Lactate-proton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots

Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition of different organic anions to the bathing solution at constant bath pH caused transient alkaline shifts in extracellular pH; withdrawal of these compounds resulted in transient acid shifts in extracellular pH. With high anion concentrations (30 mM), the largest changes in extracellular pH were observed with propionate >l-lactate ≈ pyruvate >62; 2-hydroxy-2-methylpropionate. Changes in extracellular pH induced by 10 mMl- andd-lactate were of similar size. Lactate transport inhibitors α-cyano-4-hydroxycinnamic acid and 4,4′-dibenzamidostilbene-2,2′-disulphonic acid significantly reducedl-lactate-induced extracellular pH shifts without affecting propionate-induced changes in extracellular pH. Hypoxia produced an extracellular acidification that was strongly reduced in the presence of α-cyano-4-hydroxycinnamic acid and 4,4′-dibenzamidostilbene-2,2′-disulphonic acid. In contrast, amiloride and 4,4′-di-isothiocyanostilbene-2,2′-disulphonate were without effect on hypoxia-induced acid shifts. The results indicate the presence of a lactate-proton co-transporter in rat peripheral nerves. This transport system and not Na+/H+ or C1−/HCO−3 exchange seems to be the dominant mechanism responsible for interstitial acidification during nerve hypoxia

In silico simulation of reversible and irreversible swelling of mitochondria: The role of membrane rigidity

Mitochondria have been widely accepted as the main source of ATP in the cell. The inner mitochondrial membrane (IMM) is important for the maintenance of ATP production and other functions of mitochondria. The electron transport chain (ETC) generates an electrochemical gradient of protons known as the proton-motive force across the IMM and thus produces the mitochondrial membrane potential that is critical to ATP synthesis. One of the main factors regulating the structural and functional integrity of the IMM is the changes in the matrix volume. Mild (reversible) swelling regulates mitochondrial metabolism and function; however, excessive (irreversible) swelling causes mitochondrial dysfunction and cell death. The central mechanism of mitochondrial swelling includes the opening of non-selective channels known as permeability transition pores (PTPs) in the IMM by high mitochondrial Ca2+ and reactive oxygen species (ROS). The mechanisms of reversible and irreversible mitochondrial swelling and transition between these two states are still unknown. The present study elucidates an upgraded biophysical model of reversible and irreversible mitochondrial swelling dynamics. The model provides a description of the PTP regulation dynamics using an additional differential equation. The rigidity tensor was used in numerical simulations of the mitochondrial parameter dynamics with different initial conditions defined by Ca2+ concentration in the sarco/endoplasmic reticulum. We were able to estimate the values of the IMM rigidity tensor components by fitting the model to the previously reported experimental data. Overall, the model provides a better description of the reversible and irreversible mitochondrial swelling dynamics.Funding Agency USA NIGMS NIH SC1GM128210 Institute for Functional Nanomaterials (USA NSF) 1002410 PR NASA EPSCoR (USA NASA Cooperative Agreement) NNX15AK43Ainfo:eu-repo/semantics/publishedVersio

Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity

Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca2+], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca2+. However, when GNX-4975 was added after Ca2+ it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca2+ binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its ‘c’ conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca2+]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore

Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1

The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT

Characterization of acetate transport in colorectal cancer cells and potential therapeutic implications

Acetate, together with other short chain fatty acids has been implicated in colorectal cancer (CRC) prevention/therapy. Acetate was shown to induce apoptosis in CRC cells. The precise mechanism underlying acetate transport across CRC cells membrane, that may be implicated in its selectivity towards CRC cells, is not fully understood and was addressed here. We also assessed the effect of acetate in CRC glycolytic metabolism and explored its use in combination with the glycolytic inhibitor 3-bromopyruvate (3BP). We provide evidence that acetate enters CRC cells by the secondary active transporters MCT1 and/or MCT2 and SMCT1 as well as by facilitated diffusion via aquaporins. CRC cell exposure to acetate upregulates the expression of MCT1, MCT4 and CD147, while promoting MCT1 plasma membrane localization. We also observed that acetate increases CRC cell glycolytic phenotype and that acetate-induced apoptosis and anti-proliferative effect was potentiated by 3BP. Our data suggest that acetate selectivity towards CRC cells might be explained by the fact that aquaporins and MCTs are found overexpressed in CRC clinical cases. Our work highlights the importance that acetate transport regulation has in the use of drugs such as 3BP as a new therapeutic strategy for CRC.This work was supported by Fundacao para a Ciencia e Tecnologia (FCT) by the FCT-ANR/BEX-BCM/0175/2012 and the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), as well as by the FCT fellowships: Suellen Ferro (SFRH/BD/77449/2011) and J. Azevedo-Silva (SFRH/BD/76038/2011). This work was also supported by the Marie Curie Initial Training Network: GLYCOPHARM, PITN-GA-2012-317297. This work was also supported by Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013), Fundo Europeu de Desenvolvimento Regional (FEDER), through COMPETE (FCOMP-01-0124-FEDER-037298) and Project "ON.2 SR&TD Integrated Program (NORTE-07-0124-FEDER-000017)" co-funded by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN), through (FEDER).info:eu-repo/semantics/publishedVersio

Metabolic reprogramming: a new relevant pathway in adult adrenocortical tumors

Adrenocortical carcinomas (ACCs) are complex neoplasias that may present unexpected clinical behavior, being imperative to identify new biological markers that can predict patient prognosis and provide new therapeutic options. The main aim of the present study was to evaluate the prognostic value of metabolism-related key proteins in adrenocortical carcinoma. The immunohistochemical expression of MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX was evaluated in a series of 154 adult patients with adrenocortical neoplasia and associated with patients’ clinicopathological parameters. A significant increase in was found for membranous expression of MCT4, GLUT1 and CAIX in carcinomas, when compared to adenomas. Importantly MCT1, GLUT1 and CAIX expressions were significantly associated with poor prognostic variables, including high nuclear grade, high mitotic index, advanced tumor staging, presence of metastasis, as well as shorter overall and disease free survival. In opposition, MCT2 membranous expression was associated with favorable prognostic parameters. Importantly, cytoplasmic expression of CD147 was identified as an independent predictor of longer overall survival and cytoplasmic expression of CAIX as an independent predictor of longer disease-free survival. We provide evidence for a metabolic reprogramming in adrenocortical malignant tumors towards the hyperglycolytic and acid-resistant phenotype, which was associated with poor prognosis.This work was supported by FAPESP to Maria Claudia Nogueira Zerbini (2013-26344-8). SG received the post-doctoral fellowship UMINHO/BPD/18/2014.info:eu-repo/semantics/publishedVersio

Real-Time Fluorescence Measurements of ROS and [Ca²⁺] in Ischemic / Reperfused Rat Hearts:Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2-3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2-3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia

Differential sensitivities to lactate transport inhibitors of breast cancer cell lines

This is linked to the online version of the paper at: http://dx.doi.org/10.1530/ERC-13-0132.The tumour microenvironment is known to be acidic due to high glycolytic rates of tumour cells. Monocarboxylate transporters (MCTs) play a role in extracellular acidification, which is widely known to be involved in tumour progression. Recently, we have described the upregulation of MCT1 in breast carcinomas and its association with poor prognostic variables. Thus, we aimed to evaluate the effect of lactate transport inhibition in human breast cancer cell lines. The effects of a-cyano-4-hydroxycinnamate, quercetin and lonidamine on cell viability, metabolism, proliferation, apoptosis, migration and invasion were assessed in a panel of different breast cancer cell lines. MCT1, MCT4 and CD147 were differently expressed among the breast cancer cell lines and, as expected, different sensitivities were observed for the three inhibitors. Interestingly, in the most sensitive cell lines, lactate transport inhibition induced a decrease in cell proliferation, migration and invasion, as well as an increase in cell death. Results were validated by silencing MCT1 expression using siRNA. The results obtained here support targeting of lactate transport as a strategy to treat breast cancer, with a special emphasis on the basal-like subtype, which so far does not have a specific molecular therapy.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘Programa Operacional Temático Factores de Competitividade’ (COMPETE) of ‘Quadro Comunitário de Apoio III’ and co-financed by the Fundo Europeu De Desenvolvimento Regional (FEDER)

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Background: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods: Expression of hypoxia-and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (a-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.This study was supported by Projecto Estratégico- LA 26 – 2013-2014 (PEst-C/SAL/LA0026/2013) and ON.2 SR&TD Integrated Program (NORTE-07-0124FEDER-000017)” co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER), as well as MCTI/CNPq Nº 73/2013 (Brazil). VMG received a fellowship from Fundação para a Ciência e Tecnologia (FCT) ref. SFRH/BD/51997/2012.info:eu-repo/semantics/publishedVersio