Transport characteristics of 5-aminosalicylic acid into colonic epithelium: Involvement of sodium-coupled monocarboxylate transporter SMCT1-mediated transport system

https://doi.org/10.1016/j.bbrc.2020.01.139Get rights and content

Highlights

  • Monocarboxylate transporters, SMCT1 and MCT1, were expressed in mice colon.

  • Na+-dependent nicotinate uptake in mice colon was inhibited by 5-ASA.

  • 5-ASA uptakes in murine colon was Na+-dependent and saturable.

  • 5-ASA uptake was inhibited by ibuprofen, a potent SMCT1 selective inhibitor.

  • α-CHCA, a MCTs specific inhibitor, had no effect on 5-ASA transport.

Abstract

5-Aminosalicylic acid (5-ASA) is conventionally used as a first line drug for inflammatory bowel disease (IBD). Because 5-ASA is well absorbed in the small intestine, very high dose of 5-ASA is required to deliver it to the large intestine which is a target site. Interestingly, 5-ASA is reported to be transported into the large intestine as well as the small intestine via unknown transport system. In a heterologous expression system using Xenopus oocytes, sodium-coupled monocarboxylate transporter 1 (SMCT1) has been reported to accept 5-ASA as a substrate. Although SMCT1 is found to be expressed in the large intestine, it is unknown whether SMCT1 is responsible for 5-ASA absorption from the large intestine or not. Here we determined the transport characteristics of 5-ASA in the isolated everted sac prepared from mouse large intestine. Na+-dependent uptake of [3H]nicotinate, a substrate for SMCT1, in mouse colon was competitively inhibited by 5-ASA with IC50 value of 2.8 mM. In addition to nicotinate, 5-ASA uptake in mouse colonic mucosa was Na+-dependent and saturable with Michaelis constant (Km) of 2.4 mM. Na+-activation kinetics revealed that the Na+-to-5-ASA stoichiometry was 2:1 and concentration of Na+ necessary for half-maximal transport (K0.5Na) was 36.1 mM. Na+-dependent 5-ASA uptake was competitively inhibited by nicotinate with an inhibitory constant (Ki) of 2.1 mM was comparable to the Km value of Na+-dependent nicotinate uptake (0.99 mM). Furthermore, ibuprofen, a selective SMCT1 inhibitor, was found to have a significantly inhibitory effect on the Na+-dependent 5-ASA uptake in mouse colon (IC50 = 0.19 mM). Taken collectively, these results indicated that SMCT1 in the mouse colonic mucosa is responsible for Na+-dependent 5-ASA uptake.

Introduction

5-Aminosalicylic acid (5-ASA) is conventionally used as a first choice drug for remission induction and its maintenance in inflammatory bowel disease (IBD) patients [1,2]. 5-ASA exhibits the therapeutic effect in the epithelial cells of large intestine and its effect is based on several mechanisms, such as inhibiting cyclooxygenases and lipoxygenases, scavenging effective radicals, and inhibiting NF-κB activation. When administered orally in a conventional dosage form, almost of 5-ASA is rapidly absorbed from not only small intestine but also large intestine, although 5-ASA has been classified as low passive diffusion though the plasma membrane [3]. Consequently, 5-ASA cannot reach and accumulate the colonic region. Therefore, very high dose of 5-ASA (1.5–3.0 g/day) is usually required for IBD treatment, even though several colon-selective delivery formulations for 5- ASA were developed. The resultant high transport of 5-ASA from the gastrointestinal tract to systemic circulation would cause some adverse effects, such as nephrotic syndrome, myopericarditis and fever [4]. Thus, it is important to elucidate the absorption mechanism of 5-ASA into the colonic mucosa for utilizing 5-ASA more effectively and safely.

In gastrointestinal tract, two different transport systems for monocarboxylates are expressed. One is Na+-coupled transport system that is sodium-dependent monocarboxylate transporters, SMCT1 and SMCT2. These are plasma membrane transporter for monocarboxylates such as nicotinate, pyruvate and short chain fatty acids (SCFAs) including propionate and butyrate [5]. The SMCT1-mediated transport is Na+-dependent and electrogenic. SMCT1 can be detected in various organs, including brain, thyroid, pancreas, prostate, lung, breast and colon [6,7]. On the other hand, SMCT2 is predominantly expressed in kidney and small intestine [8,9]. SMCT2-mediated transport is electroneutral with a low affinity for its substrates. Another monocarboxylate transporter is H+-coupled transport system which is monocarboxylate transporters (MCTs) [10,11]. The MCT family is mainly comprised of 14 members, of which only the first four (MCT1-MCT4) have been clearly characterized as H+-coupled monocarboxylate transporter [[12], [13], [14], [15]]. Tissue distribution of MCT1 is ubiquitous, whereas MCT2-4 is expressed in restrictive tissue. MCT1 is expressed in both apical and basolateral membrane, whereas MCT2-4 is expressed in basolateral membrane in some tissues [10,15].

Ganapathy and co-workers demonstrated that SMCT1 transports not only nicotinate but also its structurally similar drugs, such as salicylate and 5-ASA using SMCT1-expressing Xenopus oocytes [16]. In addition to SMCT1, MCT1 might accept salicylate and 5-ASA as substrates [17,18]. Therefore, both monocarboxylate transporters, SMCT1 and MCT1, might participate in the absorption of 5-ASA from the large intestine. However, the involvement of SMCT1 and MCT1 in 5-ASA transport in the large intestine has remained unclear. Here we investigated whether 5-ASA is transported via monocarboxylate transporters in large intestine. These studies showed that SMCT1 is predominantly responsible for 5-ASA absorption in isolated mouse colonic mucosa.

Section snippets

Animals

Male ICR mice (5–6 weeks; Japan SLC Co, Inc., Shizuoka, Japan) were fed a pellet diet and water ad libitum. All studies were performed with approval of the Animal Care and Use Committee of Ritsumeikan University. Mice were fasted overnight with free access to water before the experiments.

Materials

[3H]Nicotinic acid (specific activity, 50 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). l-Lactate, nicotinic acid, and 5-ASA were purchased from Wako Pure Chemical

Expression of SMCT1 and MCT1 in mouse GI tract

To determine the expression pattern of SMCT1 along the longitudinal axis of the intestinal tract, we evaluated the expression levels of SMCT1 mRNA in different regions of the mouse intestine by RT-PCR (Fig. 1A). SMCT1 mRNA was not detected in duodenum, whereas it was evident in jejunum, ileum, cecum, and colon. The mRNA levels were more abundant in the distal small intestine, cecum, and colon than in the proximal small intestine. In contrast, mRNA for MCT1 was detectable in whole small

Discussion

Because 5-ASA is a water soluble and exists as a twitter ion under the physiological condition, its intestinal absorption might be estimated to be low. However, 5-ASA is reported to be rapidly absorbed from the small and large intestine [19]. Up to now, uptake mechanism of 5-ASA in the intestine is unclear. In the present study, we investigated the involvement of SMCT1 and MCT1 in the transport of 5-ASA in colon tissues using isolated mouse colonic mucosa.

Initially, we evaluated 5-ASA transport

Author contributions

TY and TF designed the study. TY performed the experiments. YK analyzed the data. TY, YK and TF wrote the manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by a Grant from the Strategic Research Foundation at Private Universities and Grant-in-Aids for Scientific Research (C) [17K08430] from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ritsumeikan Global Innovation Research Organization (R-GIRO) Project at Ritsumeikan University.

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