Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na⁺-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na⁺-dependent and electrogenic. The Na⁺-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na⁺-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na⁺-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.     

Transport of Nicotinate and Structurally Related Compounds by Human SMCT1 (SLC5A8) and Its Relevance to Drug Transport in the Mammalian Intestinal Tract

Purpose To examine the involvement of human SMCT1, a Na⁺-coupled transporter for short-chain fatty acids, in the transport of nicotinate/structural analogs and monocarboxylate drugs, and to analyze its expression in mouse intestinal tract. Materials and Methods We expressed human SMCT1 in X. laevis oocytes and monitored its function by [¹⁴C]nicotinate uptake and substrate-induced inward currents. SMCT1 expression in mouse intestinal tract was examined by immunofluorescence. Results [¹⁴C]Nicotinate uptake was several-fold higher in SMCT1-expressing oocytes than in water-injected oocytes. The uptake was inhibited by short-chain/medium-chain fatty acids and various structural analogs of nicotinate. Exposure of SMCT1-expressing oocytes to nicotinate induced Na⁺-dependent inward currents. Measurements of nicotinate flux and associated charge transfer into oocytes suggest a Na⁺:nicotinate stoichiometry of 2:1. Monocarboxylate drugs benzoate, salicylate, and 5-aminosalicylate are also transported by human SMCT1. The transporter is expressed in the small intestine as well as colon, and the expression is restricted to the lumen-facing apical membrane of intestinal and colonic epithelial cells. Conclusions Human SMCT1 transports not only nicotinate and its structural analogs but also various monocarboxylate drugs. The transporter is expressed on the luminal membrane of the epithelial cells lining the intestinal tract. SMCT1 may participate in the intestinal absorption of monocarboxylate drugs.     

Interaction of Ibuprofen and Other Structurally Related NSAIDs with the Sodium-Coupled Monocarboxylate Transporter SMCT1 (SLC5A8)

Purpose Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na⁺-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). Methods We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [¹⁴C]-nicotinate and [³H]-ibuprofen for direct measurements of uptake in oocytes. Results In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na⁺ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 ± 16 μM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na⁺ dependent, and competitive. However, there was no uptake of [³H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [¹⁴C]-nicotinate was demonstrable under identical conditions. Conclusions Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.     

Drug discovery for hyperuricemia

Hyperuricemia is associated with an increased risk of developing gout. This increases with the degree and duration of hyperuricemia. Gout can be managed by dietary modification and pharmacologic urate-lowering therapies. The recent identification of the renal apical urate/anion exchanger URAT1 (SLC22A12) and several membrane proteins relevant to the transport of urate play an important role in gaining a better understanding of the mode of action of many drugs used to treat gout. As described in this review, therapeutics designed to modify URAT1 transport activities might be useful in treating pathologies associated with hyperuricemia such as gout and urolithiasis. Continuing studies into the urate transportsome hold promise for the development of new, more effective therapeutics for hyperuricemia.     

Transport Alteration of 4-Phenyl Butyric Acid Mediated by a Sodium- and Proton-Coupled Monocarboxylic Acid Transporter System in ALS Model Cell Lines (NSC-34) Under Inflammatory States

4-Phenyl butyric acid (PBA) has histone deacetylase inhibitory and neuroprotective effects. We aimed to examine the transport alteration activity of PBA in control (WT) and disease (MT) model cell lines of an amyotrophic lateral sclerosis (ALS) model. The transport characteristics of PBA were examined uptake rates and mRNA expression levels in NSC-34 cell lines. PBA uptake was pH, sodium, and concentration dependent. The K_m and V_max values for PBA uptake in the MT were more than two-fold higher than those in the WT. The presence of monocarboxylic acids (MA) and inhibitors of MA transporter (MCT) inhibited the uptake of PBA. PBA showed competitive inhibition in the presence of MAs in both cell lines. SiRNA transfection studies showed that PBA can be transported to NSC-34 cell lines through sodium-coupled MCT1. TNF-α and H₂O₂ increased, but LPS and glutamate reduced the uptake rate after the pretreatment of the MT cell lines. SMCT1 mRNA expression levels, in the presence of oxidative stress inducing agents, showed consistent results with the uptake results. These results demonstrate that PBA can be transported to the ALS model NSC-34 cell lines by sodium- and proton-coupled MCTs, and MA plays a vital role in the prevention of neurodegenerative diseases.     

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3‐bromopyruvate

BACKGROUND:

3‐Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3‐bromopyruvate.

METHODS:

The transport of 3‐bromopyruvate by sodium‐coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na⁺)‐coupled, electrogenic transporter for short‐chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3‐bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence‐activated cell‐sorting analysis and colony‐formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis.

RESULTS:

3‐Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na⁺‐coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3‐bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3‐bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3‐bromopyruvate.

CONCLUSIONS:

3‐Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na⁺ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Cancer 2009. © 2009 American Cancer Society.