Electrophysiological characterization of the polyspecific organic cation transporter plasma membrane monoamine transporter.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na+ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of Imax with little effect on K0.5. At most holding membrane potentials, Imax at pH 6.0 is approximately 3- to 4-fold higher than that at pH 7.5, whereas K0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: a RPE specific phenomenon.

SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.

Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy.

ATB(0,+) [SLC6A14 (solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of approximately 250 muM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that alpha-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with alpha-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.

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.

Sodium-coupled monocarboxylate transporters in normal tissues and in cancer.

SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na(+)-coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and Müller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and gamma-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.

Sodium-coupled transport of the short chain fatty acid butyrate by SLC5A8 and its relevance to colon cancer.

INTRODUCTION: SLC5A8, expressed predominantly in the colon, is a Na(+)-coupled transporter for short-chain fatty acids. In this paper, we report on the characterization of butyrate transport by SLC5A8 and the relevance of SLC5A8-mediated butyrate transport to colon cancer. RESULTS: SLC5A8 transports butyrate via a Na(+)-dependent electrogenic process. Na(+) activation of the transport process exhibits sigmoidal kinetics, indicating involvement of more than one Na(+) in the activation process. SLC5A8 is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of SLC5A8 involves DNA methylation by DNA methyltransferase 1. Reexpression of SLC5A8 in colon cancer cells leads to apoptosis but only in the presence of butyrate. SLC5A8-mediated entry of butyrate into cancer cells is associated with inhibition of histone deacetylation. The changes in gene expression in SLC5A8/butyrate-induced apoptosis include upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes. In addition, the expression of phosphatidylinositol-3-kinase subunits is affected differentially, with downregulation of p85alpha and upregulation of p55alpha and p50alpha. CONCLUSION: These studies show that SLC5A8 mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in the colon.

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain.

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.