Characterization of intracellular buffering power in human induced pluripotent stem cells and the loss of pluripotency is delayed by acidic stimulation and increase of NHE1 activity.

The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (ß) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (ßtot ), intrinsic (ßi ), and CO2 -dependent ( ßCO2 ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the ß values of ßtot and ßCO2 changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive ß during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.

Plastidial transporters KEA1 and KEA2 at the inner envelope membrane adjust stromal pH in the dark.

Photosynthesis and carbon fixation depend critically on the regulation of pH in chloroplast compartments in the daylight and at night. While it is established that an alkaline stroma is required for carbon fixation, it is not known how alkaline stromal pH is formed, maintained or regulated. We tested whether two envelope transporters, AtKEA1 and AtKEA2, directly affected stromal pH in isolated Arabidopsis chloroplasts using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). External K+ -induced alkalinization of the stroma was observed in chloroplasts from wild-type (WT) plants but not from kea1kea2 mutants, suggesting that KEA1 and KEA2 mediate K+ uptake/H+ loss to modulate stromal pH. While light-stimulated alkalinization of the stroma was independent of KEA1 and KEA2, the rate of decay to neutral pH in the dark is delayed in kea1kea2 mutants. However, the dark-induced loss of a pH gradient across the thylakoid membrane was similar in WT and mutant chloroplasts. This indicates that proton influx from the cytosol mediated by envelope K+ /H+ antiporters contributes to adjustment of stromal pH upon light to dark transitions.

Beyond the patch-clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides.

We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K+ ) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+ ) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

Standardization and Validation of Fluorescence-Based Quantitative Assay to Study Human Platelet Adhesion to Extracellular-Matrix in a 384-Well Plate.

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes.

Inhibitory Mechanisms of DHA/CQ on pH and Iron Homeostasis of Erythrocytic Stage Growth of Plasmodium Falciparum.

Malaria is an infectious disease caused by Plasmodium group. The mechanisms of antimalarial drugs DHA/CQ are still unclear today. The inhibitory effects (IC50) of single treatments with DHA/CQ or V-ATPase inhibitor Baf-A1 or combination treatments by DHA/CQ combined with Baf-A1 on the growth of Plasmodium falciparum strain 3D7 was investigated. Intracellular cytoplasmic pH and labile iron pool (LIP) were labeled by pH probe BCECF, AM and iron probe calcein, AM, the fluorescence of the probes was measured by FCM. The effects of low doses of DHA (0.2 nM, 0.4 nM, 0.8 nM) on gene expression of V-ATPases (vapE, vapA, vapG) located in the membrane of DV were tested by RT-qPCR. DHA combined with Baf-A1 showed a synergism effect (CI = 0.524) on the parasite growth in the concentration of IC50. Intracellular pH and irons were effected significantly by different doses of DHA/Baf-A1. Intracellular pH was decreased by CQ combined with Baf-A1 in the concentration of IC50. Intracellular LIP was increased by DHA combined with Baf-A1 in the concentration of 20 IC50. The expression of gene vapA was down-regulated by all low doses of DHA (0.2/0.4/0.8 nM) significantly (p < 0.001) and the expression of vapG/vapE were up-regulated by 0.8 nM DHA significantly (p < 0.001). Interacting with ferrous irons, affecting the DV membrane proton pumping and acidic pH or cytoplasmic irons homeostasis may be the antimalarial mechanism of DHA while CQ showed an effect on cytoplasmic pH of parasite in vitro. Lastly, this article provides us preliminary results and a new idea for antimalarial drugs combination and new potential antimalarial combination therapies.

Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells.

BACKGROUND/AIMS: To functionally characterize intracellular pH (pHi) regulating mechanisms, such as Na+-H+ exchanger (NHE) and Na+-HCO3- co-transporter (NBC), and further examine effects of ethanol on the pHi regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells. METHODS: OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pHi were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique. RESULTS: i) the steady-state pHi value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pHi recovery following induced-acidification was totally blocked by either removing [Na]o+ or adding HOE 694 (a NHE1 specific inhibitor), which demonstrates existence of NHE1; iii) in HCO3-/CO2-buffer system, the pHi recovery following induced-acidification was entirely blocked by either removing [Na]o+ or adding HOE 694 plus DIDS (a NBC specific inhibitor), which suggests existence of Na+- and HCO3-dependent acid-extruder, i.e. NBC; iv) the isoforms of the two acid extruders were NHE1, NBCn1, NBCe1 and NDCBE; v) ethanol (10-1000 mM) showed a biphasic and concentration-dependent effect on resting pHi (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (> 300 mM) significantly inhibited the expression of NHE1, NBCn1 and NDCBE, while up-regulated NBCe1. CONCLUSIONS: Ethanol affects pHi in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.

Acute neuroinflammation provokes intracellular acidification in mouse hippocampus.

BACKGROUND: Maintaining pH levels within the physiological norm is an important component of brain homeostasis. However, in some pathological or physiological conditions, the capacity of the pH regulatory system could be overpowered by various factors resulting in a transient or permanent alteration in pH levels. Such changes are often observed in pathological conditions associated with neuroinflammation. We hypothesized that neuroinflammation itself is a factor affecting pH levels in neural tissue. To assess this hypothesis, we examined the effects of acute LPS-induced neuroinflammation on intra- and extracellular pH (pHi and pHo) levels in the CA1 region of mouse hippocampus. METHODS: Acute neuroinflammation was induced using two approaches: (1) in vivo by i.p. injections of LPS (5 mg/kg) and (2) in vitro by incubating hippocampal slices of naïve animals in the LPS-containing media (1 µg/mL, 1 h at 35 °C). Standard techniques were used to prepare hippocampal slices. pHi was measured using ratiometric pH-sensitive fluorescent dye BCECF-AM. pHo was assessed using calibrated pH-sensitive micropipettes. The presence of neuroinflammation was verified with immunohistochemistry (IL-1ß and Iba1) and ELISA (IL-1ß and TNF-α). RESULTS: A significant reduction of pHi was observed in the slices of the LPS-injected 3-month-old (LPS 7.13 ± 0.03; Sal 7.22 ± 0.03; p = 0.043, r = 0.43) and 19-month-old (LPS 6.78 ± 0.08; Sal 7.13 ± 0.03; p = 0.0001, r = 0.32) mice. In contrast, the levels of pHo within the slice, measured in 19-month-old animals, were not affected (LPS 7.27 ± 0.02; Sal 7.26 ± 0.02; p = 0.6, r = 0.13). A reduction of pHi was also observed in the LPS-treated slices during the interval 3.5-7 h after the LPS exposure (LPS 6.92 ± 0.07; Veh 7.28 ± 0.05; p = 0.0001, r = 0.46). CONCLUSIONS: Acute LPS-induced neuroinflammation results in a significant intracellular acidification of the CA1 neurons in mouse hippocampus, while the pHo remains largely unchanged. Such changes may represent a specific protective reaction of neural tissue in unfavorable external conditions or be a part of the pathological process.

Low CO2 permeability of cholesterol-containing liposomes detected by stopped-flow fluorescence spectroscopy.

Here we ask the following: 1) what is the CO2 permeability (Pco2) of unilamellar liposomes composed of l-α-phosphatidylcholine (PC)/l-α-phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric (18)O-exchange measurements (Chol reduces Pco2 100-fold, AQP1 increases Pco2 10-fold) vs. from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a Pco2 = 0.01cm/s, and Chol-free vesicles exhibit ∼1 cm/s, and 2) the Pco2 of 0.01 cm/s is increased ≥ 10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco2 that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1.

CORM-3, a water soluble CO-releasing molecule, uncouples mitochondrial respiration via interaction with the phosphate carrier.

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O2 consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.

Fisetin-Mediated Perturbations of Membrane Permeability and Intracellular pH in Candida albicans.

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC4(3) uptake assays demonstrate membrane hyperpolarization in fisetin-treated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea.

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75µM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75µM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na(+)/H(+) exchangers, Na(+)-K(+) pump, Na(+)-driven and Na(+)-independent HCO3(-)/Cl(-) transporters and Na(+)/HCO3(-) co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4'-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3(-)/Cl(-) co-transporters present on the basolateral membrane.

Generalization of the Ratiometric Method to Extend pH Range Measurements of the BCECF Probe.

There is a variety of fluorescent probes for pH measurements and which are mainly used for biological systems. In general, they can be classified into two groups. The first group includes fluorescent pH probes which exhibit a single fluorescence emission peak. For these probes, the fluorescence excitation profile is pH-dependent and the shape of the emission spectra remains almost constant. Hence, the ratiometric pH measurement-which makes pH determination independent of the probe concentration-is implemented when the excitation is performed at two excitation wavelengths and the fluorescence emission is measured at one wavelength. The second group exhibits a dual fluorescence emission peak. Here, each protonated or deprotonated form exhibits characteristics emission and/or absorption spectra. Shifts between spectra obtained for protonated and deprotonated species can be exploited in order to perform a ratiometric measurement. In this study we present a methodology that evaluates the precision of the ratiometric measurements based on multiple wavelengths excitation to determine the optimum wavelengths combination for pH determination in biological samples. This methodology using the BCECF probe is applied to measure the pH drift in cell culture medium. It exhibits a high precision and significantly extends the range of validity for pH measurements spanning from very acidic to basic.

Characterization of the Intracellular Acidity Regulation of Brain Tumor Cells and Consequences for Therapeutic Optimization of Temozolomide.

A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.

The phosphoinositide PI(3,5)P2 inhibits the activity of plant NHX proton/potassium antiporters: Advantages of a novel electrophysiological approach.

In the present work, we discuss the way in which the parallel application of the patch-clamp technique and the 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence detection for recording luminal proton changes allows the functional characterization of nonelectrogenic potassium/proton vacuolar antiporters of the NHX (Na+/H+ exchanger) family. Moreover, we review the functional role of the tonoplast-specific phosphoinositide PI(3,5)P2, able to simultaneously inhibit the activity of NHXs and CLC-a transporters, whose coordinated action can play an important role in the water balance of plant cells.

Electrophysiology and fluorescence to investigate cation channels and transporters in isolated plant vacuoles.

The plant vacuole plays a fundamental role in cell homeostasis. The successful application of patch-clamp technique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters. The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation. In excised patch, the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels. In whole-vacuole, the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette. Both vacuolar NHXs and CLCa (chloride/nitrate antiporter) are inhibited by the phosphoinositide PI(3,5)P2, suggesting a coordinated role of these proteins in salt accumulation. Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells.

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells.

Energy Metabolism and Intracellular pH Alteration in Neural Spheroids Carrying Down Syndrome.

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.

The γ-hydroxybutyric acid (GHB) analogue NCS-382 is a substrate for both monocarboxylate transporters subtypes 1 and 4.

The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB. For this purpose, we investigated NCS-382 uptake by MCT subtypes endogenously expressed in tsA201 and MDA-MB-231 cell lines in assays of radioligand-based competition and fluorescence-based intracellular pH measurements. To further verify the results, we measured NCS-382 uptake by means of mass spectrometry in Xenopus laevis oocytes heterologously expressing MCT subtypes. As expected, we found that NCS-382 is a substrate for MCT1 with half-maximal effective concentrations in the low millimolar range. Surprisingly, NCS-382 also showed substrate activity at MCT4 as well as uptake in water-injected oocytes, suggesting a component of passive diffusion. In conclusion, transport of NCS-382 across membranes differs from GHB as it also involves MCT4 and/or passive diffusion. This should be taken into consideration when designing pharmacological studies with this compound and its closely related analogues. The combination of MCT assays used here exemplifies a setup that may be suitable for a reliable characterization of MCT ligands in general.

Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes.

Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly. Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pHi. These methods for pHi calibration and clamping are simple and reproducible.

Isoorientin Decreases Cell Migration via Decreasing Functional Activity and Molecular Expression of Proton-Linked Monocarboxylate Transporters in Human Lung Cancer Cells.

Aggressive tumor cells mainly rely on glycolysis, and further release vast amounts of lactate and protons by monocarboxylate transporter (MCT), which causes a higher intracellular pH (pHi) and acidic extracellular pH. Isoorientin, a principle flavonoid compound extracted from several plant species, shows various pharmacological activities. However, effects of isoorientin on anticancer and MCT await to explore in human lung cancer cells. Human lung cancer tissues were obtained from cancer patients undergoing surgery, while the human lung adenocarcinoma cells (A549) were bought commercially. Change of pHi was detected by microspectrofluorometry method with a pH-sensitive fluorescent dye, BCECF. MTT and wound-healing assay were used to detect the cell viability and migration, respectively. Western blot techniques and immunocytochemistry staining were used to detect the protein expression. Our results indicated that the expression of MCTs1/4 and CD147 were upregulated significantly in human lung tissues. In experiments of A549 cells, under HEPES-buffer, the resting pHi was 7.47, and isoorientin (1-300µM) inhibited functional activity of MCT concentration-dependently (up to -42%). Pretreatment with isoorientin (3-100µM) for 24h, MCT activity and cell migration were significantly inhibited (-25% and -40%, respectively), while the cell viability was not affected. Moreover, the expression of MCTs1/4, CD147, and matrix metalloproteinase (MMP) 2/9 were significantly down regulated. In summary, MCTs1/4 and CD147 are significantly upregulated in human lung adenocarcinoma tissues, and isoorientin inhibits cells-migration by inhibiting activity/expression of MCTs1/4 and MMPs2/9 in human lung cancer cells. These novel findings suggest that isoorientin could be a promising pharmacological agent for lung cancer.