Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995     

Dynamic modeling for flow-activated chloride-selective membrane current in vascular endothelial cells

In this paper, a dynamic model is proposed to quantify the relationship between fluid flow and Cl⁻-selective membrane current in vascular endothelial cells (VECs). It is assumed that the external shear stress would first induce channel deformation in VECs. This deformation could activate the Cl⁻ channels on the membrane, thus allowing Cl⁻ transport across the membrane. A modified Hodgkin–Huxley model is embedded into our dynamic system to describe the electrophysiological properties of the membrane, such as the Cl⁻-selective membrane current (I), voltage (V) and conductance. Three flow patterns, i. e., steady flow, oscillatory flow, and pulsatile flow, are applied in our simulation studies. When the extracellular Cl⁻ concentration is constant, the I-V characteristics predicted by our dynamic model shows strong consistency with the experimental observations. It is also interesting to note that the Cl⁻ currents under different flow patterns show some differences, indicating that VECs distinguish among and respond differently to different types of flows. When the extracellular Cl⁻ concentration keeps constant or varies slowly with time (i.e. oscillates at 0.02 Hz), the convection and diffusion of Cl⁻ in extracellular space can be ignored and the Cl⁻ current is well captured by the modified Hodgkin–Huxley model alone. However, when the extracellular Cl⁻ varies fast (i.e., oscillates at 0.2 Hz), the convection and diffusion effect should be considered because the Cl⁻ current dynamics is different from the case where the convection-diffusion effect is simply ignored. The proposed dynamic model along with the simulation results could not only provide more insights into the flow-regulated electrophysiological behavior of the cell membrane but also help to reveal new findings in the electrophysiological experimental investigations of VECs in response to dynamic flow and biochemical stimuli.     

Cloning and functional characterization of a cation–chloride cotransporter gene <Emphasis Type="Italic">OsCCC1</Emphasis>

Potassium (K⁺) and chloride (Cl⁻) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K⁺ and Cl⁻, it remains unclear if they actively use K⁺-coupled Cl⁻ cotransporters (KCC), as used in animals, to transport K⁺ and Cl⁻. We have cloned an Oryza sativa cDNA encoding for a member of the cation–Cl⁻ cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K⁺–Cl⁻ cotransporters than to other cation–Cl⁻ cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na⁺, K⁺, and Cl⁻ contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K⁺ and Cl⁻ than the latter but that they did not differ in terms of Na⁺ contents, suggesting OsCCC1 may be involved in K⁺ and Cl⁻ transport. Results from different tests indicated that the OsCCC1 plays a significant role in K⁺ and Cl⁻ homeostasis and rice plant development.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Postprandial acid–base balance and ion regulation in freshwater and seawater-acclimated European flounder, <Emphasis Type="Italic">Platichthys flesus</Emphasis>

The effects of feeding on both acid–base and ion exchange with the environment, and internal acid–base and ion balance, in freshwater and seawater-acclimated flounder were investigated. Following voluntary feeding on a meal of 2.5–5% body mass and subsequent gastric acid secretion, no systemic alkaline tide or respiratory compensation was observed in either group. Ammonia efflux rates more than doubled from 489 ± 35 and 555 ± 64 μmol kg⁻¹ h⁻¹ under control conditions to 1,228 ± 127 and 1,300 ± 154 μmol kg⁻¹ h⁻¹ post-feeding in freshwater and seawater-acclimated fish, respectively. Based on predictions of gastric acid secreted during digestion, we calculated net postprandial internal base gains (i.e., HCO₃⁻ secreted from gastric parietal cells into the blood) of 3.4 mmol kg⁻¹ in seawater and 9.1 mmol kg⁻¹ in freshwater-acclimated flounder. However, net fluxes of ammonia, titratable alkalinity, Na⁺ and Cl⁻ indicated that branchial Cl⁻/HCO₃⁻ and Na⁺/H⁺ exchange played minimal roles in counteracting these predicted base gains and cannot explain the absence of alkaline tide. Instead, intestinal Cl⁻/HCO₃⁻ exchange appears to be enhanced after feeding in both freshwater and seawater flounder. This implicates the intestine rather than the gills as a potential route of postprandial base excretion in fish, to compensate for gastric acid secretion.     

Current status of the role of Cl− ion in the oxygen-evolving complex

This minireview summarizes the current state of knowledge concerning the role of Cl⁻ in the oxygen-evolving complex (OEC) of photosystem II (PSII). The model that proposes that Cl⁻ is a Mn ligand is discussed in light of more recent work. Studies of Cl⁻ specificity, stoichiometry, kinetics, and retention by extrinsic polypeptides are discussed, as are the results that fail to detect Cl⁻ ligation to Mn and results that show a lack of a requirement for Cl⁻ in PSII-catalyzed H₂O oxidation. Mutagenesis experiments in cyanobacteria and higher plants that produce evidence for a correlation between Cl⁻ retention and stable interactions among intrinsic and extrinsic polypeptides are summarized, and spectroscopic data on the interaction between PSII and Cl⁻ are discussed. Lastly, the question of the site of Cl⁻ action in PSII is discussed in connection with the current crystal structures of the enzyme.     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

The Ca2+-inactivated Cl− Channel at Work: Selectivity,Blocker Kinetics and Transport Visualization

Removal of extracellular divalent cations activated a Cl⁻ channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca²⁺-inactivated Cl⁻ channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl⁻ channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl⁻-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl⁻ from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca²⁺ removal. In the absence of external Cl⁻ and Ca²⁺, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl⁻ in the millimolar range. In the presence of external Ca²⁺, no emission changes could be observed during the same time period. Chelating external Ca²⁺ in absence of Cl⁻ immediately activated Ca²⁺-inactivated Cl⁻ channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I⁻ > Br⁻ > Cl⁻≫ gluconate as it is also typical for maxi Cl⁻ channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca²⁺-inactivated Cl⁻ channels. However, this egg CaIC revealed an altered sensitivity to external Ca²⁺ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl⁻ channels described up to now. Received: 16 May 1996/Revised: 4 September 1996     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Components of depolarization-induced transmembrane ion current in isolated smooth muscle cells of the guinea pigtaenia coli

Transmembrane ion currents in isolated single smooth muscle cells (SMC) from the guinea pigtaenia coli were investigated using a whole-cell mode of the patch-clamp technique. Currents induced by depolarizing shifts in the membrane potential from its holding level of −60 mV contained an initial inward phase (Ca²⁺ current), which in 30–40 msec was followed by an outward phase. It was shown that outward current was carried by K ions and consisted at least of three components: one Ca²⁺-independent K⁺ current of delayed rectifier (KV) and two Ca²⁺-dependent K⁺ currents. The latter can be further divided into the apamin-sensitive (SK) and charybdotoxin-sensitive (BK) currents. It was found that relative contributions of these three components in total outward current at 0 mV were 35–45%, 5–15%, and 45–55% for KV, SK, and BK currents, respectively. A potential-dependent current carried by Ci ions was also found. This Cl⁻ current had inward direction within the range of potentials below the chloride equilibrium potential (E _Cl) and outward direction above theE _Cl. The magnitude of Cl⁻ current was significantly lower than the magnitude of total K⁺ current.     

Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus

Nitrite influx into crayfish showed saturation kinetics, supporting a carrier-mediated uptake. Addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS: at 10⁻⁵, 10⁻⁴ and 10⁻³M) and bumetanide (at 10⁻⁵ M and 10⁻⁴ M) to the ambient water did not significantly affect nitrite influx. Rather than suggesting that neither Cl⁻/HCO₃ ⁻ exchange nor K⁺/Na⁺/2Cl⁻ cotransport were involved in the transport, this may reflect that the gill cuticle has a low permeability to the pharmacological agents, or that the sensitivity of the transport mechanism to the inhibitors is low. Nitrite accumulation in the haemolymph was significantly decreased during hypercapnic conditions compared to normocapnic conditions. This supports the idea that an acid–base regulatory decrease in Cl⁻(influx)/HCO₃ ⁻ (efflux) induced by hypercapnia should decrease NO₂ ⁻ uptake if NO₂ ⁻ and Cl⁻ share this uptake route. The respiratory acidosis caused by exposure to hypercapnia alone was partially compensated by HCO₃ ⁻ accumulation in the haemolymph. Combined exposure to hypercapnia and nitrite improved pH recovery, mainly by augmenting the [HCO₃ ⁻] increase, but also by decreasing haemolymph PCO₂. Exposure to nitrite in normocapnic water induced an initial increase in haemolymph [HCO₃ ⁻] and later also a decrease in PCO₂. Thus, the improved acid-base compensation during combined hypercapnia and nitrite exposure was an amplification of this nitriteinduced response. Haemolymph base excess rose much more than haemolymph [Ca], suggesting that transfer of acid-base equivalents between animal and water was more important than H⁺ buffering by exoskeletal CaCO₃ in mediating the increase in haemolymph [HCO₃ ⁻]. Accepted: 27 June 2000     

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     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO₃ ⁻ secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K⁺, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO₃ ⁻. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO₃ ⁻ significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl⁻ channel located in luminal membranes) and production of cytosolic HCO₃ ⁻ induce the inward anion current and resulting depolarization. Inhibition of the Na⁺-K⁺-2Cl⁻ cotransporter and the Cl⁻-HCO₃ ⁻ exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl⁻ via the Na⁺-K⁺-2Cl⁻ cotransporter or the Cl⁻-HCO₃ ⁻ exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO₃ ⁻ produced intracellularly, but not of Cl⁻ due to lack of active Cl⁻ transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport. Received: 17 June 2000/Revised: 14 November 2000     

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.     

Electrophysiological characterization of chloride secretion across the jejunum and colon of pigs as affected by age and weaning

Hypersecretion of chloride can cause diarrhea, a disease frequently occurring in young pigs, particularly around weaning. We investigated the contribution of different channels to intestinal Cl⁻ secretion as influenced by age and weaning. Jejunal and colonic epithelia from 4-month-old pigs and 4-week-old piglets were incubated in Ussing chambers and stimulated by carbachol and forskolin. Changes in short-circuit currents were taken as measure of electrogenic net Cl⁻ secretion. DIDS or NPPB served to inhibit Ca-activated Cl⁻-channels and outwardly rectifying Cl⁻-channels (ORCC) or cystic fibrosis transmembrane regulator (CFTR), respectively. Depolarizing the basolateral membrane allowed to examine the influence of K⁺-channels on Cl⁻ secretion. Forskolin-stimulated Cl⁻ secretion was mediated by CFTR. ORCC were not involved. Carbachol-induced Cl⁻ secretion could be ascribed to an enhanced driving force due to the opening of K⁺-channels, whereas Ca-dependent Cl⁻ channels seemed not to be involved. In jejunum, piglets showed higher Cl⁻ secretion than pigs. Two days after weaning forskolin induced an I _sc overshoot and a faster increase in G _t. In colon, Cl⁻ secretion was neither influenced by age nor by weaning. The data suggest a disposition of porcine jejunum for a higher Cl⁻ secretion in young and freshly weaned piglets, which might be a natural defense mechanism as well as a predisposing factor for diarrhea.     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996