Bicarbonate secretion and acid/base sensing by the intestine.

The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the first description of "active" transport of HCO3- ions against a concentration gradient in the 1970s, the fundamental role of HCO3- transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO3- transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO3- secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells.

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

Starvation-induced regulation of carbohydrate transport at the blood-brain barrier is TGF-ß-signaling dependent.

During hunger or malnutrition, animals prioritize alimentation of the brain over other organs to ensure its function and, thus, their survival. This protection, also-called brain sparing, is described from Drosophila to humans. However, little is known about the molecular mechanisms adapting carbohydrate transport. Here, we used Drosophila genetics to unravel the mechanisms operating at the blood-brain barrier (BBB) under nutrient restriction. During starvation, expression of the carbohydrate transporter Tret1-1 is increased to provide more efficient carbohydrate uptake. Two mechanisms are responsible for this increase. Similar to the regulation of mammalian GLUT4, Rab-dependent intracellular shuttling is needed for Tret1-1 integration into the plasma membrane; even though Tret1-1 regulation is independent of insulin signaling. In addition, starvation induces transcriptional upregulation that is controlled by TGF-ß signaling. Considering TGF-ß-dependent regulation of the glucose transporter GLUT1 in murine chondrocytes, our study reveals an evolutionarily conserved regulatory paradigm adapting the expression of sugar transporters at the BBB.

Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3--coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined "transport metabolon". While transport metabolons built with HCO3--coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Experimental Approaches to Identify Selective Picomolar Inhibitors for Carbonic Anhydrase IX.

BACKGROUND: Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials, mostly due to low affinity and selectivity properties. OBJECTIVE: The current review presents the design, development, and identification of the selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. METHODS AND RESULTS: Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure- activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. CONCLUSIONS: Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.

Transport Metabolons and Acid/Base Balance in Tumor Cells.

Solid tumors are metabolically highly active tissues, which produce large amounts of acid. The acid/base balance in tumor cells is regulated by the concerted interplay between a variety of membrane transporters and carbonic anhydrases (CAs), which cooperate to produce an alkaline intracellular, and an acidic extracellular, environment, in which cancer cells can outcompete their adjacent host cells. Many acid/base transporters form a structural and functional complex with CAs, coined "transport metabolon". Transport metabolons with bicarbonate transporters require the binding of CA to the transporter and CA enzymatic activity. In cancer cells, these bicarbonate transport metabolons have been attributed a role in pH regulation and cell migration. Another type of transport metabolon is formed between CAs and monocarboxylate transporters, which mediate proton-coupled lactate transport across the cell membrane. In this complex, CAs function as "proton antenna" for the transporter, which mediate the rapid exchange of protons between the transporter and the surroundings. These transport metabolons do not require CA catalytic activity, and support the rapid efflux of lactate and protons from hypoxic cancer cells to allow sustained glycolytic activity and cell proliferation. Due to their prominent role in tumor acid/base regulation and metabolism, transport metabolons might be promising drug targets for new approaches in cancer therapy.

CAIX forms a transport metabolon with monocarboxylate transporters in human breast cancer cells.

Tumor cells rely on glycolysis to meet their elevated demand for energy. Thereby they produce significant amounts of lactate and protons, which are exported via monocarboxylate transporters (MCTs), supporting the formation of an acidic microenvironment. The present study demonstrates that carbonic anhydrase IX (CAIX), one of the major acid/base regulators in cancer cells, forms a protein complex with MCT1 and MCT4 in tissue samples from human breast cancer patients, but not healthy breast tissue. Formation of this transport metabolon requires binding of CAIX to the Ig1 domain of the MCT1/4 chaperon CD147 and is required for CAIX-mediated facilitation of MCT1/4 activity. Application of an antibody, directed against the CD147-Ig1 domain, displaces CAIX from the transporter and suppresses CAIX-mediated facilitation of proton-coupled lactate transport. In cancer cells, this "metabolon disruption" results in a decrease in lactate transport, reduced glycolysis, and ultimately reduced cell proliferation. Taken together, the study shows that carbonic anhydrases form transport metabolons with acid/base transporters in human tumor tissue and that these interactions can be exploited to interfere with tumor metabolism and proliferation.

Plasticity of Carbohydrate Transport at the Blood-Brain Barrier.

Neuronal function is highly energy demanding, requiring efficient transport of nutrients into the central nervous system (CNS). Simultaneously the brain must be protected from the influx of unwanted solutes. Most of the energy is supplied from dietary sugars, delivered from circulation via the blood-brain barrier (BBB). Therefore, selective transporters are required to shuttle metabolites into the nervous system where they can be utilized. The Drosophila BBB is formed by perineural and subperineurial glial cells, which effectively separate the brain from the surrounding hemolymph, maintaining a constant microenvironment. We identified two previously unknown BBB transporters, MFS3 (Major Facilitator Superfamily Transporter 3), located in the perineurial glial cells, and Pippin, found in both the perineurial and subperineurial glial cells. Both transporters facilitate uptake of circulating trehalose and glucose into the BBB-forming glial cells. RNA interference-mediated knockdown of these transporters leads to pupal lethality. However, null mutants reach adulthood, although they do show reduced lifespan and activity. Here, we report that both carbohydrate transport efficiency and resulting lethality found upon loss of MFS3 or Pippin are rescued via compensatory upregulation of Tret1-1, another BBB carbohydrate transporter, in Mfs3 and pippin null mutants, while RNAi-mediated knockdown is not compensated for. This means that the compensatory mechanisms in place upon mRNA degradation following RNA interference can be vastly different from those resulting from a null mutation.

Carbonic anhydrase IX and acid transport in cancer.

Alterations in tumour metabolism and acid/base regulation result in the formation of a hostile environment, which fosters tumour growth and metastasis. Acid/base homoeostasis in cancer cells is governed by the concerted interplay between carbonic anhydrases (CAs) and various transport proteins, which either mediate proton extrusion or the shuttling of acid/base equivalents, such as bicarbonate and lactate, across the cell membrane. Accumulating evidence suggests that some of these transporters interact both directly and functionally with CAIX to form a protein complex coined the 'transport metabolon'. Transport metabolons formed between bicarbonate transporters and CAIX require CA catalytic activity and have a function in cancer cell migration and invasion. Another type of transport metabolon is formed by CAIX and monocarboxylate transporters. In this complex, CAIX functions as a proton antenna for the transporter, which drives the export of lactate and protons from the cell. Since CAIX is almost exclusively expressed in cancer cells, these transport metabolons might serve as promising targets to interfere with tumour pH regulation and energy metabolism. This review provides an overview of the current state of research on the function of CAIX in tumour acid/base transport and discusses how CAIX transport metabolons could be exploited in modern cancer therapy.

Energy Dynamics in the Brain: Contributions of Astrocytes to Metabolism and pH Homeostasis.

Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a "transport metabolon" with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3 - and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme's catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy.

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.

Catalytically inactive carbonic anhydrase-related proteins enhance transport of lactate by MCT1.

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole-body acid/base regulation. The three carbonic anhydrase-related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion-sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a 'proton antenna' for MCT1, to drive proton-coupled lactate transport across the cell membrane.

Membrane-anchored carbonic anhydrase IV interacts with monocarboxylate transporters via their chaperones CD147 and GP70.

Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.

The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells.

Highly glycolytic tumor cells release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbate extracellular acidification and support the formation of a hostile environment. Transport activity of MCTs can be facilitated by non-catalytic interaction with carbonic anhydrase IX (CAIX), the expression of which has been shown to be upregulated under hypoxia. We have now studied the mechanisms that enable CAIX-mediated facilitation of proton-coupled lactate transport in breast cancer cells and Xenopus oocytes. Our results indicate that the proteoglycan like (PG) domain of CAIX could function as 'proton antenna' to facilitate MCT transport activity. Truncation of the PG domain and application of a PG-binding antibody significantly reduced proton-coupled lactate transport in MCT-expressing oocytes and hypoxic breast cancer cells, respectively. Furthermore, application of the PG-binding antibody reduced proliferation and migration of hypoxic cancer cells, suggesting that facilitation of proton-coupled lactate flux by the CAIX PG domain contributes to cancer cell survival under hypoxic conditions.

Novel fluorinated carbonic anhydrase IX inhibitors reduce hypoxia-induced acidification and clonogenic survival of cancer cells.

Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational changes as determined by X-ray crystallography. Mass spectrometric gas-analysis confirmed the CA IX-based mechanism of the inhibitors in a CRISPR/Cas9-mediated CA IX knockout in HeLa cells. Hypoxia-induced extracellular acidification was significantly reduced in HeLa, H460, MDA-MB-231, and A549 cells exposed to the compounds, with the IC50 values up to 1.29 nM. A decreased clonogenic survival was observed when hypoxic H460 3D spheroids were incubated with our lead compound. These novel compounds are therefore promising agents for CA IX-specific therapy.

Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes.

Carbonic anhydrase (CA) IX is a hypoxia inducible enzyme that is highly expressed in solid tumours. Therefore, it has been considered as an anticancer target using specific chemical inhibitors. The nitroimidazoles DTP338 and DTP348 have been shown to inhibit CA IX in nanomolar range in vitro and reduce extracellular acidification in hypoxia, and impair tumour growth. We screened these compounds for toxicity using zebrafish embryos and measured their in vivo effects on human CA IX in Xenopus oocytes. In the toxicity screening, the LD50 for both compounds was 3.5 mM. Neither compound showed apparent toxicity below 300 µM concentration. Above this concentration, both compounds altered the movement of zebrafish larvae. The IC50 was 0.14 ± 0.02 µM for DTP338 and 19.26 ± 1.97 µM for DTP348, suggesting that these compounds efficiently inhibit CA IX in vivo. Our results suggest that these compounds can be developed as drugs for cancer therapy.

A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme's intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects.

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.

Modeling of pH regulation in tumor cells: Direct interaction between proton-coupled lactate transporters and cancer-associated carbonic anhydrase.

The most aggressive tumor cells, which often reside in a hypoxic environment, can release vast amounts of lactate and protons via monocarboxylate transporters (MCTs). This additional proton efflux exacerbates extracellular acidification and supports the formation of a hostile environment. In the present study we propose a novel, data-based model for this proton-coupled lactate transport in cancer cells. The mathematical settings involve systems coupling nonlinear ordinary and stochastic differential equations describing the dynamics of intra- and extracellular proton and lactate concentrations. The data involve time series of intracellular proton concentrations of normoxic and hypoxic MCF-7 breast cancer cells. The good agreement of our final model with the data suggests the existence of proton pools near the cell membrane, which can be controlled by intracellular and extracellular carbonic anhydrases to drive proton-coupled lactate transport across the plasma membrane of hypoxic cancer cells.

Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype.

Elevated amino acid catabolism is common to many cancers. Here, we show that glioblastoma are excreting large amounts of branched-chain ketoacids (BCKAs), metabolites of branched-chain amino acid (BCAA) catabolism. We show that efflux of BCKAs, as well as pyruvate, is mediated by the monocarboxylate transporter 1 (MCT1) in glioblastoma. MCT1 locates in close proximity to BCKA-generating branched-chain amino acid transaminase 1, suggesting possible functional interaction of the proteins. Using in vitro models, we demonstrate that tumor-excreted BCKAs can be taken up and re-aminated to BCAAs by tumor-associated macrophages. Furthermore, exposure to BCKAs reduced the phagocytic activity of macrophages. This study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor-excreted BCKAs might have a direct role in tumor immune suppression. Our data further suggest that the anti-proliferative effects of MCT1 knockdown observed by others might be related to the blocked excretion of BCKAs.