Molecular Determinant of DIDS Analogs Targeting RAD51 Activity.

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.

The role of HCO3- in propionate-induced anion secretion across rat caecal epithelium.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl- secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl-- or nominally HCO3--free buffer. In the case of Cl-, the dependency was restricted to the presence of Cl- in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl--cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3--cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3- secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

Several phosphate transport processes are present in vascular smooth muscle cells.

We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.

Event DAS-444Ø6-6 soybean grown in Brazil is compositionally equivalent to non-transgenic soybean.

Soybean event DAS-444Ø6-6 is tolerant to the herbicides 2,4-D, glyphosate, and glufosinate. An investigation of potential unintended adverse compositional changes in a genetically modified crop is required to meet government regulatory requirements in various geographies. A study to meet these requirements in Brazil was completed demonstrating compositional equivalency between DAS-444Ø6-6 and non-transgenic soybean. This study supplements the extensive literature supporting transgenesis as less disruptive of crop composition compared with traditional breeding methods.

Isothiocyanatostilbenes as novel c-Met inhibitors.

The hepatocyte growth factor receptor (HGFR or c-Met) is a driver of multiple cancer subtypes. While there are several c-Met inhibitors in development, few have been approved for clinical use, warranting the need for continued research and development of c-Met targeting therapeutic modalities. The research presented here demonstrates a particular class of compounds known as isothiocyanatostilbenes can act as c-Met inhibitors in multiple cancer cell lines. Specifically, we found that 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) had c-Met inhibitory effective doses in the low micromolar range while 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS) exhibited IC50s 100 to 1000 fold higher. These compounds displayed much greater selectivity for inhibiting c-Met activation compared to similar receptor tyrosine kinases. In addition, DIDS and H2DIDS reduced hepatocyte growth factor (HGF)-induced, but not epidermal growth factor (EGF)-induced, cell scattering, wound healing, and 3-dimensional (3D) proliferation of tumor cell spheroids. In-cell and cell-free assays suggested that DIDS and H2DIDS can inhibit and reverse c-Met phosphorylation, similar to SU11274. Additional data demonstrated that DIDS is tolerable in vivo. These data provide preliminary support for future studies examining DIDS, H2DIDS, and derivatives as potential c-Met therapeutics.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Bicarbonate transport inhibitor SITS modulates pH homeostasis triggering apoptosis of Dalton's lymphoma: implication of novel molecular mechanisms.

Bicarbonate transporter (BCT) plays a crucial role in maintaining pH homeostasis of tumor cells by import of HCO3(-). This helps the tumor cells in manifesting extracellular tumor acidosis, accompanied by a relative intracellular alkalinization, which in turn promotes tumor progression. Therefore, blocking BCT-mediated HCO3(-) transport is envisaged as a promising anticancer therapeutic approach. Thus, using a murine model of a T cell lymphoma, designated as Dalton's lymphoma (DL), in the present in vitro investigation the antitumor consequences of blocking BCT function by its inhibitor 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) were explored. Treatment of DL cells with SITS resulted in an increase in the extracellular pH, associated with a decline in DL cell survival and augmented induction of apoptosis. BCT inhibition also elevated the expression of cytochrome c, caspase-9, caspase-3, Bax, reactive oxygen species, and nitric oxide along with inhibition of HSP-70 and Bcl2, which regulate tumor cell survival and apoptosis. SITS-treated DL cells displayed upregulated production of IFN-γ and IL-6 along with a decline of IL-10. Treatment of DL cells with SITS also inhibited the expression of fatty acid synthase, which is crucial for membrane biogenesis in neoplastic cells. The expression of lactate transporter MCT-1 and multidrug resistance regulating protein MRP-1 got inhibited along with hampered uptake of glucose and lactate production in SITS-treated DL cells. Thus, the declined tumor cell survival following inhibition of BCT could be the consequence of interplay of several inter-connected regulatory molecular events. The outcome of this study indicates the potential of BCT inhibition as a novel therapeutic approach for treatment of hematological malignancies.

Characterization of the Na/HCO3(-) cotransport in human neutrophils.

BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

[Effects of chloride ion channel and its blockers on myocardial ischemia reperfusion arrhythmias in rabbits].

OBJECTIVE: To explore the impact of chloride ion channel and its blockers 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), cyanato-stilbene-2, 2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB) on arrhythmias caused by myocardial ischemia reperfusion. METHODS: A total of 40 rabbits were divided into control, ischemia reperfusion, DIDS low-dose, DIDS high-dose, SITS low-dose, SITS high-dose, NPPB low-dose and NPPB high-dose groups. Myocardial ischemia reperfusion model was established by ligation of anterior descending coronary artery. And standard limb lead II of electrocardiogram (ECG) was continuously monitored during the experimental process. Then comparisons of heart rate, ECG P wave, R wave, T wave, ST segment changes and arrhythmias score were made between the above groups. RESULTS: During 30-minute ischemia, compared with the control group, all other groups showed significantly decreased heart rate ((199.8 ± 4.0) - (253.6 ± 2.1) vs (267.0 ± 3.4), all P < 0.01), elevated ECG P wave ((0.216 ± 0.019) - (0.356 ± 0.024) vs (0.186 ± 0.019), all P < 0.01), R wave ((0.564 ± 0.017) - (1.138 ± 0.048) vs (0.506 ± 0.018), all P < 0.01), T wave ((0.542 ± 0.013) - (0.856 ± 0.045) vs (0.278 ± 0.015), all P < 0.01) and ST segment ((0.326 ± 0.027) - (0.668 ± 0.054) vs (0.024 ± 0.023), all P < 0.01) and increased arrhythmia score ((1.4 ± 0.5) - (4.6 ± 0.5) vs (0.4 ± 0.5), all P < 0.01). Compared with the ischemia reperfusion group, the above indices significantly improved in the intervention groups (heart rate: (214.8 ± 3.4) - (246.8 ± 4.0) vs (199.8 ± 4.0), all P < 0.01; P wave: (0.216 ± 0.019) - (0.316 ± 0.011) vs (0.356 ± 0.024), all P < 0.01; R wave: (0.564 ± 0.017) - (0.980 ± 0.035) vs (1.138 ± 0.048), all P < 0.01; T wave: (0.542 ± 0.013) - (0.792 ± 0.026) vs (0.856 ± 0.045), all P < 0.01; ST segment: (0.326 ± 0.027) - (0.596 ± 0.018) vs (0.668 ± 0.054), all P < 0.01; arrhythmia score: (1.4 ± 0.5) - (3.8 ± 0.4) vs (4.6 ± 0.5), all P < 0.01). Among which, the DIDS group was the best, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. During 60-minute reperfusion, the decreased heart rate upswing significantly in each group and the elevated P wave, R wave, T wave and ST segment fell back gradually. The DIDS group showed the most obvious amplitude change, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. The arrhythmia score during reperfusion showed the same trend. CONCLUSION: Chloride ion channel is involved in the generation of myocardial ischemia reperfusion arrhythmia.Early application of chloride ion channel blockers DIDS, SITS and NPPB may improve the ECG manifestations and reduce the degree of reperfusion arrhythmia.

Agonist-dependent potentiation of vanilloid receptor transient receptor potential vanilloid type 1 function by stilbene derivatives.

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.

Mechanisms of chloride in cardiomyocyte anoxia-reoxygenation injury: the involvement of oxidative stress and NF-kappaB activation.

During anoxia/reoxygenation (A/R) injury, intracellular chloride ion concentration ([Cl(-)](i)) homeostasis may play a role in maintaining the normal physiological function of cardiomyocytes. Various chloride transport systems could have influenced the concentration of chloride ion, but what kinds of chloride transport systems could play an important role in cardiomyocytes subjected to A/R injury and its mechanism are unknown. The aim of our study was to clarify the contributions of various chloride transport systems to anoxia/reoxygenation in rat neonatal cardiac myocytes and further to investigate the involved mechanisms. Oxidative stress and redox-sensitive transcription factor (NF-kappaB) activation are believed to play an important role in the A/R injury. To assess whether oxidative stress and NF-kappaB involve [Cl(-)](i) changes resulting in cardiomyocytes injury, the anoxia-reoxygenation (A/R) injury model was successfully established and administered with inhibitors of various chloride transport systems. Administration with Cl(-)-substitution and Cl(-)/HCO(3) (-) exchange inhibitor(SITS) has been shown to produce a protective effect against A/R injury by decreasing [Cl(-)](i) concentration, lipid peroxidation (malondialdehyde (MDA)) levels, and NF-kappaB activity, and by increasing antioxidant enzyme (glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase(CAT)) activity. However, inhibitors for the Cl(-)-channel (9-AC) and Na(+)-K(+)-2Cl(-) co-transporter (bumetanide) had no effects. Our results indicate that Cl(-)/HCO(3) (-) exchange system plays an important role in the cardiocyte A/R injury by influencing [Cl(-)](i) concentration. The protective effects of SITS and Cl(-)-substitution on cardiomyocytes may be due to the attenuation of oxidative stress and inhibition of NF-kappaB activation.

Functional characterization of folate transport proteins in Staten's Seruminstitut rabbit corneal epithelial cell line.

PURPOSE: The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line. METHODS: [(3)H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [(3)H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [(3)H]folic acid in the presence and absence of 1 mM cold folic acid. RESULTS: Linear increase in [(3)H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent K(m) of 14.2 ± 0.2 nM, V(max) of (1.5 ± 0.1)*10(-5) micro.moles/min/mg protein, and K(d) of (2.1 ± 0.2)*10(-6) min(-1). The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [(3)H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca(2+)/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [(3)H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10(-05) cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10(-05) cm/sec. CONCLUSIONS: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.

A biochemical framework for SLC4A11, the plasma membrane protein defective in corneal dystrophies.

Mutations in the SLC4A11 protein, reported as a sodium-coup-led borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuch's CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.

Effect of anion channel blockers on L-arginine action in spermatozoa from asthenospermic men.

In earlier studies, we have established that l-arginine enhances motility and metabolic rate in spermatozoa of goat, bull and mouse. In the present study this work was extended to human sperm cells obtained from the semen samples of asthenospermic patients, which are characterised by low motility. The metabolic rate was followed by monitoring the glucose consumption (1-(13)C glucose as substrate) and the production of lactate in sperm cells, using (13)C NMR. The stimulatory effect of l-arginine was neutralised on adding an NO-synthase inhibitor like N(omega)-nitro-L-arginine methyl ester. On the other hand, the inactive d-enantiomorph did not affect the stimulatory effect of l-arginine. This strongly suggests that L-arginine acts through the NO signal pathway. We also demonstrated that the stimulatory effect of L-arginine was inhibited in the presence of anion channel inhibitors like 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Furthermore, bicarbonate supplementation was found to be essential for the action of L-arginine. These observations indicate that L-arginine induces NO synthesis and stimulates motility and metabolism only when an active anion transport system is present.

AE2 Cl-/HCO3- exchanger is required for normal cAMP-stimulated anion secretion in murine proximal colon.

Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.

Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-Jun-N-terminal kinase activation in cultured neurons.

Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K(+). Preincubation of the neurons with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T).

Barrett's specialized columnar epithelium (SCE) replaces reflux-damaged squamous epithelium. The benefits of SCE lie in its superior protection of the esophagus against further reflux damage. It was shown that this protection is dependent on ion transport and barrier function of SCE. The risks of SCE lie in its higher predisposition to malignant transformation. An understanding of underlying mechanisms of both processes would benefit considerably from greater knowledge of the structure and function of native SCE - the latter recently advanced by the availability of a telomerase-immortalized, nonneoplastic, human Barrett's cell line (BAR-T). Some of BAR-T characteristics for growth and differentiation have been described recently, but not its capacity to serve as a model for ion transport and barrier function of SCE. To determine the latter, BAR-T cells were grown in enriched media, seeded on permeable supports, and subjected to electrical, biochemical, and morphologic study. HET-1A (esophageal epithelial cell line), a nonneoplastic, human esophageal squamous cell line, was also studied for comparison. BAR-T, but not HET-1A cells in HEPES Ringer solution behaved as polarized monolayers with the capacity for ion transport and barrier function. This was evident electrically with a volt-ohm meter (EVOM),which recorded in BAR-T a resting potential difference of 2.0 +/- 0.2 mV, Isc of 17.4 +/- 3.3 microAmps/cm2 and resistance of 103 +/- 12 ohms x cm2. Further, Isc in BAR-T was inhibitable by exposure to Na-free solution, serosal ouabain, and luminal 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid. Expression of tight junction genes were determined in BAR-T and HET-1A cells using quantitative reverse transcriptase-polymerase chain reaction, with expression of zonula occludens-1 (ZO-1) set at 1 as reference. Claudins 1, 4, and 12 were prominently expressed in BAR-T (0.2-0.6 of ZO-1), while claudins 1, 11, and 12 were prominently expressed in HET-1A(0.1-0.8 of ZO-1). BAR-T, but not HET-1A, expressed claudins 4, 8, 16, 18, and 23, and HET-1A, but not BAR-T, expressed claudins 11, 15, and 20. Protein expression of prominently expressed claudins in BAR-T correlated with mRNA expression. Immunofluorescence and confocal microscopy localized claudins 1 and 4 in BAR-T to cell membranes and claudin 18, specifically to the tight junction. Membrane polarization was also documented in BAR-T by immunolocalization of NaK,ATPase to the basolateral membrane. BAR-T, but not HET-1A cells grown on permeable supports form a polarized monolayer with both ion transport and barrier function. Since a number of features of BAR-T are similar to Barrett's SCE and distinct from HET-1A, the BAR-T cell line represents a valuable resource for the study of ion transport and barrier function of nondysplastic SCE.

Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents.

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

Modulation of butyrate transport in Caco-2 cells.

The aim of this study was to investigate the putative influence of some pharmacological agents and drugs of abuse upon the apical uptake of butyrate (BT) into Caco-2 cells. The apical uptake of (14)C-BT by Caco-2 cells was (1) time and concentration dependent, (2) pH dependent, (3) Na(+) independent and Cl(-) dependent, (4) energy dependent, (5) inhibited by several BT structural analogues (acetate, propionate, alpha-ketobutyrate, pyruvate, lactate), (6) insensitive to the anion exchange inhibitors DIDS and SITS and (7) inhibited by the monocarboxylate transport (MCT) inhibitors NPPB and pCMB. These characteristics are compatible with an involvement of MCT1-mediated transport. Acutely, uptake of a low concentration of (14)C-BT (10 microM) was reduced by acetaldehyde, acetylsalicylic acid, indomethacin, caffeine and theophylline and increased by MDMA. Chronically, uptake was increased by caffeine and decreased by tetrahydrocannabinol and MDMA; reverse transcription quantitative real-time PCR analysis showed that these three compounds decreased the mRNA levels of MCT1. Acutely, acetaldehyde, indomethacin and MDMA reduced the uptake of a high concentration of (14)C-BT (20 mM), and acetylsalicylic acid increased it. Chronically, none of the compounds affected uptake. Acetaldehyde, indomethacin and propionate seem to be competitive inhibitors of (14)C-BT uptake. Acetylsalicylic acid simultaneously increased the K (m) and the V (max) of (14)C-BT uptake. In conclusion, MCT1-mediated transport of (14)C-BT in Caco-2 cells is modulated by either acute or chronic exposure to some pharmacological agents and drugs of abuse (acetaldehyde, acetylsalicylic acid, indomethacin, caffeine, theophylline and the drugs of abuse tetrahydrocannabinol and MDMA).

Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH.

Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075DKPK1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. Regulation of Cl-/Cl- and Cl-/HCO(-)3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4'-di-isothiocyanatostilbene-2,2' disulfonic acid-sensitive 36Cl- efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by approximately 1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1-920)/AE1(613-929) without affecting its low sensitivity to pHo. The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pHi.