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 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     

An Effect of Ca2+ on the Intrinsic Cl−-conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent²⁺-precipitation technique using either CaCl₂ or MgCl₂. The dependence of the initial [¹⁴C]-d-glucose (or [³H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na⁺ salt, was found to be dependent upon the precipitating divalent cation. With Mg²⁺ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca²⁺ precipitation. When the anion composition of the media was varied (uptake in Cl⁻ media in comparison to gluconate⁻-containing media) it was found that the Cl⁻-dependent component of the initial uptake was markedly depressed with Ca²⁺-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg²⁺ and Ca²⁺ prepared vesicles respectively). When Ca²⁺ was loaded into Mg²⁺ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl⁻ enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of d-glucose uptake by Ca²⁺ in Mg²⁺ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca²⁺ inhibition of d-glucose uptake and restore the enhancement due to Cl⁻ media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca²⁺-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl⁻ vs. gluconate⁻) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC₃-(5) confirmed that Cl⁻ conductance was reduced in Ca²⁺-prepared vesicles. We conclude that a Cl⁻ conductance coexists with Na⁺ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca²⁺ via stimulation of protein phosphatase (PP2B). Received: 14 December 1994/Revised: 27 November 1995     

Role of Cl− in Electrogenic H+ Secretion by Cortical Distal Tubule

The presence of an electrogenic H⁺-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V _t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10⁻⁷ m bafilomycin or 4.6 × 10⁻⁸ m concanamycin were added. No significant increase in lumen-negative V _t upon perfusion with these inhibitors as compared to control, was observed as well as when 10⁻³ m amiloride, 10⁻⁵ m benzamil or 3 mm Ba²⁺ were perfused alone or in combination. The effect of an inhibition of electrogenic H⁺ secretion, i.e., increase in lumen-negative V _t by 2–4 mV, was observed only when Cl⁻ channels were blocked by 10⁻⁵ m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm⁻².sec⁻¹ as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H⁺ ATPase in the establishment of cortical late distal tubule V _t is minor in physiological conditions, but can be demonstrated after blocking Cl⁻ channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H⁺ secretion in this segment is reduced by a Cl⁻ channel blocker, supporting coupling of H⁺-ATPase with Cl⁻ transport. Received: 6 July 1996/Revised: 27 December 1996     

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H⁺-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10⁻² to 40.6 × 10⁻²Ωm². The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10⁻² Fm⁻² compared to 0.77 × 10⁻² Fm⁻² of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system. Received: 22 October 1996/Revised: 16 January 1997     

Anion-selectivity of the Swelling-activated Osmolyte Channel in Eel Erythrocytes

Osmotic swelling of fish erythrocytes activates a broad-specificity permeation pathway that mediates the volume-regulatory efflux of taurine and other intracellular osmolytes. This pathway is blocked by inhibitors of the erythrocyte band 3 anion exchanger, raising the possibility that band 3 is involved in the volume-regulatory response. In this study of eel erythrocytes, a quantitative comparison of the pharmacology of swelling-activated taurine transport with that of band 3-mediated SO²⁻ ₄ transport showed there to be significant differences between them. N-ethylmaleimide and quinine were effective inhibitors of swelling-activated taurine transport but caused little, if any, inhibition of band 3. Conversely, DIDS was a more potent inhibitor of band 3-mediated SO²⁻ ₄ flux than of swelling-activated taurine transport. In cells in isotonic medium, pretreated then co-incubated with 0.1 mm DIDS, the band 3-mediated transport of SO²⁻ ₄ and Cl⁻ was reduced to a low level. Exposure of these cells to a hypotonic medium containing 0.1 mm DIDS was followed by the activation of a Cl⁻ permeation pathway showing the same inhibitor sensitivity as swelling-activated taurine transport. The data are consistent with swelling-activated transport of taurine and Cl⁻ being via a common pathway. A comparison of the swelling-activated transport rates for taurine and Cl⁻ with those for several other solutes was consistent with the hypothesis that this pathway is an anion-selective channel, similar to those that mediate the volume-regulatory efflux of Cl⁻ and organic osmolytes from mammalian cells. Received: 7 July 1995/Revised: 2 September 1995     

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     

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Tonoplast K⁺ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K⁺ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl⁻ on either the vacuolar side or the cytoplasmic side was partly replaced with F⁻, the reversal potential of the 48 pS channel shifted conform to the Cl⁻ equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl⁻ channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10⁻⁸ m) Ca²⁺, then no Cl⁻ channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca²⁺ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl⁻ ions induced opposite rectification properties. Our results show Ca²⁺-activated Cl⁻ channels in the tonoplast of Chara with holding potential dependent rectification. Received: 30 March 1999/Revised: 10 August 1999     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na⁺-H⁺ exchange, H⁺–K⁺ ATPase and Cl⁻/HCO⁻ ₃ exchange. In this work we studied the functional activity of a vacuolar H⁺-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H⁺ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH⁺ ₄ pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na⁺ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10⁻⁸ m (a specific inhibitor of the vacuolar H⁺-ATPase) was added to the zero Na⁺ solution. When using a solution with zero Na⁺, low K⁺ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H⁺–K⁺-ATPase. This result was confirmed by the use of 5 × 10⁻⁵ m Schering 28080. The Na⁺ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10⁻⁵ m (a specific blocker of Cl⁻ channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl⁻/normal Na⁺ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na⁺ and Cl⁻ free solution. From these results we conclude that: (i) MDCK cells have two Na⁺-independent mechanisms of pHi recovery, a concanamycin sensitive H⁺-ATPase and a K⁺ dependent, Schering 28080 sensitive H⁺–K⁺ ATPase; and, (ii) pHi recovery in Na⁺-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl⁻–free medium. This finding supports a role for chloride in the function of the H⁺ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998     

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     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Cyanide Inhibition of Chloride Conductance Across Toad Skin

The effect of cyanide (CN⁻) on voltage-activated or cAMP-induced passive chloride conductance (G ^Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN⁻ inhibited G ^Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC₅₀ was 180 ± 12 μm for voltage-activated G ^Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN⁻. Inhibition was independent of the presence of Ca²⁺. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN⁻ is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G ^Cl by CN⁻ occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN⁻] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G ^Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN⁻, but G _t recovered thereafter partially in the continuous presence of CN⁻. Dinitrophenol inhibited G ^Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN⁻ with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G ^Cl . We propose that the inhibition is directly on G ^Cl , presumably by competition with Cl⁻ at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified. Received: 8 February 1999/Revised: 22 September 1999     

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