Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney

Summary The properties of an anion-selective channel observed in basolateral membranes of microdissected, collagenase-treated, cortical thick ascending limbs of Henle's loop from mouse kidney were investigated using patch-clamp single-channel recording techniques. In basal conditions, single Cl^– currents were detected in 8% of cell-attached and excised, inside-out, membrane patches whereas they were observed in 24% of cell-attached and 67% of inside-out membrane patches when tubular fragments were preincubated with Forskolin (10^–5 m) or 8-bromo-cAMP (10^–4 m) and isobutylmethylxanthine (10^–5 m). The channel exhibited a linear current-voltage relationship with conductances of about 40 pS in both cell-attached and cell-free membrane configurations. AP _Na ⁺ P _Cl ^–ratio of 0.05 was estimated in the presence of a 142/42mm NaCl concentration gradient applied to inside-out membrane patches. Anionic selectivity of the channel followed the sequence Cl^–>Br^–>No ₃ ^– F^–; gluconate was not a permeant species. The open-state probability of the channel increased with membrane depolarization in cell-attached, i.e.,in situ membrane patches. In excised, inside-out, membrane patches, the channel was predominantly open with the open-state probability close to 0.8 over the whole range of potentials tested (–60 to +60 mV). The channel activity was not a function of internal calcium concentration between 10^–9 and 10^–3 m. We suggest that this Cl^– channel, whose properties are distinct from those in other epithelia, could account for the well-documented conductance which mediates Cl^– exit in the basolateral step of NaCl absorption in thick ascending limb of Henle's loop.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.     

Cl− channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

A unique property of basolateral membrane Cl^– channels from the mTAL is that the Cl^– concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P ₀ ). The K _1/2 for maximal activation of P ₀ by Cl^– facing intracellular domains of these channels is 10 mm Cl^–. The present experiments evaluated the nature of these Cl^–-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl^– channel faces, could augment P ₀ with a K _1/2 in the range of 10 mm isethionate without affecting conductance (g _Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P ₀ usually seen when the Cl^– concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl^– channel activity was increased by first raising the Cl^– concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl^– channel faces had no effect on P ₀ . We conclude that the intracellular aspects of these Cl^– channels contain Cl^–-interactive loci (termed [Cl] _i ) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl^– or isethionate concentrations, by interactions with PGO or TNBS.We acknoeledge the able technical assistance of Anna Grace Stewart. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veteterans Administration Merit Review Grants to T. E.Andreoli and to W. B. Reeves. C. J. Winters is a Veterans Administration Associate Investigator.     

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane

Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H⁺ (pH_i) and Cl^– (aCl_i) activities with double-barreled ion-selective microelectrodes. In a HCO ₃ ^– -free solution of pH 7.4 (HEPES/Tris buffered), pH_i was 7.25 andaCl_i was 33mm. By an exposure to a HCO ₃ ^– (25mm HCO ₃ ^– /5% CO₂, pH 7.4) solution for 15 min,aCl_i was decreased to 25mm and pH_i was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO ₃ ^– concentration [HCO ₃ ^– ]_i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO ₃ ^– -free solution reversed the changes inaCl_i and pH_i. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCl_i, pH_i, and [HCO ₃ ^– ]_i caused by admission/withdrawal of the HCO ₃ ^– , solution and decreased the buffer value. Replacement of all Cl^– with gluconate in the HCO ₃ ^– solution increased pH_i, and readmission of Cl^– decreased pH_i. The rates of these changes in pH_i were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO ₃ ^– solution, a stimulation of intracellular HCO ₃ ^– production by exposing the tissue to 25mm NH ₄ ⁺ increasedaCl_i significantly. While in the HCO ₃ ^– -free solution or in the HCO ₃ ^– , solution containing DIDS, exposure to NH ₄ ⁺ had little effect onaCl_i. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl^–/HCO ₃ ^– exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl^–/HCO ₃ ^– exchange is discussed.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Chloride Channels in Basolateral TAL Membranes. XVIII. Phenylglyoxal Induces Functional mcClC-Ka Activity in Basolateral MTAL Membranes

Cultured mouse MTAL cells contain more mRNA encoding the Cl^– channel mcClC-Ka, which mediates CTAL Cl^– absorption, than mRNA encoding the Cl^– channel mmClC-Ka, which mediates MTAL Cl^– absorption. mmClC-Ka and mcClC-Ka have three functional differences: 1) mmClC-Ka open time probability, P _o, increases with increasing cytosolic Cl^–, but variations in cytosolic Cl^– do not affect P _o in mcClC-Ka; 2) mmClC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on P _o in mcClC-Ka; and 3) mmClC-Ka channels have single-ion occupancy, while mcClC-Ka channels have multi-ion occupancy. Using basolateral vesicles from MTAL cells fused into bilayers, we evaluated the effects of 1 mM cytosolic phenylglyoxal (PGO), which binds covalently to lysine or arginine, on Cl^– channels. With PGO pretreatment, Cl^– channels were uniformly not gated either with increases in cytosolic-face Cl^– or with (ATP + PKA) at 2 mm cytosolic-face Cl^–; and they exhibited multi-ion occupancy kinetics typical for mcClC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl^– access to arginine or lysine residues on mmClC-Ka by PGO results in Cl^– channels having the functional characteristics of mcClC-Ka channels.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

Summary We evaluated the effects of vawrying aqueous Cl^– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl^– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl^– concentrations and/or PGO on the relation between holding voltageV _H -mV) and open-time probability (P _o).Reducingcis Cl^– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P _v) with a half-maximal reduction inP _o atcis Cl^–170mM. Channel activity was sustained by equimolar replacement ofcis Cl^– with F^–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl^– concentration andP ₀ at 10mm Cl^–. Reducingcis Cl^– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl^– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl^– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl^– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl^– channels.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

Effect of sulfhydryl compounds on ATP-stimulated H+ transport and Cl− uptake in rabbit renal cortical endosomes

Summary The vacuolar H⁺ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H⁺ ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H⁺ transport at 10^–5–10^–6 m, including NEM, mersalyl, aldrithiol, 5,5 dithiobis (2-nitrobenzoic acid),p-chloromercuribenzoic acid (PCMB) andp-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10^–4 m) stimulated endosomal³⁶Cl^– uptake, particularly in the presence of an inside-alkaline pH gradient (pH_in=7.6/pH_out=5.5.). This result was not an effect of PCMB on the Cl^–-conductive pathway. The less permeable PCMBS did not stimulate³⁶Cl^– uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol,. ATP-dependent³⁶Cl^– uptake was decreased by addition of PCMB. Finally, PCMB had no effect on⁴⁵Ca²⁺ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H⁺ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl^–/OH^– exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl^–/OH^– exchanger recently described in rabbit renal brush-border membranes.     

Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers ofRana pipiens

Summary The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined.Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K₂SO₄ solutions. External solutions were first changed to ones in which either Rb⁺ or Cs⁺ replaced K⁺ and then to solutions containing either NO ₃ ^– or Cl^– to replace SO ₄ ^2– . The hyperpolarizations produced by Cl^– depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO ₃ ^– were independent of external pH over a pH range from 5.5 to 9.0.In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K₂SO₄ plus Na₂SO₄. KCl or KNO₃ were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K⁺ was replaced by TEA⁺. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl^– or NO ₃ ^– depending on the final equilibrating solution. Cl^– currents during voltage pulses were both external pH and time dependent. By contrast, NO ₃ ^– currents were independent of both external pH and time.The voltage dependence of NO ₃ ^– currents could be fit by constant field equations with aP _{NO 3} of 3.7·10^–6 cm/sec. The voltage dependence of the initial or instantaneous Cl^– currents at pH 7.5 and 9.0 could also be fit by constant field equations with P_Cl of 5.8·10^–6 and 7.9·10^–6 cm/sec, respectively. At pH 5.0, no measurable instantaneous Cl^– currents were found.From these results we conclude that NO ₃ ^– does not pass through the pH, time-dependent Cl^– channels but rather passes through a distinct set of channels. Furthermore, Cl^– ions do not appear to pass through the channels which allow NO ₃ ^– through. Consequently, the measured ratio ofP _Cl/P _{NO 3} based on membrane potential changes to ionic changes made on intact skeletal muscle fibers is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.     

Activation by hyperpolarization and atypical osmosensitivity of a Cl− current in rat osteoblastic cells

During whole-cell recording of rat osteoblastic cells with high-Cl^– internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl^– channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs⁺ ions but is blocked in a voltage-dependent manner by 4,4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS) 1 mm and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mm. Cd²⁺ ions are potent blockers of this current, blocking completely above 300 m. The amplitude of the Cl^– current activated by a given hyperpolarization increases during the first 10–20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl^– channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl^– current. However, this hyposmolarity-induced Cl^– current and the hyperpolarization-activated Cl^– current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl^– current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl^– current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.We wish to thank P. Ascher and B. Barbour for useful comments.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Characterization of chloride channels in membrane vesicles from the kidney outer medulla

Summary The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na⁺/K⁺ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl^–. In order to study the Cl^– conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl^– channel activity was determined by a³⁶Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl^– gradient. The accumulation of³⁶Cl^– inside the vesicles was found to be dependent on the intravesicular Cl^– concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl^– channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl^– gradient. The anion selectivity of the channels was Cl^–>NO ₃ ^– =I^– gluconate. Among the Cl^– transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4-diisothiocyano-stilbene-2,2-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC₅₀ of 110, 200 and 550 m, respectively. Inhibition of³⁶Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4-dinitrostilbene-2,2-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 m). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency. It is proposed that NPPAB and DIDS bind to the same or functionally related site on the Cl^– channel protein.