Removal Of Cl Research Articles

Energetics of glutamate receptor ligand binding domain dimer assembly are modulated by allosteric ions

The activity of many ligand-gated ion channels and cell surface receptors is modulated by small molecules and ions, but an understanding of the underlying molecular mechanisms is scarce. For kainate, but not AMPA subtype glutamate receptors, the binding of Na(+) and Cl(-) ions to discrete, electrostatically coupled sites in the extracellular ligand binding domain (LBD) dimer assembly regulates the rate of entry into the desensitized state, which occurs when the dimer interface ruptures and the channel closes. Studies on glutamate receptors have defined the LBD dimer assembly as a key functional unit that controls activation and desensitization. Here we use analytical ultracentrifugation to probe the energetic effects of allosteric ions on kainate receptor dimer stability in solution, using a GluR6 mutant that desensitizes slowly. Our results show that sodium and chloride ions modulate kainate receptor dimer affinity as much as 50-fold, and that removal of either Cl(-) or Na(+) disrupts the dimer. The applicability of a similar allosteric mechanism for modulation of delta2 glutamate receptors by Ca(2+) was also tested. Our results indicate that ions can contribute substantial free energy to active state stabilization in both these receptors, and provide quantitative measurements of the energetic consequences of allosteric ion binding to a ligand-gated ion channel.

Fluoride inhibition of photosystem II and the effect of removal of the PsbQ subunit

Photosystem II (PSII), the light-absorbing complex of photosynthesis that evolves oxygen, requires chloride for activation of the oxygen evolving complex (OEC). In this study, fluoride was characterized as an inhibitor of Cl(-)-activated oxygen evolution in higher plant PSII. It was confirmed to be primarily a competitive inhibitor in intact PSII, with Cl(-)-competitive inhibition constant K(i) = 2 mM and uncompetitive inhibition constant K'(1) = 79 mM. A pH dependence study showed that fluoride inhibition was more pronounced at lower pH values. In order to determine the location of the fluoride effect, PSII preparations lacking various amounts of the PsbQ subunit were prepared. The competitive F(-) inhibition constant and the Michaelis constant for Cl(-) activation increased with loss of the PsbQ subunit, while the uncompetitive F(-) inhibition constant was relatively insensitive to loss of PsbQ. The S(2) state EPR signals from PSII lacking PsbQ responded to Ca(2+) and Cl(-) removal and to F(-) treatment similar to intact PSII, with enhancement of the g = 4.1 signal and suppression of the multiline signal, but the effects were more pronounced in PSII lacking PsbQ. Together, these results support the interpretation that the PsbQ subunit has a role in retaining anions within the OEC.

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO(3) transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO(3) transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO(3)(-) currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO(3)-dependent pH recovery from a CO(2)-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO(2)/HCO(3)(-)-free solution that was abolished by Cl(-) removal from the bath. In the presence of CO(2)/HCO(3)(-), however, the outward current produced by D555E decreased only slightly after Cl(-) removal. Starting from a Cl(-)-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO(2)/HCO(3)(-). The substitution of Asp(555) with an asparagine also produced a Cl(-) current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO(3)(-). Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp(555) plays a role in HCO(3)(-) selectivity.

Microwave activated electrochemical degradation of 2,4-dichlorophenoxyacetic acid at boron-doped diamond electrode

A method for improving the oxidation ability of the electrode is proposed by using microwave activation in electrochemical oxidation. The electrochemical degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) with microwave radiation (MW-EC) was carried out in a continuous flow system under atmospheric pressure. In 3 h the removal of COD, ACE (average current efficiency) and Cl − concentration was 1.63, 2.25 and 1.67 times as that without microwave radiation, respectively. The high degradation ability was resulted from the more active centers at the electrode surface due to the microwave radiation. The decay kinetics of 2,4-D followed a pseudo first-order reaction. The rate constant was increased to 2.16 × 10 −4 s −1 with the microwave radiation, while it was 8.52 × 10 −5 s −1 with electrochemical treatment only (EC). Under both conditions, the main intermediates were identified and quantified by High Performance Liquid Chromatography (HPLC). The formation rate of intermediate products and further degradation rate were increased by about 50–120% with the microwave radiation. The activation of electrochemical oxidation by microwave was discussed from the diffusion process, adsorption and the temperature at boron-doped diamond (BDD) electrode.

Water extraction with CO2 bubbling as pretreatment of melting-furnace fly ash for metal recovery

In Japan, melting-furnace fly ash (MFA) generated from ash melting and gasification/melting plants is considered an “urban mine” due to its high metal content. This study aimed to develop a novel approach to pretreating MFA for metal recovery. Water extraction with CO2 bubbling was investigated because MFA mainly consists of water-soluble salts containing elements such as Cl, Ca, Na, and K. Instead of acid addition, CO2 bubbling was applied to maintain the optimal pH for minimizing the release of target metal elements and maximizing the removal of undesirable elements during water extraction. The results revealed that CO2 bubbling effectively decreased the release of Pb, Zn, and Cd into the treatment water. This was mainly due to coprecipitation with CaCO3, which was primarily formed by the reaction of Ca2+ from the MFA with CO32− from the CO2 gas. The bubbling process also helped accelerate the removal of Cl from MFA. Furthermore, the study showed that it is possible to lower the water-to-solid ratio to 5 with only a slight reduction in water extraction effect. Finally, approximately four times the concentration of target metals (rare metals and Cu, Pb, and Zn) was achieved by removing 90% of Cl, 70%–90% of Na and K, and 30%–40% of Ca through water extraction with CO2 bubbling, resulting in a concentration of target metals that was nearly equal to that of ore.

Intracellular pH regulation in human Sertoli cells: role of membrane transporters

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Intracellular pH (pHi) is an important parameter in cell physiology regulating namely cell metabolism and differentiation. However, pHi regulation mechanisms in Sertoli cells have not yet been systematically elucidated. In this work, pHi was determined in primary cultures of human Sertoli cells. Sertoli cells were exposed to weak acids, which caused a rapid acidification of the intracellular milieu. pHi then recovered by a mechanism that was shown to be particularly sensitive to the presence of the inhibitor DIDS (4,4'-diisothiocyanostilbene disulfonic acid). In the presence of amiloride and PSA (picrylsulfonic acid), pHi recovery was also significantly affected. These results indicate that, in the experimental conditions used, pHi is regulated by the action of an Na(+)-driven HCO(3)(-)/Cl(-) exchanger and an Na(+)/HCO(3)(-) co-transporter and also by the action of the Na(+)/H(+) exchanger. On the other hand, pHi recovery was only slightly affected by concanamycin A, suggesting that V-Type ATPases do not have a relevant action on pHi regulation in human Sertoli cells, and was independent of the presence of bumetanide, suggesting that the inhibition of the Na(+)/K(+)/Cl(-) co-transporter does not affect pHi recovery, not even indirectly via the shift of ionic gradients. Finally, pHi was shown to be sensitive to the removal of external Cl(-), but not of Na(+) or K(+), evidencing the presence of a membrane Cl(-)-dependent base extruder, namely the Na(+)-independent HCO(3)(-)/Cl(-) exchanger, and its role on pHi maintenance on these cells.

Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes

Exposure of trout hepatocytes to hypertonicity induced a decrease in acridine orange (AO) fluorescence, indicating a corresponding decrease in pH inside the lumen of acidic compartments (pH(L)). Pre-exposure of cells to the specific V-ATPase inhibitor bafilomycin A1 (0.3 micromol l(-1)) increased AO fluorescence - unmasking H(+) leaks under steady-state conditions - and partially removed the hypertonicity-induced pH(L) decrease. The sustainability of the luminal acidification, but not the acidification itself, appeared to depend on a low K(+) and a high Cl(-) conductance under hypertonic conditions. Increasing K(+) conductance using the specific ionophore valinomycin (10 micromol l(-1)) or removal of extracellular Cl(-) after an instant drop in AO fluorescence resulted in a reversal of luminal acidity. The alkalinization measured under hypertonic conditions in the absence of Cl(-) was largely attenuated when cells were bathed in HCO(3)(-)-free medium, signifying the possible presence of Cl(-)/HCO(3)(-) exchange. Under steady-state conditions, while a slight and brief pH(L) increase was measured upon exposure of cells to valinomycin, Cl(-) removal, unexpectedly, induced a decrease in pH(L), indicating a role for extracellular Cl(-) in limiting luminal acidification. This was confirmed by the substantial pH(L) decrease measured upon exposure of cells to the anion exchanger inhibitor SITS (0.5 mmol l(-1)). Furthermore, hypertonicity-induced acidification was still noticeable in the presence of SITS. On the other hand, the hypertonicity-induced acidification was significantly reduced in the absence of extracellular Na(+) or Ca(2+). However, BAPTA-AM induced an increase in steady-state pH(L) that was independent of V-ATPase inhibition. Moreover, the BAPTA-induced alkalinization was still apparent after depletion of intracellular Ca(2+) using the Ca(2+) ionophore A23187 in Ca(2+)-free medium. We conclude that pH(L) of trout hepatocytes is sensitive to hypertonicity and ionic determinants of hypertonicity. Thus, changes in pH(L) should be considered when studying pH adaptations to hypertonic stress.

Bradykinin regulates human colonic ion transport in vitro

Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T(84)-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. Ion transport was measured as changes in short-circuit current (I(sc)) across colonic epithelia mounted in Ussing chambers. In intact tissue, there was a distinct polarity to BK-elicited I(sc) responses. Whereas basolateral BK stimulated sustained responses (EC(50)=0.5+/-0.1 microM), those to apical BK were more rapid and transient (EC(50)=4.1+/-1.2 nM). In T(84) cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl(-) secretion as shown by their sensitivity to bumetanide and removal of Cl(-) from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B(2) receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 microM), atropine (1 microM), capsaicin (100 microM) and piroxicam (10 microM). BK-stimulated prostaglandin (PG)E(2) release from colonic tissue. BK stimulates human colonic Cl(-) secretion by activation of apical and basolateral B(2) receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis.

Plasma Urate Level Is Directly Regulated by a Voltage-driven Urate Efflux Transporter URATv1 (SLC2A9) in Humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).

Atomic Layer Deposition of Hafnium Oxide from Hafnium Chloride and Water

Hafnium oxide (HfO2) is a leading candidate to replace silicon oxide as the gate dielectric for future generation metal-oxide-semiconductor based nanoelectronic devices. Atomic layer deposition (ALD) has recently gained interest because of its suitability for fabrication of conformal films with thicknesses in the nanometer range. This study uses periodic density functional theory (DFT) to investigate the mechanisms of both half-reactions occurring on the growing surface during the ALD of HfO2 using HfCl4 and water as precursors. We find that the adsorption energy and the preferred site of adsorption of the metal precursor are strong functions of the water coverage. As water coverage increases, the metal precursor preferentially interacts with multiple surface adsorption sites. During the water pulse the removal of Cl can be facilitated by either a ligand exchange reaction or the dissociation of Cl upon increase in coordination of the Hf atom of the precursor. Our predicted potential energy surface indicates that a more likely mechanism is hydration of the adsorbed Hf complex up to a coordination number of 7, followed by the dissociation of a chloride ion that is stabilized by solvation. Born-Oppenheimer molecular dynamics (BOMD) simulations of an adsorbed metal precursor in the presence of a multilayer of water shows that Cl dissociation is facile if sufficient water molecules are present to solvate the Cl(-) anions. Hence, solvation plays a crucial role during the water pulse and provides an alternative explanation for why ALD growth rates for this system decrease at high temperatures.

Total dissolved solids removal by electrochemical ion exchange (EIX) process

In the present investigation, synthetic wastewater was prepared by the addition of required amount of salts into deionized water. Their performance, on removal of Cl −, SO 4 2−, PO 4 3−, Ca 2+, Fe 2+ and Mg 2+ in laboratory scale plate and frame type electrochemical ion exchange (EIX) cells, were evaluated under varying operating conditions. Ruthenium dioxide coated titanium plates (RuO 2/Ti) were used as anode and stainless steel plates as cathode in all the four different EIX cells used in the present investigation. All the four EIX cells were run for a maximum of 7 h. Almost complete removal of all the above ionic solids were observed within 4.5–7 h under one or more test conditions.

Gender-Specific Protection of Estrogen against Gastric Acid-Induced Duodenal Injury: Stimulation of Duodenal Mucosal Bicarbonate Secretion

Because human duodenal mucosal bicarbonate secretion (DMBS) protects duodenum against acid-peptic injury, we hypothesize that estrogen stimulates DMBS, thereby attributing to the clinically observed lower incidence of duodenal ulcer in premenopausal women than the age-matched men. We found that basal and acid-stimulated DMBS responses were 1.5 and 2.4-fold higher in female than male mice in vivo, respectively. Acid-stimulated DMBS in both genders was abolished by ICI 182,780 and tamoxifen. Estradiol-17beta (E2) and the selective estrogen receptor (ER) agonists of ERalpha [1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole] and ERbeta [2,3-bis(4-hydroxyphenyl) propionitrile], but not progesterone, rapidly stimulated ER-dependent murine DMBS in vivo. E2 dose dependently stimulated murine DMBS, which was attenuated by a Cl(-)/HCO3(-) anion exchanger inhibitor 4,4'-didsothio- cyanostilbene-2, 2'-disulfonic acid, removal of extracellular Cl(-), and in cystic fibrosis transmembrane conductance regulator knockout female mice. E2 stimulated murine DMBS in vitro in both genders with significantly greater response in female than male mice (female to male ratio = 4.3). ERalpha and ERbeta mRNAs and proteins were detected in murine duodenal epithelium of both genders; however, neither ERalpha nor ERbeta mRNA and protein expression levels differed according to gender. E2 rapidly mobilized intracellular calcium in a duodenal epithelial SCBN cell line that expresses ERalpha and ERbeta, whereas BAPTA-AM abolished E2-stimulated murine DMBS. Thus, our data show that E2 stimulates DMBS via ER dependent mechanisms linked to intracellular calcium, cystic fibrosis transmembrane conductance regulator, and Cl(-)/HCO3(-) anion exchanger. Gender-associated differences in basal, acid- and E2-stimulated DMBS may have offered a reasonable explanation for the clinically observed lower incidence of duodenal ulcer in premenopausal women than age-matched men.

Ionic selectivity of chloride ion symport in mechanisms controlling resting potential and osmotic homeostasis in earthworm somatic muscle cells

Replacement of Cl(-) for Br(-) in bathing solution did not reduce resting potential and had no effect on modulation of transmembrane potential in hyper- and hypoosmotic solutions. Under these conditions, baclofen, an agonist of GABAergic B-type receptors, failed to activate Na(+)/K(+)-pump in earthworm somatic muscle cells. It was hypothesized that the contribution of Cl(-) symport to osmotic homeostasis is not highly selective in respect to replacement of Cl(-) to Br(-) ions, whereas in case of activation of electrogenic ion pumps, this replacement is equivalent to removal of Cl(-) ions from the bathing solution.

A glia–neuron alanine/ammonium shuttle is central to energy metabolism in bee retina

It has been proposed that glial cells may supply carbon fuel to neurons and also that there are fluxes of ammonium from neurons to glia. We have investigated both these proposals in Apis retinal slices, in which virtually all the mitochondria are in the photoreceptor neurons. Normally the superfusate contained no substrate of energy metabolism; addition of glucose or alanine did not increase oxygen consumption (QO2), confirming that the neurons received adequate substrate from glycogen in the glia. 1,4-Dideoxy-1,4-imino-D-arabinitol (DAB, 100 microm), an inhibitor of glycogen phosphorylase, progressively decreased QO2. This decrease was reversed by alanine but not glucose. Ammonium-sensitive microelectrodes did not detect significant extracellular [NH(4)(+)] ([NH(4)(+)](e)) in slices superfused with normal superfusate. Removal of Cl(-), necessary for cotransport of NH(4)(+) into the glia, increased [NH(4)(+)](e) so that at the end of 2 min photostimulation mean [NH(4)(+)](e) was 0.442 mM (S.E.M. = 0.082 mM, n = 16). In 0 Cl(-), [NH(4)(+)](e) was reduced by 2-(methylamino)isobutyrate (MeAIB) an inhibitor of alanine transport. MeAIB also blocked oxidation of alanine in the presence of DAB, but did not decrease QO2 in normal superfusate. Lactate (l and d) and pyruvate (but not glucose) increased QO2 in DAB and decreased [NH(4)(+)](e) in 0 Cl(-). These results strengthen the evidence that in superfused retinal slices, glucose is metabolized exclusively in the glia, which supply alanine to the neurons, and that ammonium returns to the glia. They also show that another fuel (perhaps lactate) can be supplied by the glia to the neurons.

Na+,K+,2Cl− Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects

Adult primary afferent neurons are depolarized by GABA throughout their entire surface, including their somata located in dorsal root ganglia (DRG). Primary afferent depolarization (PAD) mediated by GABA released from spinal interneurons determines presynaptic inhibition, a key mechanism in somatosensory processing. The depolarization is due to Cl(-) efflux through GABA(A) channels; the outward Cl(-) gradient is generated by a Na+,K+,2Cl(-) cotransporter (NKCC) as first established in amphibians. Using fluorescence imaging microscopy we measured [Cl(-)]i and cell water volume (CWV) in dissociated rat DRG cells (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and calcein, respectively. Basal [Cl(-)]i was 44.2 +/- 1.2 mM (mean +/- SE), Cl(-) equilibrium potential (E Cl) was -27.0 +/- 0.7 mV (n = 75). This [Cl(-)]i is about four times higher than electrochemical equilibrium. On isosmotic removal of external Cl(-), cells lost Cl(-) and shrank. On returning to control solution, cells reaccumulated Cl(-) and recovered CWV. Cl(-) reaccumulation had Na+-dependent (SDC) and Na+-independent (SIC) components. The SIC stabilized at [Cl(-)]i = 13.2 +/- 1.2 mM, suggesting that it was passive (E(Cl) = -60.5 +/- 3 mV). Bumetanide blocked CWV recovery and most (65%) of the SDC (IC50 = 5.7 microM), indicating that both were mediated by NKCC. Active Cl(-) uptake fell with increasing [Cl(-)]i and became negligible when [Cl(-)]i reached basal levels. The kinetics of active Cl(-) uptake suggests a negative feedback system in which intracellular Cl(-)regulates its own influx thereby keeping [Cl(-)]i constant, above electrochemical equilibrium but below the value that would attain if NKCC reached thermodynamic equilibrium.

The Chlorine Isotope Composition of Earth's Mantle

Chlorine stable isotope compositions (delta37Cl) of 22 mid-ocean ridge basalts (MORBs) correlate with Cl content. The high-delta37Cl, Cl-rich basalts are highly contaminated by Cl-rich materials (seawater, brines, or altered rocks). The low-delta37Cl, Cl-poor basalts approach the composition of uncontaminated, mantle-derived magmas. Thus, most or all oceanic lavas are contaminated to some extent during their emplacement. MORB-source mantle has delta37Cl </= -1.6 per mil (per thousand), which is significantly lower than that of surface reservoirs (approximately 0 per thousand). This isotopic difference between the surface and deep Earth results from net Cl isotopic fractionation (associated with removal of Cl from the mantle and its return by subduction over Earth history) and/or the addition (to external reservoirs) of a late volatile supply that is 37Cl-enriched.

Steam reforming of liquid hydrocarbon fuels for micro-fuel cells. Pre-reforming of model jet fuels over supported metal catalysts

The present work deals with pre-reforming of logistic hydrocarbon fuel (jet fuel) as a part of an integrated approach to developing an on-board fuel reformer for use in a micro-solid-oxide fuel cell system. The purpose of doing pre-reforming is to ensure carbon-free reformulation of JP-8 jet fuel into hydrogen and carbon monoxide for use in a micro-solid-oxide fuel cell. Several model jet fuels have been tested for the pre-reforming at low temperature (450–550 °C) in a lab-scale reforming reactor. Proper temperature control and pre-mixing of feed fuels and steam have been found to be important for the prevention of coke formation prior to pre-reforming. Both noble metal and base-metal catalysts have been prepared and tested. As compared with an Al2O3-supported Ni catalyst, supported Rh catalysts show not only high activity but also high resistance to deactivation due to carbon formation. Removal of residual Cl− from Rh/CeO2–Al2O3 improves the metal dispersion and the pre-reforming activity. The reformates from the current pre-reformer contain mainly CH4, CO, H2, in which CH4 can be further converted to H2 and CO by subsequent main-reforming.

Rutaecarpine Induces Chloride Secretion across Rat Isolated Distal Colon

The present study evaluated the effect of rutaecarpine (Rut) on Cl(-) secretion across rat distal colonic mucosa. Basolateral application of Rut elicited an increase in short-circuit current (I(SC)) response in a concentration-dependent manner. Evidence that Rut-stimulated I(SC) was due to Cl(-) secretion is based on 1) inhibition of current by bumetanide; 2) Cl(-) channel blockers diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide; and 3) removal of Cl(-) ions in bath solution. Determination of neurogenic blockers on Rut-induced I(SC) indicated that pretreatment of tissues with tetrodotoxin or indomethacin, but not atropine or hexamethonium, inhibited Rut-induced response. Treatment with Rut led to release and synthesis of prostaglandin E(2) in rat colonic mucosa. Rut-stimulated I(SC) was markedly reduced by pretreatment with MDL-12,330A [cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, bisindolylmaleimide, and thapsigargin. Elimination of the extracellular Ca(2+) also did not alter Rut response. Rut treatment resulted in the increase in intracellular cAMP levels and the activation of protein kinase A. Depolarizing the basolateral membrane with high K(+) showed that Rut-stimulated apical Cl(-) current was largely prevented by cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors. Permeabilizing apical membrane with nystatin revealed that Rut-stimulated basolateral K(+) current was specifically inhibited by Ba(2+) ions and chromanol 293B. The evidence derived from present study suggests that Rut-stimulated Cl(-) secretion is mediated by generation of endogenous prostaglandin E(2) and that it also involves the stimulation of cAMP and protein kinase A pathways, which subsequently lead to the activation of apical Cl(-) channels, mostly the CFTR and basolateral cAMP-dependent K(+) channels.

Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl− secretion in HT‐29 human colon cancer cells

TSPO (translocator protein), known previously as PBR (peripheral-type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over-expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized. We investigated the expression and function of TSPO in the human colon cancer cells HT-29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam-binding inhibitor, in the cytosol. Radioligand binding studies using the specific high-affinity drug ligand [(3)H]PK 11195 and membrane fraction demonstrated saturable binding, with K(d) and B(max) values of 13.5+/-1.5 nM and 10.1+/-1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose-dependent rise in intracellular [Ca(2+)], which was unaffected by extracellular Ca(2+), but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT-29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca(2+)-dependent transepithelial Cl(-) secretion. This secretion was inhibited: (i) after removal of extracellular Cl(-); (ii) by apical addition of the Cl(-) channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate]; and (iii) by basolateral addition of the Na(+)-K(+)-2Cl(-) co-transporter inhibitor bumetanide. Furthermore, the intracellular Ca(2+) chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195-induced Cl(-) secretion. These findings indicate that TSPO is located in mitochondrial membranes of HT-29 and reveal that its activation induces a rise in cytosolic Ca(2+), leading to the stimulation of Cl(-) secretion.

Enhanced Formation of a HCO−3 Transport Metabolon in Exocrine Cells of Nhe1–/– Mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.