Cl- transport by gastric mucosa: cellular Cl- activity and membrane permeability

The mechanism of Cl- secretion in the isolated, resting (i.e. cimetidine-treated) gastric mucosa of Necturus has been investigated with radioisotopic and electrophysiological techniques. Measurement of transepithelial 36Cl- fluxes (mucosal to serosal (M leads to S), Jms Cl-; S leads to M, Jsm Cl-) during control conditions show that at open circuit, when the transepithelial potential difference psi ms = 20 mV (S ground), Jms Cl- = Jsm Cl-, i.e. Jnet Cl- = 0, but during short-circuit current conditions Jnet Cl- = I sc = 2 mu equiv cm-2 h. Experiments with low [Cl-] solutions indicate that Cl- exchange diffusion does not contribute significantly to either Jms Cl- or Jsm Cl-. Double-barrelled, Cl- -selective microelectrodes showed that in open circuit, the cellular (C) chemical potential for Cl-, psi c Cl- = 31 mV (apparent [Cl-] = 29 mM), the electrical potential across the M membrane, psi m = -34 mV (mucosa ground) while that across the S membrane, psi s = -52 mV (serosa ground). During short-circuit current conditions, psi m = psi s = -49 mV and [Cl-]c = 30 mM. The permeability of the M membrane to Cl- (Pm Cl-) was calculated both from the tracer experiments and the electrode measurements by using the constant-field equation. Short-term (45 s) uptake of 36Cl- at [Cl]m = 96 mM during short circuit conditions gave Pm Cl- = 2.6 x 10(-5) cm s-1. Measurement of [Cl-]c by means of the electrodes when [Cl-]m was changed from 96 to 2 mM or from 2 to 96 mM gave Pm Cl- = 2.9-5.7 x 10(-5) cm s-1. Our results indicate that during open circuit conditions Cl- is accumulated across the S membrane into gastric cells in an energy-requiring step, but since Jnet Cl- = 0, Cl- must leak back into the S solution at a rate equal to the entry rate. When the tissue is short-circuited, Cl- secretion occurs (Jnet Cl- = Isc) owing to the same energy-requiring accumulation of Cl- by the cells and a passive (apparently electrodiffusive) movement across the mucosal membrane.     

Molecular Physiology of an Extra-renal Cl- Uptake Mechanism for Body Fluid Cl- Homeostasis

The development of an ion regulatory mechanism for body fluid homeostasis was an important trait for vertebrates during the evolution from aquatic to terrestrial life. The homeostatic mechanism of Cl^- in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Unlike in mammals, in which the kidney plays a central role, in most fish species, the gill is responsible for the maintenance of Cl^- homeostasis via Cl^- transport uptake mechanisms. Previous studies in zebrafish identified Na⁺-Cl^- cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl^- uptake, similar to distal convoluted tubular cells in mammalian kidney. However, the mechanism by which basolateral ions exit from NCC cells is still unclear.Of the in situ hybridization signals of twelve members of the clc Cl^- channel family, only that of clc-2c exhibited an ionocyte pattern in the gill and embryonic skin. Double in situ hybridization/immunocytochemistry confirmed colocalization of apical NCC2b with basolateral CLC-2c. Acclimation to a low Cl^- environment increased mRNA expression of both clc-2c and ncc2b, and also the protein expression of CLC-2c in embryos and adult gills. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl^- content in zebrafish embryos, a phenotype similar to that of ncc2b mutants; this finding suggests a role for CLC-2c in Cl^- uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl^- homeostasis. Further comparative genomic and phylogenetic analyses revealed that zebrafish CLC-2c is a fish-specific isoform that diverged from a kidney-predominant homologue, in the same manner as NCC2b and its counterparts (NCCs).Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl^- uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl^- uptake during the evolution of vertebrates in an aquatic environment.     

Control of Plasma Membrane Cl- Fluxes in Chara corallina by External Cl- and Light

The efflux of Cl^– at the plasma membrane of Chara wasstudied in relation to two treatments known to affect the flux:that of removal of external Cl^– and of light. It is shownthat although removal of external Cl^– results in a rapidreduction in Cl^– efflux (consistent with a direct effectof external Cl^– on the transport system) the magnitudeof this reduction in the dark is greater than the measured darkinflux. Therefore, in the dark at least, it is proposed that1:1 exchange diffusion cannot account for the trans-stimulationof efflux by external Cl^–. Light induces an inhibition of efflux and a concomitant stimulationof Cl^– influx at 20 °C, but at 10 °C the responsesto light of the two fluxes can be separated temporally. It istherefore suggested that the fluxes are not reciprocally dependenton the same factor which mediates in the light response. Furtherconsiderations show that it is unlikely that the cytoplasmicCl^– concentration mediates in the light response of eitherflux, but that changes in cytoplasmic pH may do so.     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

Electrogenic Cl- pump in Acetabularia

Measurements of this transmembrane potential difference (V) under various conditions have demonstrated the operation of an electrogenic Cl- pump in the outer plasma membrane (plasmalemma) of the unicellular marine alga Acetabularia. In preparations of partly purified membranes (containing plasmalemma), there is Cl- stimulated, N,N'-dicyclohexylcarbodiimide-insensitive, vanadate-sensitive ATPase activity with a pH optimum around pH 6.5. These properties are consistent with the assumption that the electrogenic Cl- pump is an ATPase. In order to investigate electrical details of the "Mitchellian" type of charge-translocating enzyme, steady-state current-voltage curves of the electrogenic pump (Ip(V)) were measured in vivo under dark and light conditions and analysed by two-state reaction kinetic model. This model with the resulting parameters predicts V-sensitive, undirectional Cl- effluxes through the pump. The predictions of this model agree with the experimental results. Green light causes a fast decrease of V, which is explained as a disturbance of the pump cycle. Relaxation studies on this effect and reaction kinetic analysis of Ip(V) under different external Cl- concentrations are used to develop a consistent three-state model of the pump that includes the order of and absolute rate constants of individual reactions, states of charge, stoichiometry, voltage-sensitivity and density of the pump molecules in the membrane.     

Inhibition of protein synthesis by Cl-

Optimum K+ concentration for protein synthesis in four eukaryotic cell-free systems is obtained with 70 to 80 mM added KCl or with 110 to 150 mM added K(OAc). The different K+ optima are due to inhibition of protein synthesis by Cl- at concentrations higher than those present in the cytoplasm of eukaryotic cells. Initiation of protein synthesis is severely inhibited with 150 mM added KCl. This inhibition results from an impairment of mRNA binding to ribosomes. The binding of initiator Met-tRNAt, however, is only slightly inhibited by 150 mM KCl.     

Swelling activated Cl- channels in microglia

Microglia have a swelling-activated Cl- current (which we call IClswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of IClswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I->Br->Cl- (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTP?S or GDP?S, or physiological levels of internal Mg2+. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. IClswell was reversibly inhibited by known Cl- channel blockers (NPPB, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na+/K+/Cl- symport inhibitor, bumetanide. Together, these results confirm the similarity of IClswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.     

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.     

Cl- Fluxes during Osmoregulation in Chara

Measurements of Cl^– influx in cells of Chara corallinashow that control of this flux contributes to the ability ofthis cell to regulate its osmotic pressure. Transcellular osmosiswas used to generate cell fragments with abnormally high ₁,(H-cells), and with abnormally low ₁, (L-cells). Plasmalemmainflux (_oc) was very high in L-cells, and markedly reduced inH-cells. Influx was not affected by the presence of sucrosein the pond water and the consequent reduction in turgor. InH-cells the chloride flux from cytoplasm to vacuole (_cv) wasalso strongly inhibited. It is suggested that control of Cl^–fluxes at both plasmalemma and tonoplast is involved in osmoregulationin these cells. Key words: Chara corallina, osmoregulation, Cl^– flux     

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.     

Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor

When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.     

An ATP-driven Cl- pump in the brain

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.     

Dipeptide-induced Cl- secretion in proximal tubule cells

During a survey of dipeptides that might be transported by therenal PEPT2 transporter in proximal tubule cells, we discovered thatacidic dipeptides could stimulate transient secretory anion current andconductance increases in intact cell monolayers. The stimulatory effectof acidic dipeptides was observed in several proximal tubule cell linesthat have been recently developed by immortalization of early proximaltubule primary cultures from the Wistar-Kyoto and spontaneouslyhypertensive rat strains and humans, suggesting that this phenomenon isa characteristic of proximal tubule cells. The electrical currentinduced in intact monolayers by Ala-Asp, a representative of theseacidic dipeptides, must representCl secretion rather thanNa⁺ orH⁺ absorption, because1) it wasNa⁺ independent,2) it showed a pH dependencedifferent from that of the PEPT2 cotransporter, and3) it correlated with anAla-Asp-induced increase inCl conductance of theapical membrane in basolaterally amphotericin B-permeabilizedmonolayers. The secretory current could be inhibited by stilbenedisulfonates, but not diphenylamine-2-carboxylates, suggesting anon-cystic fibrosis transmembrane conductance regulator type ofCl conductance. The effectof Ala-Asp was dose dependent, with an apparent 50% effectiveconcentration of ~1 mM. Ala-Asp also produced intracellularacidification, suggesting that acidic dipeptides are also substratesfor an H⁺-peptide cotransporter.

     

Cl- sensitive biosensor used electrolyte-solution-gate diamond FETs

We have investigated the electrolyte-solution-gate field effect transisitors (SGFETs) used hydrogen terminated (H-terminated) or partially oxygen terminated (O-terminated) polycrystalline diamond surface in the Cl- and Br- ionic solutions. The H-terminated channel SGFETs are insensitive to pH values in electrolyte solutions. The threshold voltages of the diamond SGFETs shift according to the density of Cl- and Br- ions about 30 mV/decade. One of the attractive biomedical applications for the Cl- sensitive SGFETs is the detection of chloride density in blood or in sweat especially in the case of cystic fibrosis. The sensitivities of Cl- and Br- ions have been lost on the partially O-terminated diamond surface. These phenomena can be explained by the polarity of surface change on the H-terminated and the O-terminated surface.