Injection of carbonic anhydrase isoform II (CA) into Xenopus frog oocytes increased the rate of H+ flux via the rat monocarboxylate transporter isoform 1 (MCT1) expressed in the oocytes. MCT1 activity was assessed by changes of intracellular H+ concentration measured by pH-selective microelectrodes during application of lactate. CA-induced augmentation of the rate of H+ flux mediated by MCT1 was not inhibited by ethoxyzolamide (10 microM) and did not depend on the presence of added CO2/HCO3- but was suppressed by injection of an antibody against CA. Deleting the C terminus of the MCT1 greatly reduced its transport rate and removed transport facilitation by CA. Injected CA accelerated the CO2/HCO3(-)-induced acidification severalfold, which was blocked by ethoxyzolamide and was independent of MCT1 expression. Mass spectrometry confirmed activity of CA as injected into the frog oocytes. With pulldown assays we demonstrated a specific binding of CA to MCT1 that was not attributed to the C terminus of MCT1. Our results suggest that CA enhances MCT1 transport activity, independent of its enzymatic reaction center, presumably by binding to MCT1.
"Monocarboxylate cotransporters (MCTs) 1, 2, and 4 facilitate lactate transport across the HCEn by employing a lactate-H+ cotransport mechanism . MCT lactate transport activity is increased by interaction with carbonic anhydrase 2 (CA2) , ,  and is further augmented by the Na/H+ exchanger 1 (NHE1) . Although MCT4 expression was decreased in HCEnC-21 and HCEnC-21T cells, MCT1 and MCT2 were expressed at levels similar to those in corneal endothelial tissue (Figure 5H). "
[Show abstract][Hide abstract] ABSTRACT: Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness-the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.
PLoS ONE 12/2012; 7(12):e51427. DOI:10.1371/journal.pone.0051427 · 3.23 Impact Factor
"CAII has been shown to enhance the activity of various acid–base transporters like the chloride/bicarbonate exchanger AE1 (Vince & Reithmeier, 1998; McMurtrie et al. 2004), the sodium bicarbonate cotransporter NBCe1 (Pushkin et al. 2004; Becker & Deitmer, 2007), and the sodium/hydrogen exchanger NHE1 (Li et al. 2002), an interaction coined 'transport metabolon' . By using heterologous protein expression in Xenopus oocytes, we could show that CAII enhances transport activity of MCT1 and MCT4, but not of MCT2 (Becker et al. 2005, 2010; Klier et al. 2011). In contrast to other transport metabolons described so far, the interaction between MCT and CAII does not depend on catalytic activity of CAII, but requires the enzyme's intramolecular H + shuttle with the residue H64 playing a central role (Becker & Deitmer, 2008; Becker et al. 2011). "
[Show abstract][Hide abstract] ABSTRACT: Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a ‘proton collecting antenna' for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate.
The Journal of Physiology 03/2012; 590(Pt 10):2333-51. DOI:10.1113/jphysiol.2011.220152 · 5.04 Impact Factor
"In vitro, expression of both CAIX and MCT4 is increased by HIF-1αA common upregulatory mechanism, perhaps acting via HIF-1α, might lead to the observed spatial correlation. A close spatial association between a pair of other isoforms of these proteins, MCT1 and CAII, has been reported , . CAXII as well as CAIX is upregulated in tumors, and six other known isoforms are present in the brain , so our sampling of only CAIX tells us little about the distribution of total CA activity. "
[Show abstract][Hide abstract] ABSTRACT: Tumors create a heterogeneous acidic microenvironment which assists their growth and which must be taken into account in the design of drugs and their delivery. In addition, the acidic extracellular pH (pHe) is itself exploited in several experimental techniques for drug delivery. The way the acidity is created is not clear. We report here the spatial organization of key proton-handling proteins in C6 gliomas in rat brain. The mean profiles across the tumor rim of the Na+/H+ exchanger NHE1, and the lactate-H+ cotransporter MCT1, both showed peaks. NHE1, which is important for extension and migration of cells in vitro, showed a peak 1.55 times higher than in extratumoural tissue at 0.33 mm from the edge. MCT1 had a broader peak, further into the tumor (maximum 1.76 fold at 1.0 mm from the edge). In contrast, MCT4 and the carbonic anhydrase CAIX, which are associated with hypoxia, were not significantly upregulated in the rim. The spatial distribution of MCT4 was highly correlated with that of CAIX, suggesting that their expression is regulated by the same factors. Since protons extruded by NHE1 diffuse away through extracellular clefts, NHE1 requires a continuous source of intracellular protons. From the stoichiometries of metabolic pathways that produce or consume H+, and the greater availability of glucose compared to oxygen in most parts of a tumor, we support the classic view that most of the net proton efflux from C6 gliomas originates in glycolytic formation of lactate and H+ inside the tumor, but add that some lactate is taken up into cells in the rim on MCT1, and some lactate diffuses away, leaving its associated protons available to re-enter cells for extrusion on NHE1. Therapeutic inhibition of NHE1, MCT1 or CAIX is predicted to affect different parts of a tumor.
PLoS ONE 02/2011; 6(2):e17416. DOI:10.1371/journal.pone.0017416 · 3.23 Impact Factor
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