Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Mechanisms of transport of Na+ and Cl- in the lizard colon

1. Ionic fluxes of sodium and chloride across lizard colon mucosa were measured and compared with the electrical characteristics of the tissue under voltage-clamped conditions. 2. In a Ringer-bicarbonate solution there was both a net sodium flux (JNanet) and a net chloride flux (JClnet) from mucosa to serosa. The net flux residual (JR) was near zero, indicating that net sodium and chloride transport is the result of an electrically neutral transport mechanism. 3. In the presence of sodium, the net chloride flux was abolished and the short-circuit current (Isc) and the electrical potential difference (PD) were unchanged. In the absence of chloride the net sodium flux was abolished and the short-circuit current and electrical potential difference were not modified. 4. From an analysis of the effects of the inhibitors, furosemide, amiloride and disulfonic stilbene (DIDS), a plausible model was developed to explain the characteristics of sodium and chloride absorption.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Differential Effects of Aldosterone and Vasopressin on Chloride Fluxes in Transimmortalized Mouse Cortical Collecting Duct Cells

The effects of aldosterone and vasopressin on Cl⁻ transport were investigated in a mouse cortical collecting duct (mpkCCD) cell line derived from a transgenic mouse carrying the SV40 large T antigen driven by the proximal regulatory sequences of the L-pyruvate kinase gene. The cells had features of a tight epithelium and expressed the amiloride-sensitive sodium channel and the cystic fibrosis transmembrane conductance regulator (CFTR) genes. dD-arginine vasopressin (dDAVP) caused a rapid, dose-dependent, increase in short-circuit current (I _sc ). Experiments with ion channel blockers and apical ion substitution showed that the current represented amiloride-sensitive Na⁺ and 5-nitro-2-(3-phenylpropylamino)benzoate-sensitive and glibenclamide-sensitive Cl⁻ fluxes. Aldosterone (5 × 10⁻⁷ m for 3 or 24 hr) stimulated I _sc and apical-to-basal ²²Na⁺ flux by 3-fold. ³⁶Cl⁻ flux studies showed that dDAVP and aldosterone stimulated net Cl⁻ reabsorption and that dDAVP potentiated the action of aldosterone on Cl⁻ transport. Whereas aldosterone affected only the apical-to-basal ³⁶Cl⁻ flux, dDAVP mainly increased the apical-to-basal Cl⁻ flux and the basal-to-apical flux of Cl⁻ to a lesser extent. These results suggest that the discrete dDAVP-elicited Cl⁻ secretion involves the CFTR and that dDAVP and aldosterone may affect in different ways the observed increased Cl⁻ reabsorption in this model of mouse cultured cortical collecting duct cells. Received: 8 January 1998/Revised: 25 March 1998     

Pharmacological Evidence that Calcium is Not Required for P2-Receptor-Stimulated Cl− Secretion in HT29-Cl.16E

Extracellular ATP at micro- to millimolar concentrations activates Cl⁻ conductance and increases cytosolic calcium ([Ca] _i ) in many epithelial cells, including the colonic epithelial cell line HT29-Cl.16E. Therefore, [Ca] _i has been postulated to be the intracellular messenger for Cl⁻ channel activation. HT29-Cl.16E is a highly differentiated cell line that forms confluent monolayers and secretes mucins and Cl⁻. The involvement of [Ca] _i in the purinergically-stimulated Cl⁻ secretion was investigated pharmacologically in this cell line by whole-cell patch-clamp and Ussing chamber techniques, as well as [Ca] _i measurements in fura-2 loaded cells. The calmodulin inhibitors W13 (5 μm) and chlorpromazine (50 μm) abolished increases in ATP-stimulated [Ca] _i -increases by 90% and 80%, respectively. However, these inhibitors had no effect on the ATP-stimulated Cl⁻ conductance measured in either individual cells or confluent monolayers. As controls, the effects of W13 and chlorpromazine on Ca²⁺-ionophore stimulated Cl⁻ conductance was measured. In this case, the two compounds inhibited whole cell Cl⁻ conductance and monolayer Isc by 90% and 100%, respectively. These data demonstrate: (1) The purinergically-stimulated increase in Cl⁻ current does not require an increase in [Ca] _i , suggesting the involvement of either another signaling pathway or direct activation of Cl⁻ channels by purinergic receptors. (2) A calmodulin or a calmodulinlike binding site that is sensitive to W13 and chlorpromazine participates in the regulation of the [Ca] _i increase by purinergic receptors in HT29-Cl.16E. Received: 4 December 1995/Revised: 16 August 1996     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

The effect of ethanol on ion transport in frog skin

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of ²²Na⁺ influx. Ethanol did not alter sodium outflux when bathing the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na⁺ and Cl^? were increased. The movement of Cl^? was evaluated by comparison of Cl^? flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect on Na⁺ transport.     

Evidence that Basolateral But Not Apical Membrane Localization of Outwardly Rectifying Depolarization-Induced Cl− Channel in Airway Epithelia

The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl⁻ (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl⁻ current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br⁻≒ I⁻ > Cl⁻. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I _sc ) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl⁻ current is present on the basolateral membrane of airway epithelia. Received: 7 October 1999/Revised: 24 April 2000     

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.     

The inhibitory effect of strophanthidin on secretion by the isolated gastric mucosa

The unidirectional fluxes of Na⁺ and Cl^- were measured across the isolated gastric mucosa of the bullfrog (R. catesbiana). The addition of strophanthidin, a cardiac aglycone, resulted in marked reductions of the spontaneous potential and short-circuit current. Associated with these changes, the isolated gastric mucosa ceased secreting chloride and hydrogen ion. Although the active component of chloride transfer was inhibited, the exchange diffusion component seemed to increase. No significant changes in membrane conductance or sodium flux were noted. Possible mechanisms of strophanthidin inhibition were discussed in view of its effect on chloride transport across the gastric mucosa and on sodium and potassium transfer in other tissues. It was concluded that the cardiac glycosides may not be specific inhibitors of sodium and potassium transport. This non-specific inhibition suggests that active chloride transport is affected by strophanthidin directly and/or anion secretion is dependent upon normal functioning of cation transport systems in the tissue.     

Ionic components of electric current at rat corneal wounds

Background

Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents?

Methodology/Principal Findings

Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca²⁺ efflux increased steadily whereas K⁺ showed an initial large efflux which rapidly decreased. Surprisingly, Na⁺ flux at wounds was inward. A most significant observation was a persistent large influx of Cl⁻, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl⁻, Na⁺ and K⁺. Injury to the cornea caused significant changes in distribution and expression of Cl⁻ channel CLC2.

Conclusions/Significance

These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca²⁺ and Cl⁻ fluxes appear to be mainly actively regulated, while K⁺ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.     

Efflux und Transport von Cl− und Rb+ in Maiswurzeln. Wirkung von Außenkonzentration,Ca++, EDTA und IES

Summary The efflux of ³⁶Cl and ⁸⁶Rb and the fluxes of these ions into the xylem were investigated using the device shown in Fig. 1.Efflux of ³⁶Cl is stimulated by external KCl while transport into the xylem is inhibited. Stimulation of the efflux appears to be stronger than inhibition of the transport.The stimulation of the efflux of ³⁶Cl was also observed with roots of intact seedlings.Assuming that the mode of transfer of Cl^– into the xylem (flux 3, Fig. 8) is diffusion exhibiting a linear isotherm (Luttge and Laties, 1966), these results suggest that the primary action of external salts is on the efflux across the plasma-lemma (Weigl, 1967, 1968). We were unable, however, to find a linear relationship between concentration and rate of chloride transport to the shoots of intact seedlings.With respect to the mode of ion transfer to the xylem (Weigl and Lüttge, 1965; Luttge and Laties, 1966) we have to be aware of the following facts:A linear isotherm cannot be taken to signify diffusive permeation (Torii and Laties, 1966; Luttge and Laties, 1966). If the Michaelis constant is extremely high relative to the ion concentration, the relationship between the ion concentration and the rate of a metabolic or mediated transport approaches linearity.The isotherm of the transport into the xylem may primarily reflect the difference of two large fluxes (4 and 5; Fig. 8).The transport data of Luttge and Laties (1966) need not be presented as a straight line (Fig. 6).If at high external ion concentrations the ratio of the ion concentration in the exudation sap to the external ion concentration approaches unity, diffusive permeation into the stele is still not proved to be the mode of migration, since at high stelar ion concentration flux 6 tends to become equal to flux 3.Considerations on radial ion transfer into the xylem depend on contemporary knowledge of the location of transport systems. Cl^–-uptake into root tips (2 mm) from solutions of 1–10 mM KCl did not exhibit a linear isotherm. These results are unpublished since the discrepancy to the results of Torii and Laties (1966) may be due to a higher content of vacuoles in our root tips. We feel it unlikely, however, that a linear isotherm of Cl^–-uptake into root tips is adequately explained by assuming that it is due to a lack of vacuoles while the sensibility to inhibitors is assumed to be due to the presence of vacuoles in root tips.Transport of Cl^– into the xylem is susceptible to inhibitors of oxydative phosphorylation, suggesting that this process, even at high external ion concentrations, is dependent on metabolic energy in contrast to the passive efflux from the cortical cells across the plasmalemma into the environment of the root. The precise location of the metabolic step(s) on the pathway of ions from the environment of the root to the xylem is unknown.The observed effects of Ca⁺⁺, EDTA and IAA may be considered in relation to the theory that auxin exerts its influence on growth by altering the diffusion potential across cell membranes (Brauner and Diemer, 1967). Growth is susceptible to the effect of Ca⁺⁺ and EDTA (Adamson, 1962; Setterfield, 1963; Thimann, 1963). Nevertheless, since IAA exerts no influence on ion fluxes in corn roots, it is not clear whether IAA really exerts its influence on growth by altering the diffusion potential across plant cell membranes. We might be dealing with occasional effects of secondary importance.     

Time-resolved release of calcium from an epithelial cell monolayer during mucin secretion

A significant amount of Ca²⁺ is contained in secretory mucin granules. Exchange of Ca²⁺ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²⁺ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²⁺ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²⁺ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875–2881, 2005). The results argue that Ca²⁺ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²⁺ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²⁺ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²⁺ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.     

Electrochemical gradients and k and cl fluxes in excised corn roots

The compartmental analysis method was used to estimate the K⁺ and Cl⁻ fluxes for cells of excised roots of Zea mays L. cv. Golden Bantam. When the measured fluxes are compared to those calculated with the Ussing-Teorell flux-ratio equation, an active inward transport of Cl⁻ across the plasmalemma is indicated; the plasmalemma K⁺ fluxes are not far different from those predicted for passive diffusion, although an active inward transport cannot be precluded. Whether fluxes across the tonoplast are active or passive depends upon the vacuolar potential which is unknown. Assuming no electropotential gradient, the tracer flux ratios are fairly close to those predicted for passive movement. However, if the vacuole is positive by about 10 millivolts relative to the cytoplasm, the data suggest active inward transport for K⁺ and outward transport for Cl⁻.     

Studies of the chloride transport in the gastric mucosa of the frog

The unidirectional fluxes of Cl^- and Na⁺ across the frog gastric mucosa in vitro were investigated with radioactive isotopes, and related to the secretory and electrical properties of the normal, and metabolically inhibited, mucosa. The flux of Cl^- from nutrient to secretory surface of the mucosa was observed to rise sharply with increasing acid secretion, while the corresponding flux of Na⁺ did not change appreciably. Lowering [NaCl] in the secretory solution caused a proportional drop in the fluxes from secretory to nutrient surface, of both Cl^- and Na⁺. Under the same conditions, the flux of Cl^- from nutrient to secretory surface fell by nearly the same amount as did the flux of Cl^- in the opposite direction, while the flux of Na⁺ from nutrient to secretory surface remained essentially unchanged. Electrical and hydrodynamic causes for this observation could be excluded. Metabolic inhibitors, including cyanide, azide, DNP, and anaerobiosis depressed Cl^- flux in both directions distinctly below the corresponding values observed with the normal, non-secreting mucosa. At the same time, a decrease in electrical potential difference and conductance was observed under inhibition. The flux of Na⁺ was little changed by metabolic inhibition. The relationship between fluxes and conductance of Cl^- during metabolic inhibition differs markedly from that observed under normal conditions, and is consistent with the view that during metabolic inhibition most of the Cl^- moving across the mucosa does so as a free ion. From the above data it is concluded that Cl^- is normally transported across the mucosa in combination with a carrier, the supply of which is impaired under metabolic inhibition. According to the behavior of the Na⁺ flux, the passive permeability of the mucosa appeared to be little affected by the metabolic inhibition applied, but seemed to rise considerably after death of the mucosa, probably due to structural damage.     

Radial Water and Solute Flows in Roots of Zea mays: II. ION FLUXES ACEOSS ROOT CORTEX

Unidirectional fluxes of Na⁺, K⁺, Cl^–, and phosphate weremeasured across a preparation of hollow cylinder of a segmentof maize root. Cation fluxes are 2–3 orders of magnitudelarger than those of the anions. From the Ussing flux ratio,it was concluded that cations are moving passively while anionsare pumped actively across the root. The anion pumps are acceleratedby the uncoupler DNP and inhibited by cyanide. It is suggestedthat they are coupled directly to the electron transport system,rather than to ATP. From analysis of the diffusion coefficientsof the ions it was concluded that radial ion movement in theroot cortex must be through, rather than between, the cells.     

Effect of inhibitors on chloride-dependent transmural potential in the rectal wall of Schistocerca gregaria

Previous experiments in vitro revealed a transmural potential difference (PD) and a short-circuit current (Isc) across the rectal wall of Schistocerca gregaria, which were dependent on chloride ions in lumen. The present report shows that anoxia, dinitrophenol and cyanide inhibit DP and Isc, proving that the required energy derives from oxidative metabolism. Acetazolamide also inhibits DP and Isc. Ouabain, when in haemocoele, has also an inhibitory effect, not ascribable to a Na⁺K⁺ pump blocking action. It is suggested that HCO₃^? plays an important rôle in the active transport of Cl^? from lumen to haemocoele and that ouabain may in some way inhibit chloride pumping.     

Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl⁻-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic β cells. In ClC-3^−/− mice, insulin secretion evoked by membrane depolarization (high extracellular K⁺, sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a 80% reduction in depolarization-evoked β cell exocytosis (monitored as increases in cell capacitance) in single ClC-3^−/− β cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl⁻ fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.     

Calcium modulation of the effects of serotonin,carbachol, and histamine on rabbit ileal ion transport.

In mammalian intestine, a number of secretagogues have been shown to work through either cyclic nucleotide or calcium mediated pathways to elicit ion secretion. Because excessive intestinal electrolyte and fluid secretion is central to the pathogenesis of a variety of diarrheal disorders, understanding of these processes is essential to the development of future clinical treatments. In the current study, the effects of serotonin (5HT), histamine, and carbachol on intestinal ion transport were examined in in vitro preparations of rabbit ileum. All three agonists induced a rapid and transient increase short-circuit current (delta Isc) across ileal mucosa. Inhibition of the delta Isc response of all three agents in chloride-free solution or in the presence of bumetanide confirmed that chloride is the main electrolyte involved in electrogenic ion secretion. Pretreatment of tissue with tetrodotoxin or atropine did not effect secretagogue-mediated electrolyte secretion. While tachyphylaxis of delta Isc response was shown to develop after repeated exposure of a secretagogue to tissue, delta Isc responses after sequential addition of different agonists indicate that cross-tachyphylaxis between agents did not occur. Serotonin, histamine, and carbachol have previously been reported to mediate electrolyte secretion through calcium-dependent pathways. To access the role of extracellular calcium in regulating ion secretion, the effect of verapamil on each agent was tested; verapamil decreased 5HT-induced delta Isc by 65.2% and histamine response by 33.5%, but had no effect on carbachol-elicited chloride secretion. An additive secretory effect was found upon simultaneous exposure of 5HT and carbachol to the system; no other combination of agents produced a significant additive effect. Findings from this study support previous work which has suggested that multiple calcium pathways may be involved in mediating chloride secretion in mammalian intestine.     

Epithelial cell specific properties and genetic complementation in a ΔF508 cystic fibrosis nasal polyp cell line

Summary Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a “tight epithelium”in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, ΔF508 homozygote, cell line that has tight junctions pre-crisis. The cells (ΣCFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl⁻ transport as measured by efflux of radioactive Cl⁻, short circuit current (I_sc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K⁺- and Ca⁺²-dependent Cl⁻ transport. Measurement of³⁶Cl⁻ efflux following stimulation with A23187 and ionomycin indicate a Ca⁺²-dependent Cl⁻ transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26° C for 48 h there is no indication of cAMP-dependent Cl⁻ as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells. Gene complementation studies with an episomal vector containing wild-type CF transmembrane conductance regulator cDNA showed correction of the cAMP-dependent Cl⁻ transport defect. This cell line contributes unique phenotypic features to the store of transformed CF epithelial cells already available.