þ Vehicle (6) þ L-Cysteine (4) þ L-Serine (5) þ BCH (4) þ PAG (3)

100% 100 100 100 100 100

15

104 7 6

1037 9 a

1377 4 110 77b 1017 5b 1027 5b 997 12b

a

1387 9 125 78a 1107 9 126 79a 1197 14

20

997 4

1087 6 a

1407 11 1527 12a 1197 12 1307 10a 1447 13a

148 7 7a 1567 11a 1087 13b 1347 12a 1417 11a

Ileum preparations from fasted mice were treated with 1 mM AOAA and ES-induced contractions were then measured for 20 min. In the assay mixtures, 1 mM Lcysteine, 1 mM L-serine, 1 mM BCH, and 1 mM PAG were further supplemented. Data are means 7 S.D. of the indicated number (n) of independent experiments. a

P o0.05, significantly different from the first ES-induced contraction (time 0). Po 0.05, significantly different from the respective values with AOAA alone at each time. b

ES-induced contractions (% of control)

AOAA response for 20 min (Table 2). Not only the co-treatment with 1 mM L-leucine, but also that with 1 mM L-alanine inhibited the appearance of the AOAA response for 20 min in typical experiments (data not shown). The co-treatment with 1 mM BCH delayed the appearance of the AOAA response without changing the responses observed 15 and 20 min after ES (Table 2). These results suggested that AOAA, similar to L-cysteine, was mainly taken up into target cells via BCH-sensitive amino acid transporters in the initial phase (5 min) after the treatment and AOAA then enhanced ES-induced contractions because of the inhibition of H2S synthesis in ileum preparations. The L-cysteine-induced inhibition of ES-induced contractions was mediated via AOAA-sensitive CBS, and the treatment with PAG did not change the L-cysteine response in the ileum preparations from fasted mice (Yamane et al., 2014). In the present study, the co-treatment with 1 mM PAG significantly decreased the AOAA response 5 min after ES (Fig. 1 and Supplementary Fig. 2C). In some preparations, PAG inhibited the AOAA response for 20 min (Fig. 1); however, the effects of PAG from 10 min after ES were not significant (Table 2). The inhibitory effects of PAG on the AOAA response suggested that PAG inhibited the L-cysteine response in a manner that depended on the experimental conditions. In the present study, the effects of 1 mM PAG were examined with 5 mM L-cysteine (Fig. 2). ES-induced contractions 10 min after ES were 106 78% with PAG, which was significantly greater than the

Pretreatment

100

50

0

PAG

*

*

- + 10 min

+

* * - + 20 min

Fig. 2. Inhibition of the L-cysteine response by a co-treatment, but not pre-treatment with PAG. ES-induced contractions with 5 mM L-cysteine were measured for 20 min in ileum preparations from fasted mice. Vehicle (open column) or 1 mM PAG (hatched column) was further supplemented in the assay mixture. In some cases, the preparations were pre-treated with 1 mM PAG for 10 min, and ES-induced contractions were then measured after washing of the preparations with PAG-free buffer. Data are means 7 S.D. of four independent experiments. * Po 0.05, significantly different from the control.

response observed without PAG, 53 713% (n ¼3, P o0.05). In contrast, ES-induced contractions at 20 min with and without PAG were reduced to a similar degree by L-cysteine, as reported previously (Yamane et al., 2014). In preparations that were pre-treated with 1 mM PAG for 10 min and then washed with PAG-free buffer, the 5 mM L-cysteine response 10 min after ES was almost the same as that observed without the PAG treatment (Fig. 2). Thus, PAG appeared to delay the appearance of the L-cysteine response, and this may have been due to the inhibition of L-cysteine uptake, and not the inhibited formation of H2S. The potentially inhibitory effect of 1 mM PAG on the responses observed with 1 mM L-cysteine was not detected and was attributed to a time lag in the appearance of the L-cysteine response (Fig. 1); however, the response with 10 mM L-cysteine 10 min, but not 20 min after ES was slightly decreased by PAG (Yamane et al., 2014). The results obtained suggested that PAG was taken up into target cells, at least partially, via the amino acid transporters responsible for the uptake of AOAA and L-cysteine. 3.3. Inhibition of L-[3H]leucine uptake by AOAA and PAG in PC12 cells Our previous findings suggested the involvement of enteric neurons in the formation of H2S in ileum preparations with L-cysteine (Yamane et al., 2014). Therefore, we herein examined the effects of AOAA and PAG on amino acid transport activity in neuronal cells. A system L transporter is composed of 4F2 light chains (LAT1-4) and heavy chain (4F2hc, also referred to as CD98), whereas LAT3/4 alone exhibits transport activities (Fotiadis et al., 2013; Bodoy et al., 2013). The existence of LAT1, LAT2, and 4F2hc mRNA transcripts was confirmed in PC12 cells, and the uptake of L[3H]leucine was shown to be inhibited by a co-treatment with 1 mM BCH (Nemoto et al., 2003). Thus, we selected PC12 cells as a model of neurons. The co-treatment with 1 mM L-cysteine decreased L-[3H]leucine uptake (Fig. 3), as reported previously (Nemoto et al., 2003), while the co-treatment with AOAA and PAG at 1 mM significantly decreased L-[3H]leucine uptake in PC12 cells (Fig. 3). 3.4. Effects of diet on ES-induced contractions with L-cysteine and D, L-homocysteine in the mouse ileum The activities of amino acid transporters, including systems L and B°, þ , were previously shown to be regulated by various stimuli including diet and hormones (Luo et al., 2009; Hamdi and Mutungi, 2011; Rosario et al., 2012; Poncet and Taylor, 2013). Therefore, we compared L-cysteine- and D,L-homocysteine-induced responses between fasted and fed mice. In ileum preparations

uptake into PC12 cells (% of control)

Table 2 Time-dependent enhancement in ES-induced contractions by AOAA and its delay with L-cysteine, L-serine, BCH, and PAG.

L-[3H]Leucine

474

100 75 50 25 0

-

L-Cys

AOAA

PAG

Fig. 3. Inhibition of L-[3H]leucine uptake via system L by AOAA and PAG in PC12 cells. PC12 cells were incubated with L-[3H]leucine for 8 min in the presence of 1 mM L-cysteine, AOAA, or PAG. Data are means 7 S.E.M of four independent experiments performed in duplicate. * Po 0.05, significantly different from the control.

ES-induced contractions (% of control)

S. Yamane et al. / European Journal of Pharmacology 764 (2015) 471–479

140 120 100 80 60 40 20 0

475

fed (4)

fasted (4) 0

10

20

ES-induced contractions (% of control)

Time (min) 140 120 100 80 60 40 20 0

**

*

fasted (3) 0

*

*

fed (3)

*

***** * *

10

20

ES-induced contractions (% of control)

Time (min) 140 120 100 80 60 40 20 0

fed (5)

****

fasted (3) 0

10

20

Time (min)

20 min

100

* 50

* L-Cys

D, L-Homocys

50

*

L-Cys + D, L-Homocys

L-Cys

D, L-Homocys

3

2

Feeding

1

3

2

Feeding

100

0

1

0

ES-induced contractions (% of control)

ES-induced contractions (% of control)

9 min

L-Cys + D, L-Homocys

Fig. 4. Effects of L-cysteine and D,L-homocysteine on ES-induced contractions in ileum preparations from fasted and fed mice. Ileum preparations were prepared from fasted (○) and fed (●) mice, and ES-induced contractions were then assayed for 20 min with 3 mM L-cysteine (A), 5 mM D,L-homocysteine (B), a combination of L-cysteine and D,Lhomocysteine (C). The selected data showing the responses at 9 min and 20 min were shown in (D) and (E), respectively, as bar charts. L-Cysteine (L-Cys); D,L-Homocysteine (D,L-Homocys). Data are means 7S.D. of the indicated number of experiments. * P o 0.05, significantly different from the values obtained from preparations from fed mice.

from fasted mice, the treatment with 3 mM L-cysteine decreased ES-induced contractions from approximately 5 min after ES in a time-dependent manner (Fig. 4A and Table 3). However, the appearance of the L-cysteine response was delayed in ileum preparations from fed mice. Although the difference observed in the amplitudes of contractions was not significant in the preparations from fasted and fed mice because of wide variations, the time required to show 50% of the ES-induced contraction (T1/2) in the preparations with L-cysteine from fed mice was 15.1 73.7 min (n ¼4), which was greater than the T1/2, 8.2 71.8 min, in those from fasted mice. L-Cysteine responses 20 min after ES in

preparations from fasted and fed mice were the same. The 3 mM Lcysteine responses at 20 min were markedly reduced in preparations treated with 1 mM BCH; the values obtained were 977 12 and 93 714% in preparations from fasted and fed mice, respectively (n ¼3). The inhibitory effects of 10 mM L-cysteine on ES-induced contractions, including the degree of inhibition and timedependency, in preparations from fed mice (data not shown) were similar to those from fasted mice (Yamane et al., 2014). The treatment with 5 mM D,L-homocysteine significantly decreased ESinduced contractions from 7 min after ES in the preparations from fasted mice as reported previously (Yamane et al., 2014), while this

476

S. Yamane et al. / European Journal of Pharmacology 764 (2015) 471–479

Table 3 Diverse effects of L-lysine on ES-induced contractions with L-cysteine in ileum preparations from fasted and fed mice. Time (min)

5 10 15 Contraction (% of control)

3 mM L-Cysteine Fasted (4) 747 11 437 26 Fed (4) 917 25 847 32 3 mM L-Cysteine þ 5 mM L-Lysine Fasted (4) 108 79a 797 22 Fed (3) 102 78 617 19 5 mM L-Lysine Fasted (2) 106, 104 103, 92 Fed (3) 103 712 1017 7

20

T1/2

257 26 467 17

187 26 307 21

8.2 7 1.8 15.1 73.7b

617 21 247 13

49 719 97 25

20.4 7 4.3a 9.2 7 3.5b

94, 108 937 11

109, 89 987 12

N.D. N.D.

Ileum preparations were prepared from fasted and fed mice, and ES-induced contractions were then assayed for 20 min with 3 mM L-cysteine and/or 5 mM Llysine. The time required to show 50% of the contraction was evaluated as T1/2. Data are means 7 S.D. of the indicated number (n) of experiments. a b

P o0.05, significantly different from the values without L-lysine. Po 0.05, significantly different from values in preparations from fasted mice.

amino acid did not change ES-induced contractions for 20 min in those from fed mice (Fig. 4B and Supple. Fig. 3). The combinatory effect of 3 mM L-cysteine and 5 mM D,L-homocysteine appeared to be additive in preparations from fasted and fed mice (Fig. 4C). The ES-induced contractions with 5 mM D,L-homocysteine at 9 min (Fig. 4D) and at 20 min (Fig. 4E) in fasted mice were significantly less than those in fed mice. In the presence of 10 mM D,L-homocysteine, ES-induced contractions at 10 min were 69 712% (n ¼4) in the preparations from fed mice, and 26% and 38% (n ¼2) in those from fasted mice; however, the contractions at 20 min in the two groups were almost the same at 16–34%. The treatment with 5 mM L-cystine did not change ES-induced contractions in preparations from fasted and fed mice (Supple. Fig. 4). ES-induced contractions were not changed by the 5 mM L-cystathionine treatment in preparations from fasted and fed mice: the values were within 95–106% (n¼ 2, respectively). These results suggested that the contractile responses induced by L-cysteine and D,Lhomocysteine in ileum preparations differed between fasted and fed mice. In preparations from fed mice, the contractile responses induced by 1 mM AOAA varied depending on the preparations (data not shown).

preparations from fasted and fed mice (n ¼2, respectively, data not shown).

4. Discussion In the present study, we showed that the inhibition of ES-induced contractions by L-cysteine and D,L-homocysteine was dependent on BCH-sensitive amino acid transporters in mouse ileum preparations. The L-cysteine/D,L-homocysteine-induced response was modulated by the co-addition of other amino acids in vitro and by nutrient conditions in vivo. A proposed model in fasted mice is shown in Fig. 5. 4.1. Role of amino acid transport systems on L-cysteine-induced inhibition and AOAA-induced enhancement of ES-induced contractions The L-cysteine response was inhibited by BCH and other amino acids such as L-leucine, L-alanine, and L-serine (Table 1). The treatment with D-cysteine and D-serine also inhibited the L-cysteine response. Previous studies established that BCH selectively inhibited system L and system B°, þ transporters (Sloan and Mager, 1999; Fotiadis et al., 2013; Bodoy et al., 2013). LAT1 and LAT2 form a heterodimer with 4F2hc (CD98) and exhibit the transport activity characteristics of system L (Kanai et al., 1998; Pineda et al., 1999; Segawa et al., 1999), while LAT3 (Babu et al., 2003) and LAT4 (Bodoy et al., 2005) do not require 4F2hc to express functional activity. The activities of all members of system L were inhibited by BCH. Previous studies reported that L-serine and L-alanine were transported by system L having LAT2, but not by the system having LAT1, in the functional expression system in Xenopus oocytes (Kanai et al., 1998; Pineda et al., 1999; Segawa et al., 1999). Previous studies established that LAT1 mRNA or protein was absent from or quite limited in the jejunum and ileum of rats and humans, and LAT2 existed homogenously across digestive tissues including the ileum (Segawa et al., 1999; Prasad et al., 1999; Fraga et al., 2005). L-Cysteine, L- and D-serine, and L-alanine are not substrates for LAT3 (Babu et al., 2003), while L-serine and L-alanine are not substrates for LAT4 (Bodoy et al., 2005). These findings in combination with our results suggested that system L with LAT2 was involved in the L-cysteine response under our experimental conditions. L-Methionine was shown to be taken up via system L

3.5. Combinatory effects of L-lysine and L-cysteine on ES-induced contractions and its modification by diet We compared the effects of several amino acids at 5 mM on ESinduced contractions with and without 3 mM L-cysteine between preparations from fasted and fed mice. The co-treatment with 5 mM L-lysine, a substrate for system B°, þ , delayed the L-cysteine response in preparations from fasted mice (Table 3): T1/2 was 20.4 74.3 min with L-lysine (n ¼4), which was significantly greater than that without L-lysine (8.271.8 min, P o0.05). The treatment with 3 mM L-cysteine decreased ES-induced contractions with and without L-lysine to a similar degree 20 min after ES, as observed for the 1 mM L-cysteine/5 mM L-lysine response shown in Table 1. However, the co-treatment with L-lysine accelerated the onset of the inhibitory effect of 3 mM L-cysteine in preparations from fed mice (Table 3): T1/2 was 9.2 73.5 min with L-lysine (n ¼3), which appeared to be less than that without L-lysine, 15.1 73.7 min. The treatment with L-lysine by itself did not change ES-induced contractions from the start to 20 min after ES in preparations from fasted and fed mice. Thus, modifications to the L-cysteine response by L-lysine appeared to be dependent on the dietary conditions of the mice. The effects of L-leucine or L-alanine at 5 mM on the 3 mM L-cysteine response 10 and 20 min after ES were changeable in

L-Cys, D,L-Homocys

AOAA, PAG

L-Lys

BCH System L (LAT2)

System B0,+

L-Cys, L-Homocys

AOAA

CBS H 2S decrease in contractions

Fig. 5. A proposed model for actions of amino acids, AOAA, and PAG on the regulation of motility with L-cysteine in ileum preparations. L-Cysteine (L-Cys) and D,Lhomocysteine (D,L-Homocys) are taken up via BCH-sensitive amino acid transporters, that were likely system L with LAT2 and system B°, þ , into H2S-forming cells, and both amino acids inhibited ES-induced contractions via the AOAA-sensitive CBS-mediated formation of H2S. The treatment with AOAA alone enhanced ES-induced contractions because of the inhibition of H2S synthesis.

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with LAT1/2 (Pineda et al., 1999; Segawa et al., 1999); therefore, this amino acid may have an inhibitory effect on the L-cysteine response. We previously showed that L-methionine accelerated the appearance of the L-cysteine response in ileum preparations, and this may have been due to the formation of S-adenosylmethionine, an activator of CBS (Yamane et al., 2014). Thus, the possible L-methionine-induced inhibition of L-cysteine uptake by competition with system L may have been masked under our experimental conditions. System B°, þ can transport not only charged amino acids including L-lysine, but also L-cysteine, L-alanine, Lleucine, and BCH (Sloan and Mager, 1999). In the ileum preparations from fasted mice, the co-treatment with 5 mM L-lysine delayed the 3 mM L-cysteine response (Table 3); the co-treatment significantly increased the T1/2 value without changing the L-cysteine response 20 min after ES. Thus, L-cysteine and D,L-homocysteine appeared to be taken up, at least partially, by system L possibly with LAT2 and system B°, þ , and then inhibited ES-induced contractions via H2S in ileum preparations from fasted mice. 4.2. AOAA and PAG as substrates of amino acid transporters The treatment of ileum preparations with AOAA, an inhibitor of H2S synthesis, alone enhanced ES-induced contractions (Table 2), whereas that with PAG did not (Yamane et al., 2014). In the present study, we showed that the appearance of the AOAA response was delayed by the co-treatment with BCH, L-cysteine, L-serine, and PAG (Table 2 and Supplementary Fig. 2). Since AOAA and PAG both have similar chemical structures to amino acids, we speculated that these reagents may have been taken up by the ileum preparations via BCH-sensitive amino acid transporters. In the present study, we examined the effects of AOAA and PAG on [3H]leucine uptake in PC12 cells as a model of neurons. Not only L-cysteine, but also AOAA and PAG inhibited [3H]leucine uptake in PC12 cells (Fig. 3). As shown in Fig. 2, PAG inhibited the L-cysteine response under specified conditions (5 mM L-cysteine, 10 min after ES), and the reagent inhibited the AOAA response within 5–10 min of ES (Table 2). Thus, PAG may have decreased L-cysteine- and AOAAinduced responses by inhibiting the uptake of L-cysteine and AOAA. Previous studies proposed that PAG had limited cellmembrane permeability (Szabó, 2007; Asimakopoulou et al., 2013), but was effective as an inhibitor of CSE in gastrointestinal tissues (Teague et al., 2002; Gil et al., 2011; Nagao et al., 2011). Our results suggested that AOAA and PAG were both taken up by preparations/cells via BCH-sensitive amino acid transporters. Therefore, pharmacological analyses using AOAA and PAG need to take their uptake via amino acid transporters into consideration. 4.3. Changes in contractile responses in ileum preparations by feeding Regarding the sensitivity of contractile activity to the amino acids tested, two marked changes were observed between ileum preparations from fasted and fed mice. The response induced by 3 mM L-cysteine was delayed more in preparations from fed mice than in those from fasted mice (Table 3), and the response induced by 5 mM D,L-homocysteine was abolished in preparations from fed mice (Figs. 4D and E and Supplementary Fig. 3). The 3 mM L-cysteine response at 20 min and 10 mM L-cysteine response were not changed by feeding in vivo. Gene-expression profiling previously showed that the expression of CBS was not modified after 24 and 72 h of fasting in the murine small intestine (Sokolović et al., 2007). Thus, upstream pathways before H2S synthesis appeared to be modified by diet. Diet and/or nutrients have been shown to regulate the expression of amino acid transport systems including L in various cells (Poncet and Taylor, 2013), and system B°, þ mRNA levels were found to be increased in intestinal tissues from

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weaned piglets (He et al., 2013). L-Arginine supplementation enhanced the intestinal expression of SLC7A7 and SLC7A1 in pigs (Yin et al., 2014), and the up-regulation of system A transporters and resulting L-cysteine uptake occurred within 4 h in hydrogen peroxide-treated rat cardiomyocytes (King et al., 2011). Thus, transporters for the uptake of L-cysteine and D,L-homocysteine may have been down-regulated in ileum preparations from fed mice under our conditions. Furthermore, the activity of system L was shown to be modulated by adhesion molecules such as 4F2hc (CD98) and intracellular adhesion molecule I (Liu et al., 2003) as well as by TAT1 (SLC16A10 product, system T, Ramadan et al., 2007), while the activity of system B°, þ was modified by protein kinase C (Samluk et al., 2012) and tryptophan derivatives (Karunakaran et al., 2008). The precise mechanism(s) responsible for diet-dependent changes in the L-cysteine-induced response including the identification of molecules regulating the transport activity of L-cysteine and cell types producing H2S need to be elucidated in more detail in future studies. A second difference noted between preparations from fasted and fed mice was sensitivity to L-lysine. In the preparations from fasted mice, the treatment with 5 mM L-lysine decreased and/or delayed the 3 mM L-cysteine response (Table 3) while the treatment with 10 mM L-lysine decreased the 1 mM L-cysteine response (Table 1). In contrast, the treatment with 5 mM L-lysine enhanced the 3 mM L-cysteine response from 10 min in the preparations from fed mice; however, the stimulatory effect of L-lysine was not detected at 5 min, and this may have been because of a time lag in the L-cysteine response. Our results showed the diverse effects of L-lysine on the L-cysteine-induced response; an inhibition in preparations from fasted mice and stimulation in those from fed mice. The L-lysine-induced inhibition of the L-cysteine response may be explained as follows. The co-treatment with L-lysine, similar to other amino acids such as L-leucine and Lalanine, may have inhibited L-cysteine uptake, particularly within 5 min of ES. System L with LAT1/2 may act as an obligatory amino acid exchanger, and a treatment with several amino acids including L-lysine has been shown to activate the efflux of L-cysteine via system L (Pineda et al., 1999; Poncet and Taylor, 2013; Fotiadis et al., 2013). Thus, L-lysine taken up via system B°, þ may stimulate the efflux of L-cysteine from intracellular spaces via system L. The L-lysine-induced enhancement of the L-cysteine response in the preparations from fed mice may be explained by the decreased efflux of L-cysteine via system L and/or decreased uptake of L-lysine via system B°, þ ; however, the uptake of L-cysteine from fed mice may have been less than that from fasted mice. In conclusion, our results suggested that BCH-sensitive amino acid transporters in H2S-forming cells regulated L-cysteine/D,Lhomocysteine-induced contractile responses in mouse ileum preparations. The dietary treatment of mice affected L-cysteine/D,Lhomocysteine responses and the sensitivity of the L-cysteine response to L-lysine. Amino acid transport systems have been reported to vary in gastrointestinal tissues from model animals and patients with chronic intestinal inflammation including Crohn's disease (Sundaram et al., 2007; Eriksson et al., 2008) and following massive bowel resection (Welters et al., 2001; Ray et al., 2003). In future studies, possible pathophysiological changes in the L-cysteine- and/or H2S-induced regulation of motility need to be elucidated in more detail.

Conflicts of interest statement The authors declare that there are no conflicts of interest.

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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.07. 042.

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