Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Summary This paper provides the results of studies which characterized conductive³⁶Cl^– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive³⁶Cl^– uptake was studied under two different experimental conditions. In the first,³⁶Cl^– flux was driven by an inside positive voltage created with oppositely directed Cl^– and gluconate gradients. In the second, an inwardly direct K⁺ gradient was used to drive³⁶Cl^– uptake. By these two methods, voltage-sensitive³⁶Cl^– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total³⁶Cl^– flux. Separate paired studies demonstrated that the conductive³⁶Cl^– uptake was inhibited by the Cl^– channel blocker diphenylamine-2-carboxylate (DPC) with an IC₅₀ for DPC of 154 m. The voltagedependent³⁶Cl^– uptake had an activation energy of 6.4 kcal/mole. This³⁶Cl^– conductance had an anion selectivity sequence of I^–>Cl^–NO ₃ ^– gluconate.     

Cl− channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl− or cAMP-dependent protein kinase

Summary We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl^– concentrations in modulating the activity of Cl^– channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl^– channels, respectively. Raising thetrans Cl^– concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990.J. Membrane Biol. 118:269–278). With 2mm trans KCl,trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)trans Cl^– concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl^– channel activity by phosphorylation at or near the Cl^–-sensitive site on the intracellular face of these channels.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

HCO 3 − /Cl− exchange across the human erythrocyte membrane: Effects of pH and temperature

Summary Changes in extracellular pH (pH_o) in red cell suspensions were monitored in a stopped-flow rapid reaction apparatus under conditions wheredpH_o/dt was determined by the rate of HCO ₃ ^– /Cl^– exchange across the membrane. Experiments were performed at 5°C<T<40°C using either untreated cells or cells exposed to 0.11mm SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid). Although SITS exposure reduced the rate of exchange by 90%, both untreated and SITS-treated cells are similarly affected by changes in pH₀ and temperature. The rate of HCO ₃ ^– /Cl^– exchange exhibits a minimum at about pH_o 5 and a maximum at about pH₀ 7.4 at all temperatures. A transition temperature of 17°C was observed in the Arrhenius relationship for all pH₀. The activation energies (E _a) in kcal/mol are 19.6 below and 11.7 above 17°C for 50<8. These findings, similar to those reported for Cl^– self-exchange, suggest that: (i) a change in the rate-limiting step for HCO ₃ ^– /Cl^– exchange occurs at 17°C, possibly due to an altered interaction between the transport pathway and membrane lipids; (ii) the carrier system can be titrated by either H⁺ or SITS from the outside of the membrane, but the untitrated sites continue to transport normally; (iii) the pH₀ dependence of the rate of exchange is consistent with the titratable carrier having its most alkaline pK in the range expected for amino groups; and (iv) below pH₀ 5, the nature of the exchange is markedly altered.     

The mechanism of Cl− transport at the plasma membrane ofChara corallina I. Cotransport with H+

Summary Cl^– transport into cells ofChara corallina was studied in relation to that of other ions which have been proposed as cosubstrates for the Cl^– transport system. Although there appears to be a partial mutual dependence between K⁺ and Cl^– for transport in intact cells, this is not apparent in cells which have been perfused internally. Moreover, in intact cells, the fluxes of K⁺ and Cl^– show a large degree of independence in their responses to Cl^– starvation. Cl^– transport is electrogenic in a direction indicating the transport of excess positive charge into the cell. In the absence of any other likely counter ion, it is suggested that Cl^– is cotransported with H⁺. Response of Cl^– influx to internal and external pH in perfused cells is consistent with this suggestion. There appears, in addition, to be a role for ATP in transport as judged by fourfold stimulation of Cl^– influx in perfused cells when 1mm ATP is incorporated in the perfusion medium.     

A Ca2+-activated Cl− current in sheep parotid secretory cells

Previous studies have shown that the whole-cell current-voltage (I-V) relation of unstimulated sheep parotid cells is dominated by two K⁺ conductances, one outwardly and the other inwardly rectifying. We now show that once these K⁺ conductances are blocked by replacement of pipette K⁺ with Na⁺ and by the addition of 5 mmol/liter CsCl to the bath, there remains an outwardly rectifying conductance with a reversal potential of 0 mV. Replacement of 120 mmol/liter NaCl in the pipette solution with an equimolar amount of Na-glutamate shifted the reversal potential of this residual current to -55 mV, indicating that the conductance was Cl^? selective. The Cl^? current was activated by increasing the free Ca²⁺ in the pipette solution from 10 to 100 nmol/liter. When the Ca²⁺ concentration in the pipette solution was 10 nmol/liter, the relaxations observed in response to membrane depolarization could be fitted with a single exponential, whose time constant increased from 81 to 183 ms as the pipette potential was increased from -30 to +60 mV. Relaxation analysis showed that the current was activated by membrane depolarization. Reversal potential measurements in experiments in which external Cl^? was replaced with various anions, gave the following relative permeabilities: SCN^- (1.80) > I^- (1.09) > CI^- (1) > NO ₃ ^- (0.92) > Br^- (0.75). The relative conductances were: SCN^- (2.18) > I^- (1.07) > Cl^? (1.00) > Br^- (0.91) > NO ₃ ^- (0.50). The Cl^? current was blocked by NPPB (ID₅₀ ≈ 10 μm), DIDS (10 or 30 μmol/liter) and furosemide (100 μmol/liter).     

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.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

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.     

Reconstituted Cl− pump protein: A novel ion(Cl−)-motive ATPase

Cl^– absorption by theAplysia californica foregut is effected through an active Cl^– transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl^–-stimulated ATPase and ATP-dependent Cl^– transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg²⁺, Cl^–, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl^– accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl^– accumulation and electrical potential while an outwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl^– accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl^– accumulation, intraliposomal negative potential difference, and also Cl^–-stimulated ATPase activity. Both aspects of Cl^– pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl^–-ATPase actively transports Cl^– by an electrogenic process.     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: II. Na+ and Cl− effect of inward movements of Cl−

Summary At low concentration (1mm) of Cl^– in the outer solution, the influx of chloride through the isolated skin (J ₁₃ ^Cl ) of the South American frogLeptodactylus ocellatus (L.) seems to be carried by two mechanisms: (i) a passive one that exhibits the characteristics of an exchange diffusion process, and (ii) an active penetration. Studies of the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl ) indicate, that the presence of a high (107mm) concentration of Cl^– in the outer solution activates the translocation of this ion through the cells. Studies of the unidirectional flux of Cl^– across the outer barrier (J ₁₂ ^Cl ) indicate that Na⁺ out stimulates the penetration of Cl^– at this level. Cl^– out, in turn, stimulates, theJ ₁₂ ^Na , but this effect is only detected at low concentrations of Na⁺ out.     

A. A. Ukhtomskiĭ and I. P. Pavlov: a controversy or a dialogue?

Pavlov's concept of conditioned reflexes and Ukhtomskii theory of dominanta fall within the biological line in physiology. They unravel the integral adaptive and active nature of the organism behavior in the environment. It is impossible to develop modern concepts about the determinants of goal-directed behavior of animals and voluntary activity of humans without in-depth study of the achievements of these Russian physiological schools which not only formed the methodological basis for the current studies but also directed the way for their further development.     

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.     

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⁺.     

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.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.