Diisothiocyanatostilbene-2,2 ' - Disulfonate Research Articles

The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine.

The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.

Transition From Proto-Kranz-Type Photosynthesis to HCO3 - Use Photosynthesis in the Amphibious Plant Hygrophila polysperma.

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3–) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3– for photosynthesis. H. polysperma transports HCO3– into the leaf by a DIDS-sensitive HCO3– transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3– use photosynthesis in the submerged environments.

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm.

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3-) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3- use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3- use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3- use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3- transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3- to CO2 at the plasmalemma by α-CA1 and transport of HCO3- across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

Isothiocyanate groups of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibit cell penetration of octa-arginine (R8)-fused peptides.

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine-a well-known cell-penetrating peptide (CPP)-is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide-ie, mitochondrial targeting domain (MTD)-to the cytosol. We found that 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonate (DIDS)-an anion channel blocker-inhibited the ability of octa-arginine (R8)-fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di-sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di-sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.

Redox-dependent gating of VDAC by mitoNEET

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites.

Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO2 permeability (PM,CO2), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces PM,CO2. We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O2 scavenger, and determined the rate constant of Hb deoxygenation (kHbO2). Because kHbO2 depends on Hb kinetics as well as diffusion of O2 to the extracellular space, we used a mathematical model to estimate effects on membrane O2 permeability (PM,O2). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease PM,CO2. We find that, in RBCs from wild type (WT) mice, pCMBS reduces kHbO2 by 64% (est. PM,O2 by ~84%) and DIDS, by 41% (est. PM,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O2, at least in part, crosses RBC membranes via the same membrane proteins that conduct CO2. Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers kHbO2 by 11% (est. PM,O2 by ~27%); RhAG, by 16% (est. PM,O2 by ~35%); the double knockout (dKO), by 33% (est. PM,O2 by ~59%). The combination dKO + pCMBS lowers kHbO2 (vs. WT without inhibitors) by 77% (est. PM,O2 by ~91%), whereas dKO + DIDS lowers kHbO2 by 52% (est. PM,O2 by ~76%). The effects of pCMBS on estimated PM,O2 are very similar when we compare dKO vs. RhAG−/−, or when we compare WT vs. AQP1−/−. Thus, the deletion of AQP1 may have little effect on the sensitivity of PM,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O2 permeability, and point towards the existence of other O2 channels that account for at least 30%.Support or Funding InformationSupported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to ROThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The First N2 Channels Identified by a Novel Technique for Assessing Nitrogen Gas Efflux from Oocytes

Decompression illness (DCI) is a serious problem for both divers and pilots. Divers must schedule their ascents to minimize the risk of nitrogen (N2) bubble formation as the partial pressure of N2 (PN2) decreases. Pilots can experience the same effects via decreases in cabin pressure. In order to approach management or even prevention of DCI, it would be beneficial to understand the mechanism of N2 transport across cell membranes. Specific aquaporins and rhesus (Rh) proteins can conduct CO2, NH3, NO, and probably O2 across membranes. Certain solute transporters (e.g., NBCe1) may also be able to conduct gases. Whereas several assays exist for CO2, O2 and NH3 permeability, an effective assay for N2 movements across membranes has been elusive. Here we present the first such assay, based on maintaining the neutral buoyancy of a Xenopus oocyte previously injected with a 200‐nl air bubble (79% N2, 21% O2). We apply pressure to the air above a saline solution, causing the bubble to constrict sufficiently that the oocyte falls to a depth of ~5 cm. As air molecules dissolve in and diffuses through the cytoplasm, and eventually exit the cell, the bubble tends to collapse. A feedback system (webcam, computer, digitally controlled pressure regulator) reacts by lowering the neutral‐buoyancy air pressure (PNB) to maintain the oocyte at a 5‐cm depth. The pressure inside the bubble (PBubble > PNB by fixed amount) falls proportionally with the decreasing number of air molecules. The rate at which PNB falls thus reflects gas efflux from the bubble. For control oocytes injected with H2O rather than cRNA, PNB falls slowly, reflecting a slow exit of N2 (+ O2) from the bubble (kPNB = 1.16 × 10−3 ± 0.02 s−1, n=47). We estimate that 10–50% of kPNB represents N2 dissolving in cellular water/lipids. For oocytes injected with cRNA to express NOD26—a plant AQP in cell membranes of root nodules of the N2‐fixing soybean plant, Glycine max—PNB falls 13.0% more rapidly (kPNB = 1.31 × 10−3 ± 0.04 s−1, p=0.018, n=5), presumably reflecting an increased N2 (+ O2) efflux across the oocyte membrane. kPNB for oocytes expressing human AQP5 or the plant AQP, AtPIP 2;1—AQPs known to function as CO2 channels—were not significantly different from H2O‐injected control oocytes (n=20 & 5, respectively). However, kPNB was 8.6% greater in oocytes expressing human RhAG (kPNB = 1.26 × 10−3 ± 0.03 s−1, p=0.003, n=26) vs. H2O‐injected control oocytes. 200 mM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) decreased the RhAG‐dependent component of kPNB by 89% (kPNB = 1.17 × 10−3 ± 0.03 s−1, p=0.04, n=12). DIDS had no effect on H2O‐injected controls (p=0.65, n=11). Together these experiments demonstrate a simple and robust assay for the efflux of N2 across a biological membrane, as well as the first examples of N2 channels in a biological membrane.Support or Funding InformationFunding: Office of Naval Research (N00014‐15‐1‐2060 & N00014‐16‐1‐2535)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of VDAC1 Protects Against Glutamate-Induced Oxytosis and Mitochondrial Fragmentation in Hippocampal HT22 Cells.

The involvement of glutamate in neuronal cell death in neurodegenerative diseases and neurotrauma is mediated through excitotoxicity or oxytosis. The latter process induces oxidative stress via glutamate-mediated inhibition of cysteine transporter xCT, leading to depletion of the cellular glutathione pool. Mitochondrial damage, loss of mitochondrial membrane potential (MMP), and depletion of energy metabolites have been shown in this process. The Voltage-Dependent Anion Channel-1 (VDAC1) is one of the main components of the mitochondrial outer membrane and plays a gatekeeping role in mitochondria-cytoplasm transport of metabolites. In this study, we explored the possible participation of VDAC-1 in the pathophysiology of oxytosis. Administration of glutamate in HT22 cells that lack the glutamate ionotropic receptors induced an upregulation and oligomerization of VDAC1. This was associated with an increase in ROS and loss of cell survival. Glutamate-mediated oxytosis in this model also decreased MMP and promoted ATP depletion, resulting in translocation of cytochrome c (cyt C) and apoptosis inducing factor (AIF) from mitochondria into the cytosol. This was also accompanied by cleavage of AIF to form truncated AIF. Inhibition of VDAC1 oligomerization using 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), significantly improved the cell survival, decreased the ROS levels, improved mitochondrial functions, and decreased the mitochondrial damage. Notably, DIDS also inhibited the mitochondrial fragmentation caused by glutamate, indicating the active role of VDAC1 oligomerization in the process of mitochondrial fragmentation in oxytosis. These results suggest a critical role for VDAC1 in mitochondrial fragmentation and its potential therapeutic value against glutamate-mediated oxidative neurotoxicity.

DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) directly inhibits caspase activity in HeLa cell lysates.

Apoptosis is an important mechanism of cell demise in multicellular organisms and Cl− transport has an important role in the progression of the apoptotic volume decrease (AVD). DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) is one of the most commonly used Cl− transport inhibitors that eliminates or reduces different apoptotic hallmarks such as AVD, caspase-3 activity and DNA fragmentation. DIDS is also a protein crosslinker that alkylates either amino or thiol groups. Since caspases are thiol proteases, our aim was to study whether DIDS could directly inhibit the activity of these proteases. Here, we show that caspase activity induced by 4 h incubation with staurosporine was inhibited by DIDS in HeLa cells that were maintained in the absence of serum for 24 h. Interestingly, the caspase-inhibitory effect of DIDS is downstream to the inhibition of cytochrome c release, suggesting that DIDS might be also acting at the apoptosome. Moreover, DIDS was able to inhibit capase-3, -9, and -8 activities in cell lysates, implying that DIDS can react with and directly block caspases. Our data suggest that antiapoptotic activity of DIDS involves not only inhibition of the voltage-dependent anion channel (VDAC) at the mitochondria and Cl− channels at the plasma membrane, but also a third mechanism based on the direct inhibition of caspases.

Acquired cisplatin resistance in human ovarian A2780 cancer cells correlates with shift in taurine homeostasis and ability to volume regulate.

Cisplatin resistance is a major challenge in the treatment of cancer and develops through reduced drug accumulation and an increased ability to avoid drug-induced cell damage, cell shrinkage, and hence initiation of apoptosis. Uptake and release of the semiessential amino acid taurine contribute to cell volume homeostasis, and taurine has been reported to have antiapoptotic effects. Here we find that volume-sensitive taurine release in cisplatin-sensitive [wild-type (WT)] human ovarian cancer A2780 cells is reduced in the presence of the phospholipase A2 inhibitor bromenol lactone, the 5-lipoxygenase (5-LO) inhibitor ETH 615-139, and the cysteine leukotriene receptor 1 (CysLT1) antagonist zafirlukast and impaired by the anion channel blocker DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate). Comparing WT and cisplatin-resistant (RES) A2780 cells we also find that evasion of cisplatin-induced cell death in RES A2780 cells correlates with an increased accumulation of taurine, due to an increased taurine uptake and a concomitant impairment of the volume-sensitive taurine release pathway, as well an inability to reduce cell volume after osmotic cell swelling. Downregulation of volume-sensitive taurine release in RES A2780 cells correlates with reduced expression of the leucine-rich repeat-containing protein 8A (LRRC8A). Furthermore, acute (18 h) exposure to cisplatin (5-10 μM) increases taurine release and LRRC8A expression in WT A2780 cells whereas cisplatin has no effect on LRRC8A expression in RES A2780 cells. It is suggested that shift in LRRC8A activity can be used as biomarker for apoptotic progress and acquirement of drug resistance.

The Role of K+and Cl−Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

This study tested the role of K(+) and Cl(-) channels in the regulation of retinal blood flow. Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion-channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameter were measured in anesthetized animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the potent vasoconstrictor Endothelin 1 (Et1). Voltage-activated K(+) current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+) current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-) current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10 μL) of DIDS (estimated intraocular concentration 10 mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1 μM) and iberiotoxin (4 μM), and the IKv-inhibitor correolide (40 μM) all decreased resting flow by approximately 10%. Endothelin 1 (104 nM) reduced flow by approximately 65%. This effect was completely reversed by DIDS, but was unaffected by Penitrem A, iberiotoxin, or correolide. These results suggest that Cl(-) channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-) channels may be appropriate molecular targets in retinal pathologies characterized by increased Et1 activity and reduced blood flow.

Lactate-H+Transport Is a Significant Component of the In Vivo Corneal Endothelial Pump

To confirm the expression of monocarboxylate transporters (MCT) 1, 2, and 4 in rabbit CE and to test the hypothesis that cellular buffering contributed by HCO₃⁻, NBCe1, and carbonic anhydrase (CA) activity facilitates lactate-H⁺ efflux thereby controlling corneal hydration in vivo. MCT1-4 expression of rabbit endothelium was examined by Western blotting and immunofluorescence staining. Lactate-induced acidification (LIA) was measured in perfused CE in the presence and absence of HCO₃⁻ and acetazolamide (ACTZ) using tissue treated with siRNA specific to MCT1, 2, and 4. Corneal thickness and lactate concentration were measured in New Zealand White rabbits treated with the topical CA inhibitor Azopt, and from eyes that were injected intracamerally with ouabain, disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), and shRNA specific to the 1Na⁺:2HCO₃⁻ cotransporter NBCe1. MCT1 and MCT4 are localized to the lateral membrane, while MCT2 is apical. Cell pH measurements showed LIA in response to 40 mM lactate in bicarbonate free (BF) Ringer's that was inhibited by niflumic acid and by MCT siRNA knockdown, and significantly reduced in the presence of HCO₃⁻. Lactate-dependent proton flux in vitro was not significantly greater in the presence of HCO₃⁻ or reduced by ACTZ. However, when active transport, NBCe1, or CA activity was disrupted in vivo, corneal edema ensued and was associated with significant corneal lactate accumulation. MCT1, 2, and 4 are expressed in rabbit CE on both the apical and basolateral surfaces and function to transport lactate-H⁺. Lactate-H⁺ flux is facilitated by active transport, HCO₃⁻ transport and CA activity, disruption of which causes corneal edema in vivo and indicates that facilitation of lactate efflux is a component of the endothelial pump.

ABCA1 upregulating apolipoproein M expression mediates via the RXR/LXR pathway in HepG2 cells

We have previously reported that liver X receptor (LXR) agonist, TO901317, could significantly inhibit hepatic apolipoprotein M (apoM) expression. It has been reported that TO901317 could activate the ATP-binding cassette transporter A1 (ABCA1) that mediates cholesterol efflux to the lipid-poor apoAI, which is an essential step for the high-density lipoprotein (HDL) formation. It is unknown if ABCA1 may regulate hepatic apoM expression. In the present study, HepG2 cells were cultured with the synthetic LXR agonists, TO901317 or GW3965 in the presence or absence of ABCA1 antagonist, disodium 4,4′-diisothiocyanatostilbene- 2,2′-disulfonate (DIDS). The mRNA levels of ABCA1, apoM and liver receptor homolog-1 (LRH-1) determined by the real-time RT-PCR. It demonstrated that both TO901317 and GW3965 could significantly enhance ABCA1 expression, and simultaneously, inhibit LRH1 expression. However, TO901317 alone could significantly inhibit apoM expression, while GW3965 alone did not influence apoM expression. ABCA1 antagonist, DIDS, have no effects on GW3965 induced upregulation of ABCA1 and downregulation of LRH1. However, apoM mRNA level was significantly decreased when the cells cultured with GW3965 together with DIDS. The present study demonstrated that apoM expression could be elevated by ABCA1 via the RXR/LXR pathway and LRH1 does not involve in the regulation of apoM by the activation of ABCA1, although the direct regulative pathway(s) between ABCA1 and apoM gene is still unknown yet. The detailed mechanism needs further investigation.

Na+/HCO3 − Cotransporter Immunoreactivity Changes in Neurons and Expresses in Astrocytes in the Gerbil Hippocampal CA1 Region After Ischemia/Reperfusion

The maintenance of intracellular pH is important in neuronal function. Na(+)/HCO(3)(-) cotransporter (NBC), a bicarbonate-dependent acid-base transport protein, may contribute to cellular acid-base homeostasis in pathophysiological processes. We examined the alterations of NBC immunoreactivity and its protein levels in the hippocampal CA1 region after transient cerebral ischemia in gerbils. In the sham-operated group, moderate NBC immunoreactivity was detected in CA1 pyramidal neurons, and, 12 h after I/R, the immunoreactivity in the pyramidal neurons was markedly increased over controls. Three days after I/R, NBC immunoreactivity nearly disappeared in the CA1 pyramidal neurons. However, NBC immunoreactivity was detected in the non-pyramidal neurons of the ischemic CA1 region at 3 days after I/R. From double immunofluorescence study with glial markers, NBC immunoreactivity was detected in astrocytes, not in microglia, at 4 days after I/R. NBC protein level in the CA1 region was significantly increased at 12 h post-ischemia and significantly decreased at 2 days post-ischemia. Thereafter, NBC protein level was again increased and returned to the level of the sham-operated group at 4 days post-ischemia. On the other hand, treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an inorganic anion exchanger blocker including Cl-bicarbonate exchanger, protected CA1 pyramidal neurons from I/R injury at 4 days post-ischemia. These results indicate that changes in NBC expressions may play an important role in neuronal damage and astrocytosis induced by transient cerebral ischemia.

Dual Role of CO2/HCO3− Buffer in the Regulation of Intracellular pH of Three-dimensional Tumor Growths

Intracellular pH (pHi), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H+ ions (e.g. produced by respiration) are extruded by transporters such as Na+/H+ exchange, or neutralized by HCO3− taken up by carriers such as Na+-HCO3− cotransport. Using fluorescence pHi imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H+ efflux and HCO3− influx, largely sensitive to dimethylamiloride and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), respectively. The magnitude of HCO3− influx was comparable among the cell lines and may represent a constitutive element of tumor pHi regulation. In contrast, H+ efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO3− flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (∼10,000 cells) was highly non-uniform and produced low pHi at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H+ flux more than HCO3− flux. HCO3− flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO2/HCO3−, acid extrusion by H+ flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H+ transporters require extracellular mobile pH buffers, such as CO2/HCO3−, to overcome low H+ ion mobility and chaperone H+ ions away from cells. CO2/HCO3− exerts a dual effect: as substrate for membrane-bound HCO3− transporters and as a mobile buffer for facilitating extracellular diffusion of H+ ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO2/HCO3− is important for maintaining uniformly alkaline pHi in small, non-vascularized tumor growths and may be important for cancer disease progression.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Na+/HCO3- cotransporter immunoreactivity changes in neurons and expresses in astrocytes in the gerbil hippocampal CA1 region after ischemia/reperfusion

The maintenance of intracellular pH is important in neuronal function. Na+/HCO3− cotransporter (NBC), a bicarbonate-dependent acid–base transport protein, may contribute to cellular acid–base homeostasis in pathophysiological processes. We examined the alterations of NBC immunoreactivity and its protein levels in the hippocampal CA1 region after transient cerebral ischemia in gerbils. In the sham-operated group, moderate NBC immunoreactivity was detected in CA1 pyramidal neurons, and, 12 h after I/R, the immunoreactivity in the pyramidal neurons was markedly increased over controls. Three days after I/R, NBC immunoreactivity nearly disappeared in the CA1 pyramidal neurons. However, NBC immunoreactivity was detected in the non-pyramidal neurons of the ischemic CA1 region at 3 days after I/R. From double immunofluorescence study with glial markers, NBC immunoreactivity was detected in astrocytes, not in microglia, at 4 days after I/R. NBC protein level in the CA1 region was significantly increased at 12 h post-ischemia and significantly decreased at 2 days post-ischemia. Thereafter, NBC protein level was again increased and returned to the level of the sham-operated group at 4 days post-ischemia. On the other hand, treatment with 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an inorganic anion exchanger blocker including Cl-bicarbonate exchanger, protected CA1 pyramidal neurons from I/R injury at 4 days post-ischemia. These results indicate that changes in NBC expressions may play an important role in neuronal damage and astrocytosis induced by transient cerebral ischemia.

Pharmacological Characterization of Pannexin-1 Currents Expressed in Mammalian Cells

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation.

Non-transferrin Iron Reduction and Uptake Are Regulated by Transmembrane Ascorbate Cycling in K562 Cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [(55)Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.

HPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes

Here, we examined hPepT1 expression in the monocytic cell line, KG-1. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that hPepT1 is expressed in KG-1 cells, while cDNA cloning and direct sequencing confirmed the sequence of KG-1 hPepT1 (accession number, AY634368). Immunoblotting of cell lysates from KG-1 cells or macrophages isolated from human peripheral blood revealed a ∼100 kDa immunoreactive band mainly present in the membrane fraction. Uptake experiments showed that the transport of 20 μM radiolabeled Gly-Sarcosine ([14C]Gly-Sar) in KG-1 cells was Na+, Cl− dependent and disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS)-sensitive. In addition, hPepT1 activity was likely to be coupled to a Na+/H+ exchanger, as evidenced by the fact that [14C]Gly-Sar uptake was not affected by the absence of Na+ when cells were incubated at low pH (5.2). Interestingly, hPepT1-mediated transport was reduced in KG-1 cells incubated at low pH as it was also observed in nonpolarized Caco2-BBE cells. This pattern of pH-dependence is due to a disruption of the driving force of hPepT1-mediated transport events. This was supported by our finding that nonpolarized cells, Caco2-BBE cells and KG-1 cells, have an increased permeability to H+ when compared to polarized Caco2-BBE cells. Finally, we showed that hPepT1 is responsible for transporting fMLP into undifferentiated and differentiated (macrophage-like) KG-1 cells. Together, these results show that hPepT1 is expressed in nonpolarized immune cells, such as macrophages, where the transporter functions best at the physiological pH 7.2. Furthermore, we provide evidence for hPepT1-mediated fMLP transport, which might constitute a novel immune cell activation pathway during intestinal inflammation.