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Impressive work has been completed in recent decades on the transmembrane anion transport capability of small synthetic transporters from many different structural classes. However, very few predicting models have been proposed for the fast screening of compound libraries before spending time and resources on the laboratory bench for their synthesis. In this work, a new approach is presented which aims at describing the transport process by taking all the steps into explicit consideration, and includes all possible experiment-derived parameters. The algorithm is able to simulate the macroscopic experiments performed with lipid vesicles to assess the ion-transport ability of the synthetic transporters following a non-electrogenic uniport mechanism. While keeping calculation time affordable, the final goal is the curve-fitting of real experimental data-so, to obtain both an analysis and a predictive tool. The role and the relative weight of the different parameters is discussed and the agreement with the literature is shown by using the simulations of a virtual benchmark case. The fitting of real experimental curves is also shown for two transporters of different structural type.

Durability improvement is always important for steel–concrete structures exposed to chloride salt environment. The present research investigated the influence of a novel nano-precursor inhibiting material (NPI), organic carboxylic acid ammonium salt, on the mechanical and transport properties of concrete. The NPI caused a slight reduction in the strength of concrete at later ages. NPI significantly decreased water absorption and slowed down the speed of water absorption of concrete. In addition, the NPI decreased the charge passed and the chloride migration coefficient, and the results of the natural chloride diffusion showed that the NPI decreased the chloride concentration and the chloride diffusion coefficient. The NPI effectively improved the resistance of chloride penetration into testing concrete. The improvement in the impermeability of concrete was ascribed to the incorporation with the NPI, which resulted in increasing the contact angle of cement pastes. The contact angle went up from 17.8° to 85.8° for 0% and 1.2% NPI, respectively, and cement pastes became less hydrophilic. Some small pore throats were unconnected. Besides, the NPI also optimized the pore size distribution of hardened cement paste. [ABSTRACT FROM AUTHOR]

• The mechanism and measurement for chloride transport of AAC are reviewed. • Relation between chloride binding capacity and mix proportion of AAM are studied. • The characterization of steel corrosion process in AAC are discussed. • Effects of steel corrosion on microstructure and properties of AAC are provided. Reinforcement corrosion reduces the service life of concrete buildings and infrastructures, resulting in significant worldwide economic losses every year. Alkali-activated materials (AAMs) has been widely considered as a type of greener alternative to Portland cement due to its comparable mechanical properties and much lower energy and resource consumption and emissions. The high alkalinity of pore solution and unique microstructure endow AAM a very promising materials to manufacture structures with high resistance against Cl- penetration. This paper reviews the recent progress of studies on steel corrosion behavior of alkali-activated concretes (AACs), the transport of chloride and chloride binding in AACs are highlighted. The purpose of this paper is to demonstrate the steel corrosion resistance of AACs considering the chloride penetration process and chloride binding in comparison to Portland cement concrete, the advantages and disadvantages of AACs are also discussed. [ABSTRACT FROM AUTHOR]

• Cl ion diffusion is fully coupled with heat transfer, mass transport and aging. • Pore solution chemistry, concentration, and pore structure aging are considered. • Thermal effects on diffusivity and Cl binding capacity are included. • Cl profile is predicted with varying time, temperature, concentration, and humidity. • A critical balance between modeled physics and model robustness is achieved. Chloride-induced corrosion of steel reinforcement is one of the major long-term deterioration mechanisms for reinforced concrete infrastructures. Chloride transport through cement-based materials is a complex chemo-physical process involving ionic diffusion in concentrated solution, pore structure, chemistry, membrane permeability of the matrix, cracking, and the variation of the internal and external environmental conditions. Although in the literature there are plenty of both simplistic phenomenological models and sophisticated models, in this study, a new model is developed taking aim at capturing the fundamental physics and, at the same time, having a formulation as simple as possible that it can be effectively calibrated and validated using available limited experimental data. The model couples the ionic diffusion process with the concrete micro-structure evolution due to continued hydration accounting for hygro-thermal variations and their effects on both the diffusion and hydration processes. The formulation is implemented in a semi-discrete conduit transport network that mimics the internal heterogeneity of the cementitious material by connecting the matrix space between coarse aggregate pieces. This allows the model to replicate naturally the meso-scale tortuosity effect which is an important feature towards representing realistically the heterogeneity-induced variations of chloride concentration within the concrete. The limited model parameters are carefully calibrated and the formulation is validated by simulating multiple experiments ranging from diffusion through pastes to large concrete cylinders. The results of numerical simulations show the ability of the model to describe spatial and temporal evolution of the chloride concentration within the samples under varying chloride concentrations and temperature boundary conditions within both saturated and unsaturated concrete. [ABSTRACT FROM AUTHOR]

• Early CO 2 curing enhances the water infiltration and chloride permeability resistance of cement mortar. • Early CO 2 curing decreases the chloride binding capacity of cement paste. • The addition of fly ash reduces the chloride binding of early CO 2 cured cement paste. Early CO 2 curing has attracted a lot of attentions due to sequestration of carbon dioxide and rapid strength development for cement-based materials. This study investigates the chloride transport and binding properties of fly ash-blended cement-based materials with and without early CO 2 curing. A comparison is made between the specimens exposed to early CO 2 curing followed by subsequent water curing and those continuously cured in water. Results show that early CO 2 curing decreases the water sorption and chloride migration coefficient of cement mortar. The replacement of cement with fly ash decreases the strength and permeability of mortar regardless of the curing regime. CO 2 cured cement pastes show lower chloride binding capacity than water cured cement pastes after immersion in 0.1 M, 0.5 M and 1 M sodium chloride solutions due to the consumption of calcium hydroxide, presence of carbonates as well as lower contents of C-S-H gel. The presence of fly ash increases the contents of bound chloride in water cured cement pastes but decreases them in CO 2 cured cement pastes. [ABSTRACT FROM AUTHOR]

This paper draws attention to an important unsolved problem in our understanding of the processes of ion transfer to plant cell vacuoles, which emerged from consideration of unexpected features of the measured kinetics of the transfer of tracer chloride from the outside solution to the vacuole of Characean cells. Over labelling times of 1-2 h the kinetics of vacuolar transfer of chloride are consistent with the two-phase model of the cell, of cytoplasm and vacuole in series, and the cytoplasmic exchange is characterized by a single rate constant for exchange. However, for shorter times the vacuolar transfer is biphasic, and there appear to be two components of vacuolar transfer, a fast component important at short times, and a slow component whose contribution increases with time. Half-cell labelling experiments, measuring the rate of transfer of tracer in the flowing cytoplasm, make it clear that the ground cytoplasm must be part of the slow phase; the view that the slow phase represents slowly exchanging organelles within the cytoplasm is not tenable. It is argued that the fast component is most likely to arise by transfer to the vacuole associated with action potentials on cutting the cell for analysis. There are two unexpected features of the kinetics of the slow component of vacuolar transfer. The first is the complete lack of discrimination between Cl- and Br- in the vacuolar transfer, in spite of strong discrimination in favour of Cl- at the plasmalemma. The vacuolar transfer is uphill, and if it arises from single ion transfer through an appropriate transporter, such lack of discrimination is unexpected. The property is even more surprising in the light of the second peculiarity of the kinetics, the relation between vacuolar transfer and influx, whether influx is manipulated by metabolic conditions, inhibitors or external concentration. Increased Cl- influx is associated with increased transfer of both Cl- and Br- to the vacuole. It is difficult to explain this in terms of independent processes of single ion transfer at the plasmalemma and tonoplast. An explanation in terms of transfer of salt-filled vesicles from cytoplasm to vacuole, by budding from the endoplasmic reticulum at a rate related to the influx to the cell, may be more consistent with the observed kinetics. The question of whether vesicle trafficking plays a role in vacuolar ion accumulation deserves to be re-examined by modern techniques. Finally some evidence is considered that might suggest (even more speculatively) that vesicle trafficking may also play a role in the dramatic stimulus-triggered losses of vacuolar solute in certain types of cell, such as stomatal guard cells and pulvinar motor cells. Again the question deserves to be investigated. [ABSTRACT FROM PUBLISHER]

The colon cancer cell line HT29 is a useful model to study intestinal chloride secretion. These cells have both cAMP-activated and calcium-activated chloride channels. Changes in elemental content of the cells after stimulation with agonists were determined by X-ray microanalysis in the scanning or scanning transmission electron microscope. Exposure of HT29 cells to pituitary adenylate cyclase activating polypeptide-27 (PACAP) caused a transient decrease in the cellular Cl and K concentrations, indicating (net) efflux of chloride. The effect of PACAP is inhibited by somatostatin, which is known to inhibit cAMP-activated as well as calcium-activated chloride secretion and by U-73122, an inhibitor of phospholipase C. Alloxan, an inhibitor of adenylate cyclase, did not significantly affect the PACAP-induced loss of chloride. The calcium-chelating agent EGTA inhibited the PACAP-induced loss of chloride, indicating the need for extracellular calcium ions. Also vasointestinal polypeptide (VIP) caused a decrease of the cellular chloride concentration in HT29 cells. VIP-induced loss of chloride could be inhibited by pre-treating the cells with somatostatin or UK14,304, an alpha-2 adrenergic agonist that has been shown previously to inhibit purinergically activated chloride efflux. Our results indicate that there is cross-talk between the cAMP- and the calcium-activated pathways for chloride secretion in HT29 cells. [ABSTRACT FROM AUTHOR]

Blue light is one of the most important environmental signals regulating monovalent anion transport in plant cells. In the unicellular freshwater chlorophyte Monoraphidium braunii, blue light is essential for the activation of HCO3-, NO3-, NO2- and Cl-transport systems. These anions are taken up when blue light is present but the uptake ceases when this radiation is suppressed, indicating that blue light is a switch signal for the monovalent anion transport system(s) of this alga. Similar results have been obtained in other green algae and higher plants. The action spectra for the uptake of NO3- and Cl- in M. braunii are very similar and resemble the absorption spectra of flavins or a combination of flavins and pterins. It is proposed that both anions share the same transport system(s). The uptake of monovalent anions consists of a cotransport with H+, thus producing alkalinization of the external medium. The time between the onset of blue light and the beginning of alkalinization can be as short as 2 s. Taken together, the results suggest that the photoreceptor mediating the blue light activation of monovalent anion uptake in this green alga is a plasma membrane-bound flavoprotein. [ABSTRACT FROM AUTHOR]

Chloride transport in sheath and blade tissue and the cellular distribution of Cl- were investigated in an attempt to determine the physiological basis of the preferential accumulation of Cl- in sheaths of salt-stressed sorghum (Sorghum bicolor L.). Import and export of 36Cl- in leaf sheaths and blades of intact sorghum were followed over a 2 week period. X-ray microanalysis of frozen-hydrated bulk tissue samples was used to determine the accumulation of Cl- and other elements in the vacuoles of sheath and blade cells. Sheath tissue accumulated Cl- despite a relatively high Cl- turnover rate. Chloride was shown to accumulate in most cell types of the sheath, particularly in adaxial epidermal cells. After an initial increase in the concentration of Cl-, blade tissue regulated Cl- levels within certain limits. Chloride levels in blades were greater in the abaxial and adaxial epidermal cells than in other cell types. The epidermal cells of blades accumulated Cl- to approximately the same concentration as sheath epidermal cells. The Cl- concentration in the photosynthetically active mesophyll and bundle sheath cells, however, remained low. Thus, the partitioning of Cl- previously observed in the leaves of salinized sorghum apparently results from the ability of bundle sheath and mesophyll cells to maintain concentrations of Cl- at lower levels than do epidermal cells. In addition, the relatively large sheath parenchyma cells tend to serve as reservoirs for the storage of Cl-. [ABSTRACT FROM AUTHOR]

The kinetics of the light-driven Cl- uptake pump of Synechococcus R-2 (PCC 7942) were investigated. Th kinetics of Cl- uptake were measured in BG-11 medium (pHo, 7.5; [K+]o, 0.35 mol m-3; [Na+]o, 18 mol m-3; [Cl-]o, 0.508 mol m-3) or modified media based on the above, Net 36Cl- fluxes (ϕCl-o,i) followed Michaelis-Menten kinetics and were stimulated by Na+ [18 mol m-3 Na+ BG-11 ϕCl-max = 3.29 ±0.60(49) nmol m-2S-1 versu Na+-free BG-11 ϕCl-max = 1.02±0.13 (54) nmol m-2s-1] but the Km was not significantly different in he presence or absence of Na+ at pHo 10; the Km was lower, but not affected by the presence or absence of Na+ [Km = 22.3±3.54 (20) mmol m-3]. Na+ is a non-competitive activator o net ϕcl-o,i High [K+]o (18 mol m-3) did not stimulate net ϕCl-o,i or change the Km in Na+-free medium. High [K+]o (18 mol m-3) added to Na+ BG-11 medium decreased net ϕCl-o,i[18 mol m-3 K+ BG-11; ϕCl-max = 2.50±0.32 (20) nmol m-2s-1 versus BG-11 medium; ϕCl-max = 3.35±0.56 (20) nmol m-2s-1] but did not affect the Km 55.8 ±8.00 (40) mmol m-3]. Na+-stimulation of net ϕCl-o,i followed Michaelis-Menten kinetics up to 2-5 mol m-3[Na+]o but higher concentrations were inhibitory. The Km for Na+-stimulation of net ϕCl-o,i[K½(Na+)] was different at 47 mmol m-3[Cl-]o (K½[Na+] = 123 ± 27 (37) mmol m-3]. Li+ was only about one-third as effective as Na+ in stimulating Cl- uptake but the activation constant was similar [½(Li+) = 88±46 (16) mmol m-3]. Br- was a competitive inhibitor of Cl- uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na+. The overall Ki was 297±23 (45) mmol m-3. The discrimination ratio of Cl- over Br-(δCl-/δBr-) was 6.38±0.92 (df = 147). Synechococcus has a single Na+-stimulated Cl- pump because the Km of the Cl- transporter and its discrimination between Cl- transporter and its discrimination between Cl- and Br- are not significantly different in the presence and absence of Na+. The Cl- pump is probably driven by ATP. [ABSTRACT FROM AUTHOR]

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely in- activate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues. [ABSTRACT FROM AUTHOR]

The characteristics of Cl transport in isolated tonoplast vesicles from red-beet ( Beta vulgaris L.) storage tissue have been investigated using the Cl-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K and valinomycin, increased the initial rate of Cl transport, demonstrating that Cl could be electrically driven into the vesicles. Chloride influx was unaffected by SO, but was competitively blocked by NO, indicating that both Cl and NOmay be transported by the same porter. In some preparations, increases in free-Ca concentration from 10 to 10 mol·dm caused a significant decrease in Cl influx, which may indicate that cytosolic Ca concentration has a role in controlling Cl fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles. [ABSTRACT FROM AUTHOR]

A fluorescence method for the direct measurement of Cl transport in isolated tonoplast vesicles is described. This technique utilises the Cl-sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl, Br, I, SCN, NOand tetraphenylborate but not by NO, SO, iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet ( Beta vulgaris L.) storage roots or from barley ( Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl transport in tonoplast vesicles, how these were overcome and some characteristics of Cl transport at the tonoplast as measured by the probe. [ABSTRACT FROM AUTHOR]

Active Cl uptake by Chlorella fusca was examined by using Cl as a label. Under light/air conditions chloride influx from a 2.4·10 M solution was 4.0±0.04 nmol ms. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl uptake had a broad optimum around pH 7 and showed saturation kinetics with a K of 1.25·10 M and a v of 7.0 nmol ms in light/air. Br inhibited Cl uptake strongly, J, ClO, SO, and NOhad no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide resulted in a good correlation between Cl uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level. The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl transport cannot proceed via secondary active H/Cl cotransport. In addition, 2H/Cl cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl/OH exchange pump is proposed. [ABSTRACT FROM AUTHOR]

A possible role of the charasome in terms of chloride transport into Chara corallina Klein ex. Willd., em. R.D.W. is examined. The branches of Chara contain the most charasome material and are shown to be very effective in acquiring Cl to support continued shoot growth. The early maturation of the branches, the rather large Cl fluxes into these cells, and their ability of translocate Cl to growing cells of the shoot indicate a special role of these branches in Cl accumulation. The structure of the charasome, with its extensive periplasmic space, appears especially suited as a site for H−Cl cotransport (influx). We show, by histochemical assay, that the charasomes of mature cells contain ATPase activity; such activity is absent in growing charasomes of very young cells. ATPase activity is also associated with the plasmodesmata of C. corallina. Charasome ATPase activity and Cl uptake are both inhibited by p-chloromercuribenzenesulfonic acid (1 mM) or diethylstibestrol (40 μM; 45 min). The anion transport inhibitor, 4,4-diisothiocyano-2,2-disulfonic acid stilbene (1 mM) had no effect on Cl transport and inhibited ATPase activity only when applied after chemical fixation of the cells. Results of an attempt to demonstrate the presence of Cl within the cytoplasmic tubules of the charasome, using a silver precipitation technique, proved difficult to interpret because of a reaction between the silver and a cellular substance produced in the light. [ABSTRACT FROM AUTHOR]

Methods are described for establishing primary monolayer cultures from chloride-secreting epithelial cells of the dogfish shark ( Squalus acanthias) rectal gland (SRG). After stimulation, cultures display exceptionally high rates (150 to 250 μA/cm) of transepithelial chloride secretion. Hormones and neurotransmitters which increase cytoplasmic cyclic AMP, cyclic GMP, calcium or phospholipid-derived second messengers are potent secretagogues. Cultures can be analyzed using a variety of morphologic, biochemical, and electrophysiologic methods. These characteristics make SRG cultures a powerful model for delineating the molecular basis of the transmembrane and intracellular signaling networks that stimulate or inhibit secondary active chloride transport in epithelia. Secondary active chloride transport occurs in a variety of epithelia, including those comprising both the proximal and distal segments of vertebrate nephrons. [ABSTRACT FROM AUTHOR]

The round window has been used as a route for introducing ototoxic substances into the inner ear in order to bypass barriers between the systemic circulation and the inner ear. We have used this method to administer locally sodium potassium-ATPase inhibitors and chloride transport inhibitors to the inner ear of the chinchilla. Drugs dissolved in saline solution were applied on the round window membrane. Endocochlear potential (EP) was recorded from the basal turn using the round window approach. The EP was not altered following application of saline solution as a control. Following application of ouabain (1 mM), the EP steadily declined. After vanadate (27 mM), the EP initially increased, and subsequently declined. Sanguiarine did not alter the EP. The loop diuretics furosemide and piretanide caused a marked decline in the EP after local application. However, the stilbene derivative DIDS did not alter the EP after topical application. These findings raise questions about whether the loop diuretics have any effect on chloride transport in the cochlea and make appear unlikely that active chloride transport contributes to the normal EP. [ABSTRACT FROM AUTHOR]

Chloride uptake by the cyanobacterium Anacystis nidulans at 38°C is energy dependent showing maximum rate (around 5.10 mol Clxml cell waterxmin) and accumulation (up to 160 fold) in light and air. The respective values in air and darkness were 40-70% lower. In the dark under N no uptake was found. Chloride transport had an optimum at pH 6.7 and a K of 2.10 M which was pH-independent. It was inhibited by carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide in the light and in the dark, and also to a lesser extent by valinomycin. 3-(3,4-dichlorophenyl)-1,1-dimethylurea in the light caused a moderate stimulation. To obtain information about the energy source of active chloride transport the action of the four inhibitors on membrane potential (determined through the distribution of triphenylmethylphosphonium) and ATP level (determined by the firefly method) was examined. It was found that a high negative membrane potential was unfavorable for chloride accumulation probably by stimulating passive efflux. On the other hand a good correlation between ATP level and chloride transport activity was obtained. Attempts to induce chloride uptake by sudden acidification of the external medium in presence of N,N′-dicyclohexyl-carbodiimide or during anaerobiosis were not successful. Two mechanisms of chloride uptake are discussed: [ABSTRACT FROM AUTHOR]

Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 µm). Short-circuit current (I) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed I equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 µm. Responses in I to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology. [ABSTRACT FROM AUTHOR]

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell. [ABSTRACT FROM AUTHOR]

We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE2, but that in the presence of 10(-5) M indomethacin both the conversion to PGE2 and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.U [ABSTRACT FROM AUTHOR]

The chloride conductance of the basolateral cell membrane of the Necturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl-. When extracellular Cl- activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl- conductance of the paracellular shunt but a low Cl- conductance of the cell membranes. Reduction of Cl- in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl- perfusion was abolished by exposure to HCO-3-free solutions and SITS treatment. In control conditions, intracellular Cl- activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl- in only the basolateral solution caused a decreased in intracellular Cl-. From an estimate of the net Cl- flux across the basolateral cell membrane and the electrochemical driving force, a Cl- conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl- conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl- by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl- transport across the basolateral cell membrane of the Necturus proximal tubule cell. [ABSTRACT FROM AUTHOR]

We examined the direct effects of isoproterenol (ISO) and L-norepinephrine (NE) on electrolyte transport in isolated rabbit cortical collecting tubules (CCT) perfused in vitro. The addition of either ISO (10(-6) M) or NE (10(-6) M) to the bath decreased transepithelial potential difference (PD), on average by 51 and 25%, respectively. These effects of ISO and NE were abolished by prior addition of the beta-adrenergic blocker, L-propranolol. ISO (10(-5) M) had no effect from lumen. Also, osmotic water permeability was not influenced by ISO. Ouabain and ISO had additive effects on PD. Elimination of chloride from both perfusate and bath, or addition of acetazolamide, abolished the effect of ISO on PD. Although isotopic sodium flux for lumen to bath was not influenced by ISO, chemical net chloride absorption increased from 1.1 +/- 0.4 to 2.7 +/- 0.6 peq . cm-1 . sec-1 (n = 8, p less than 0.005). In conclusion, both ISO and NE and capable of decreasing PD in rabbit CCT perfused in vitro. This effect is mediated by beta-adrenergic receptors and is accompanied by the increase in net chloride absorption. Although the mechanism responsible for this decrease in PD with ISO in unclear, active chloride absorption, active hydrogen secretion, or membrane chloride permeability changes may account for the effects of ISO. [ABSTRACT FROM AUTHOR]

Sodium and chloride influxes across the nonshort-circuited isolated skin of Rana esculenta were measured at widely varying external ionic concentrations. The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed. At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na/H; Cl/HCO) functioning at low external concentrations. At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components. The physiological significance of these results is discussed. [ABSTRACT FROM AUTHOR]

The ionic permeability of the basolateral membrane of Necturus gallbladder epithelium was studied with intracellular microelectrode techniques. After removal of most of the subepithelial tissue (to reduce unstirred layer thickness), impalements were performed from the serosal side, and ionic substitutions were made in the serosal solution while a microelectrode was kept in a cell. Thus, it was possible to obtain continuous (and reversible) records of transepithelial and cell membrane potentials and to measure intermittently the transepithelial resistance and the ratio of cell membrane resistances. From these data and the mean value of the equivalent resistance of the cell membranes in parallel (obtained from cable analysis in a different group of tissues), absolute cell membrane and shunt resistances and equivalent electromotive forces (emf's) were calculated. From the changes of basolateral membrane emf ( E) produced by the substitutions, the conductance ( G) and permeability ( P) of the membrane for K, Cl and Na were estimated. Potassium-for-sodium substitutions produced large reductions of both cell membrane potentials, of E, and of the resistance of the basolateral membrane ( R), indicating high G and P. Chloride substitution with isethionate or sulfate resulted in smaller changes of cell membrane potentials and E and in no significant change of R, indicating small but measurable values of G and P. Sodium substitutions with N-methyl- d-glucamine (NMDG) resulted in cell potential changes entirely attributable to the biionic potential produced in the shunt pathway ( P> P), and in no significant changes of P or E, indicating that G and P are undetectable. The question of the mechanism of Cl transport across the basolateral membrane was addressed by comparing the mean rate of transepithelial Cl transport ( J) and the predicted passive Cl flux across the basolateral membrane (from the membrane Cl conductance, potential, and Cl equilibrium potential). The conclusion is that only a very small fraction of the Cl flux across the basolateral membrane can be electrodiffusional. Since the paracellular Cl conductance is also too low to account for J, these results suggest the presence of a neutral mechanism of Cl extrusion from the cells. This could be a NaCl pump, a downhill KCl transport mechanism, or a Cl−HCO exchange mechanism. [ABSTRACT FROM AUTHOR]

The influence of four opioid antagonists on short circuit current (I), transepithelial potential difference (Pd) and tissue conductance (G) in the guinea pig colonic mucosa was investigated in vitro under both basal and PGE plus theophylline-stimulated conditions. The experiments aimed at identifying the opioid receptor type(s) endogenously activated to control chloride secretion. Under blockade of sodium-dependent I by amiloride (100 µmol/I), net anion secretion was regarded to equal the lumen-negative shift in I upon addition of 1 µmol/l PGE plus 100 gmol/l theophylline. It was significantly elevated by 100 nmol/l of the ϰ-selective antagonist nor-binaltorphimine (nor-BNI). This augmenting effect was totally abolished in amiloride-free buffer or by omission of chloride. 1 µmol/l TTX completely prevented the effect of both PGE plus theophylline and nor-BNI. Both the ϰ agonist U 69593 (10 nmol/l) and the calcium channel agonist Bay K 8644 (1 µmol/l) significantly depressed net anion secretion stimulated by PGE plus theophylline. Nor-BNI at 10 nmol/l prevented the suppressive effect of both Bay K 8644 and U 69593. This suggests release of endogenous opioids by the calcium channel agonist Bay K 8644 and competition between the ϰ agonist U 69593 and the ϰ antagonist nor-BNI. In contrast to the ϰ antagonist nor-BNI, the µ antagonist CTOP-NH at 100 nmol/l significantly impaired, while the µ-selective agonist DAGO at 0.2 nmol/l augmented, net anion secretion stimulated by PGE plus theophylline. The effect of CTOP-NHn2 was abolished in chloride-free buffer. The δ selective antagonist ICI 174.864 (up to 1 µmol/l) and the non-selective antagonist naloxone (up to 1 µmol/l) failed to influence stimulated net anion secretion. The failure of naloxone may be due to superimposition of the contrasting effects of µ and ϰ receptor blockade by a high naloxone concentration. The antagonist effect of nor-BNI considered above was obtained at 9.6 nmol/l potassium but was no longer observed when the potassium concentration in the bathing solution was reduced to 4.8 mmol/l. It is suggested that both elevated potassium concentration and calcium channel activation by Bay K 8644 release endogenous ϰ agonists in the guinea pig colonic mucosa under the present conditions. Since CTOP-NH impaired stimulated net anion secretion at both of the potassium concentrations, release of endogenous µ agonists and, hence, activation of µ opioid receptors seem to take place under both conditions. In conclusion, the present work demonstrates for the first time that activation of ϰ opioid receptors by endogenous opioid peptides impair, while activation of µ opioid receptors augment, stimulated chloride secretion in this tissue. [ABSTRACT FROM AUTHOR]

Internodal cells of Chara, grown in culture either at pH 5.7, 6.5 or 7.5, were studied to determine their chloride influx capability, the quantitative aspects of charasome morphology and the degree to which these two parameters could be correlated. In cells grown at pH 5.7 the charasomes were relatively small, were widely spaced on the plasma membrane, and contributed only a 0.6% increase to the surface area of the plasma membrane in the acid region of the cell. In contrast, the charasome membrane surface area of cells grown at pH 7.5 had increased × 19, the density of charasomes on the cell surface increased × 42, thus producing a × 3.57 increase in the acid region plasma membrane surface area. Chloride influx in cells grown at pH 7.5 was × 8.7-12.7 greater than in cells grown at pH 5.7. Cells that had been starved of chloride exhibited a × 2.4 average increase in the rate of chloride influx. Our observations establish the existence of a positive correlation between the rate of chloride influx and the increase in membrane surface area due to charasomes, although other factors, such as the effect of pH on transport-related enzymes, and the effect of charasome structure on chemical equilibria, may also be of importance. [ABSTRACT FROM AUTHOR]

The effect of total system pressure on the uniformity of epitaxial GaAs layers grown at 1023 K in the LP-VPE system has been studied with respect to layer thickness and dopant uptake. It was found that the uniformity of both of these quantities is distinctly improved when the hydrogen pressure is lowered from 3.3×10 to 6.5×l0 Pa. This behaviour is explained by the increase in gas phase diffusivity and the reduction in depletion of the gas phase during GaAs growth at reduced total pressure. [ABSTRACT FROM AUTHOR]

The mechanism of uphill Cl accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl-selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential ( V) was −39.2±0.4 mV and intracellular Cl activity ( A) was 34.6±0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na-free solution, A decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level of A in 5 min. In the presence of furosemide (1 mmol/l), Cl uptake induced by Na-readmission was inhibited by 44%. Superfusion with a K-free solution gradually decreased A until it was close to the equilibrium level after 75 min; superfusion with a high-K (29.5 mmol/l) solution increased A significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K-free to high-K and from high-K to K-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (−7 mmol/l) in A. These changes in A were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl uptake after cessation of acetylcholine (ACh 1 μmol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na−K−Cl cotransport. The additional inhibitory effect of DIDS to furosemide inhibition suggests an involvement of anion exchange transport, in parallel with the cotransport, in uphill Cl uptake into the cells. [ABSTRACT FROM AUTHOR]

Short-circuit current (SCC) aross isolated pelvic skin of the toad Bufo arenarum has been shown to be reflected by the algebraic sum of net sodium and chloride transport. After the animals had been maintained in tap water, amiloride - an apical sodium channel blockerled to a reversal of potential difference (rPD) across this preparation, to which corresponded a reversed short-circuit current (rSCC). Both rSCC and rPD were abolished by dermal treatment of skins with the metabolic inhibitor dinitrophenol, or by omission of chloride ion from the Ringer solution bathing both sides of the skin. There was a significant positive correlation between rSCC and isotopically determined net chloride transport after amiloride. An inhibitory action of amiloride on unidirectional chloride fluxes was detected, but only early after drug addition. rSCC was absent in skins of toads exposed to 110 mmol/l NaCl in tap water during 10 days. Together, our results suggest that amiloride addition -by inhibiting active sodium movement - can in certain conditions reveal the existence of an inward active chloride transport. [ABSTRACT FROM AUTHOR]

Fluphenazine (FPZ) and trifluoperazine (TFP) are phenothiazine derivatives commonly used as antipsychotic transquilizers. Their mechanism of action is incompletely understood. Epidermal addition of each drug promoted biphasic short-circuit current (SCC) changes across isolated pelvic skin of Bufo arenarum toads. By means of radiotracers fluxes, SCC was found to be given by the algebraic sum of net sodium and chloride transport. A readily stimulant effect was detected a low concentrations (from 1·10 mol/l up to 1·10 mol/l for FPZ, from 1·10 mol/l up to 3.2·10 mol/l for TFP) above which inhibition prevailed. Dermal FPZ also stimulated SCC. A higher concentration and time threshold were required. Epidermal 1·10 mol/l FPZ stimulation was partially reversible, with a diminished membrane resistance and enhancement of sodium influx, without alteration of sodium efflux or net chloride transport. It could be prevented by amiloride pretreatment, or diminished by dermal sodium removal. Variation of epidermal bulk pH from 5.8 to 8.7 demonstrated that ionized and nonionized molecules contribute to FPZ's effect. Our results suggest that SCC stimulation elicited by FPZ and TFP may be a consequence of direct or indirect modifications on apical sodium conductance. [ABSTRACT FROM AUTHOR]

Cells were isolated from the thick ascending limb of Henle's loop of rabbit kidney outer medulla and a plasma membrane fraction was prepared by differential centrifugation. Sodium and rubidium uptake into the plasma membrane vesicles were determined by a rapid filtration technique. In the presence of a 100 mM KCl gradient and 0.5 mM sodium the vesicles took up 252±82 pmoles sodium/mg protein x 15s; 52% of the uptake was dependent on the presence of chloride or inhibited by 10 M bumetanide. If KCl was stepwise replaced by choline chloride, sodium uptake decreased in the absence of bumetanide but was only insignificantly altered in the presence of bumetanide. Potassium exerted a halfmaximum stimulation at 22.3±9.7 mM. In tracer exchange experiments under zero salt gradient conditions, sodium uptake was also strongly reduced in the absence of potassium. Rubidium uptake into the same membrane fraction was highest in the presence of a NaCl gradient, and decreased 41% when sodium was replaced by choline or when 10 M bumetanide was present. Replacement of sodium chloride by sodium nitrate also inhibited rubidium uptake. When the sodium, chloride and potassium dependence of the bumetanide sensitive sodium uptake was investigated in more detail, Hill coefficients for sodium of 1.0±0.03, for chloride of 1.8±0.2 and for potassium of 0.98±0.03 were obtained. These results are consistent with the presence of a Na−Cl−K cotransport system in the medullary thick ascending limb of Henle's loop which may operate with a stoichiometry of 1/2/1. [ABSTRACT FROM AUTHOR]

Isolated cells were prepared from the medullary thick ascending limb of Henle's loop (TALH) and the response of oxygen consumption was correlated with the active chloride transport system found in these cells. Oxygen consumption was 31.6 μl O/mg protein·h and inhibited 50% by the absence of either sodium or chloride in the incubation medium. The absence of both sodium and chloride produced no further inhibition of oxygen consumption. Ouabain (10 M) inhibited oxygen consumption by 50% and the inhibitory effect depended on the presence of both sodium and chloride in the incubation medium. Further, furosemide inhibited oxygen consumption by a maximum of 50% at 10 M and also had no inhibitory effect if either sodium or chloride were absent. Furosemide had no effect on the Na, K-ATPase activity or ATP levels of the TALH cells. Thus, the data suggest that 50% of the oxygen consumption of the TALH cells is related to the movement of sodium and chloride into the cell and that the ions may be transported in a coupled manner. In addition the effect of various diuretics on oxygen consumption in the isolated TALH cells was tested. The diuretics could be grouped in three categories: (1) highly effective in inhibiting chloride-dependent oxygen consumption with an apparent inhibitory constant ( K) of around 10 M, including the diuretics furosemide, bumetanide, ethacrynic acid-cysteine and piretanide, (2) diuretics which were less effective in inhibiting oxygen consumption with an apparent K of around 10 M, HOE 740 and ethacrynic acid, and (3) diuretics which were ineffective in inhibiting chloride-dependent oxygen consumption, amiloride and hydrochlorothiazide. [ABSTRACT FROM AUTHOR]

A membrane fraction, rich in basal-lateral plasma membranes, was prepared from the rectal gland of the spiny dogfish, Squalus acanthias, and the uptake ofNa into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of a chloride gradient directed into the vesicles; it was strikingly reduced when chloride was replaced with nitrate and was even slower with gluconate. If the membrane vesicles were pre-equilibrated with potassium chloride or potassium nitrate plus valinomycin, to minimize any electrical driving forces on sodium movement, the uptake of sodium was still greatest in the presence of chloride and remarkably decreased in the presence of nitrate. Furosemide, 10 and 10 M, decreased sodium uptake into the vesicles in a dose dependent manner only in the presence of chloride. Furthermore, saturation of sodium uptake by increasing sodium chloride concentrations was observed. The above results provide direct evidence for a coupling of sodium and chloride fluxes across the plasma membrane of the rectal gland via a cotransport system sensitive to furosemide. They support the hypothesis that chloride secretion of the rectal gland is a secondary active transport and is driven by the sodium gradient across the basal-lateral membranes of the cell. [ABSTRACT FROM AUTHOR]

To define the luminal agent(s) responsible for the reduction of nephron filtration rate following increases of loop of Henle flow rate early proximal flow rate (EPFR) during loop perfusion with 17 different salt solutions were compared to the non-perfused fubules. During orthograde microperfusions a reduction of EPFR as indication of a feedback response was noted with a number of nonovalent Cl and Br salts (LiCl, KCl, NaCl, RbCl, CsCl, NHCl, choline Cl, NaBr, KBr), with Na salts except Na acetate (NaHCO, NaNO, NaF, NaI, NaSCN), and with CaCl and MgCl. These latter 2 solutions where used in a concentration of 70 mM while all other solutions had a concentration of 140 mM. During retrograde perfusion from the distal to the proximal end of the loop of Henle EPFR fell significantly with Cl and Br salts with percentage changes of EPFR ranging from −8.0 to −44.3%. In contrast, Cl free salts and Cl salts of divalent cations were associated with percentage changes of EPFR ranging from +7.1 to −6.2%, significance being reached only during perfusion with NaSCN. When furosemide (5×10M) was added to NaBr or KBr a feedback response was not observed. During orthograde perfusion with NaNO distal Cl concentrations were 44.2±5.08 mM (mean±S.E.) at a perfusion rate of 10nl/min and 59.1±3.93 mM at a rate of 40 nl/min. CaCl perfusion induced a marked elevation of distal Cl concentrations to levels higher than 140 mM. Loop chloride handling was normal during RbCl perfusion. The magnitude of the feedback response during retrograde perfusion was not changed by lowering NaCl concentration from 140 to 60 mM, but fell when NaCl concentration was further reduced. In contrast to orthograde perfusions it was insensitive to changes in flow rate. Our results are compatible with the thesis that feedback responses depend critically upon the rate of Cl transport probably across the macula densa cells. Br ions can replace Cl because they appear to share a common transport pathway which can be inhibited with furosemide. Unspecificity of feedback responses during orthograde microperfusions is due to presence of Cl ions in the macula densa region even when solutions are initially Cl free. Cl salts of divalent cations do not elicit a feedback response because Cl transport is severely curtailed. [ABSTRACT FROM AUTHOR]

Tannin, a polydisperse polyphenol extracted from cotton bracts (CBE), has been implicated in the pathogenesis of byssinosis, a lung disease of mill workers. CBE tannin inhibits chloride secretion in airway epithelial cells by means of an unknown mechanism(s). Activation of protein kinase C (PKC) by PMA (phorbol 12-myristate 13-acetate) in airway cells increases chloride secretion. The effect of tannin on this PKC pathway was examined, using canine tracheal epithelium mounted in Ussing chambers. PMA addition (10 nM) to the mucosal bath resulted in a 0.36 ± 0.07 μEq/cm · h (mean ± SEM, n = 20) increase in short-circuit current (I) and a 0.38 ± 0.17 μEq/cm · h increase in net chloride secretion (J). The inactive 4α-phorbol had no effect. Tannin addition to the mucosal bath produced a dose-dependent decrease in I and J. In tissues pretreated with 2-50 μg/ml tannin, and subsequently stimulated with PMA, tannin inhibited PMA stimulation of chloride secretion beginning at a tannin concentration of 10 μg/ml (0.09 ± 0.05 μEq/cm · h [ n = 10] increase in I and 0.08 ± 0.03 μEq/cm · h increase in J with PMA after tannin pretreatment). At 50 μg/ml tannin, the stimulatory effect of PMA was completely abolished. The known PKC inhibitor, H-7 (20 μM), inhibited PMA stimulation, while chelerythrine (2 μM) had not effect on PMA-stimulated I and J, and calphostin C was toxic to the airway epithelium. In membrane fragments, 2.5 μg/ml tannin inhibited the rate of histone III phosphorylation by PMA from 32.1 ± 4.4 nmol/mg protein per min to 20.1 ± 2.7 nmol/mg protein per min ( n = 7). In bovine airway cells, tannin pretreatment (2.5 μg/ml) decreased the cytosolic activity of PKC but had no effect on PKC translocation to the membrane. We conclude that tannin inhibits chloride secretion in airway epithelial cells in part by inhibiting PKC. [ABSTRACT FROM AUTHOR]

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues. [ABSTRACT FROM AUTHOR]

To study C1 conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was greater than 100 omega.cm2, and short circuit current (Isc = 2-20 microA/cm2), representing active secretion of Cl, increased greater than threefold with addition of 10 microM isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the C1-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mM, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360 +/- 5 nm, emission greater than 410 nm). SPQ leakage from the cells was less than 10% in 60 min at 37 degrees C. Intracellular calibration of SPQ fluorescence vs. [C1] (0-90 nM) was carried out using high-K buffers containing the ionophores nigericin (5 microM) and tributyltin (10 microM); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 M-1. Intracellular Cl activity was 43 +/- 4 mM. Cl flux was measured in response to addition and removal of 114 mM Cl from the bathing solution. Addition of 10 microM isoproterenol increased Cl efflux from 0.10 to 0.27 mM/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mM). In the absence of isoproterenol, removal of external Na or addition of 0.5 mM furosemide, reduced Cl influx by greater than fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mM barium diminished Cl influx by greater than twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the real-time measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism. [ABSTRACT FROM AUTHOR]

The effect of the sulfhydryl reagent parachloromercuribenzoic acid (PCMB) on chloride transport was examined in rabbit renal brush-border membrane vesicles (BBMV). PCMB had no effect on the chloride conductive pathway. In the presence of an inside-alkaline pH gradient and a K/valinomycin voltage clamp, the addition of PCMB stimulatedCl uptake and induced a threefold overshoot above the equilibrium value, indicating Cl/OH exchange. The effect of PCMB was reversed by dithiothreitol. Cl/OH exchange was not observed in the absence of PCMB. PCMB-activated Cl/OH exchange persisted even when the membrane potential was made inside-negative relative to the controls, thus, demonstrating that PCMB's effect onCl uptake under pH-gradient conditions is not mediated by parallel Cl and H conductive pathways. PCMB-activated Cl/OH exchange was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) with IC values of 290 and 80 μ m, respectively. These results demonstrate that modification of sulfhydryl groups by PCMB activates Cl/OH exchange in BBMV. [ABSTRACT FROM AUTHOR]

The effects of bathing solution HCO/CO concentrations on baseline cell membrane voltages and resistances were measured in Necturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO/CO Ringer's solutions (2.4 mm HCO/air or 1 mm HEPES/air) or a high HCO/CO Ringer's (10 mm HCO/1% CO). The principal finding of these studies was that the apical membrane fractional resistance ( fR) was higher in tissues bathed in the 10 mm HCO/CO Ringer's, averaging 0.87±0.06, whereas fR averaged 0.63±0.07 and 0.48±0.08 in 2.4 mm HCO and 1 mm HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical ( R) and basolateral membrane ( R) resistances in tissues bathed in 10 mm HCO/1% CO Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO/CO Ringer's, the higher value of fR was found to be due to both an increase in R and a decrease in R. The higher values of fR and lower values of R confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K and Cl substitutions. The results of these studies suggest that an electrodiffusive Cl transport mechanism exists at the basolateral membrane of tissues bathed in a 10 mm HCO/1% CO Ringer's, which can explain in part the fall in R. The above observations are discussed in terms of a stimulatory effect of solution [HCO/PCO on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes. [ABSTRACT FROM AUTHOR]

The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent Cl conductance. Volume increase and conductance activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at -100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5 mM, but was independent of whether Ki was 2.4, 5, or 10 mM. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

Sodium (22Na) transport was studied in a basolateral membrane vesicle preparation from rabbit parotid. Sodium uptake was markedly dependent on the presence of both K+ and Cl- in the extravesicular medium, being reduced 5 times when K+ was replaced by a nonphysiologic cation and 10 times when Cl was replaced by a nonphysiologic anion. Sodium uptake was stimulated by gradients of either K+ or Cl (relative to nongradient conditions) and could be driven against a sodium concentration gradient by a KCl gradient. No effect of membrane potentials on KCl-dependent sodium flux could be detected, indicating that this is an electroneutral process. A KCl-dependent component of sodium flux could also be demonstrated under equilibrium exchange conditions, indicating a direct effect of K+ and Cl on the sodium transport pathway. KCl-dependent sodium uptake exhibited a hyperbolic dependence on sodium concentration consistent with the existence of a single-transport system with Km = 3.2 mM at 80 mM KCl and 23 degrees C. Furosemide inhibited this transporter with K0.5 = 2 X 10(4) M (23 degrees C). When sodium uptake was measured as a function of potassium and chloride concentrations a hyperbolic dependence on [K] (Hill coefficient = 0.87 +/- 0.09) and a sigmoidal dependence on [Cl] (Hill coefficient = 1.31 +/- 0.07) were observed, consistent with a Na/K/Cl stoichiometry of 1:1:2. Taken together these data provide strong evidence for the electroneutral coupling of sodium and KCl movements in this preparation and strongly support the hypothesis that a Na+/K+/Cl cotransport system thought to be associated with transepithelial chloride and water movements in many exocrine glands is present in the parotid acinar basolateral membrane. [ABSTRACT FROM AUTHOR]

The leukotriene, LTC4, exerts a stimulatory effect on chloride transport in the frog cornea. In the work described here, the mechanism of action of LTC4 to stimulate chloride transport was studied. In corneas pretreated with indomethacin, the effect of LTC4 was abolished, suggesting the involvement of cyclo-oxygenase products in the response. Incubation of corneas with LTC4 resulted in a significant stimulation in PGE2 synthesis, as determined by TLC-autoradiography and radioimmunoassay. In addition, LTC4 was found to stimulate cAMP synthesis in the cornea, and this stimulation was blocked with indomethacin. PGE2 was previously shown by us to be the dominant cyclo-oxygenase product formed in the frog cornea, and is capable of stimulating cAMP and chloride transport. We suggest that LTC4 stimulation of chloride transport is mediated via activation of the cyclo-oxygenase pathway, resulting in enhanced PGE2 synthesis. Elevated PGE2 levels induce cAMP synthesis, and ultimately, the stimulation of chloride transport. Further, the activation of cyclo-oxygenase was found to be dependent on phospholipase A2 activity. This was shown by the inhibition of the LTC4 effect in the presence of quinacrine. Similarly, inhibition of the LTC4 effect in the presence of trifluoperazine suggests that cyclo-oxygenase activation by LTC4 may be mediated via calmodulin. We have previously demonstrated that the frog cornea has the biosynthetic capacity to produce LTC4. Therefore LTC4 may function as an endogenous regulator of chloride transport in this tissue. [ABSTRACT FROM AUTHOR]