Control of Cl− influx inChara by internal pH

Summary The possible regulation of Cl^– influx inChara by the cytoplasmic Cl^– concentration and cytoplasmic pH was investigated using both intact and intracellularly perfused cells. In perfused cells Cl^– influx was sensitive to changes in the internal Cl^– concentration but only when the concentration was less than 1mm.In intact cells the metabolic inhibitors, CCCP, DCMU, and oligomycin which inhibit Cl^– influx also reduced the cytoplasmic pH. A correlation between ATP concentration and cytoplasmic pH was shown to apply when the ATP concentration was lowered using these inhibitors. The possible relationships between ATP status, cytoplasmic pH, and Cl^– influx are discussed.     

Proton/chloride cotransport in Chara: mechanism of enhanced influx after rapid external acidification

Rapid lowering of the external pH (pH jump) enhances Cl^– influx in Chara. Experiments were conducted to distinguish between two factors which have previously been proposed to mediate in the response: raised cytoplasmic pH and lowered cytoplasmic Cl^– concentration. It is concluded that the latter alternative is more likely because: i) Cl^– influx is reduced at high external pH; ii) influx following the pH jump is never greater than that following pretreatment in Cl^–-free solution, which reduces cytoplasmic Cl^– concentration (Cl^– starvation); iii) the joint application of pH jump and Cl^– starvation does not result in a greater Cl^– influx than does Cl^– starvation alone; and iv) addition of NH ₄ ⁺ , which increases cytoplasmic pH, does generate an additional stimulation of Cl^– influx following a pH jump. It is suggested that the increased cytoplasmic pH at the end of pretreatment at high external pH decays rapidly during the pH jump, and thus any effect on Cl^– influx is so transient as to be undetectable by the methods used. The results are discussed in terms of a reaction kinetic model for 2H⁺/Cl^– cotransport (Sanders, D. and Hansen, U.-P, 1981, J. Member. Biol. 58, 139–153) which describes quantitatively; i) the effects of NH ₄ ⁺ on Cl^– influx in terms involving only a change in cytoplasmic pH; and ii) the combined effects of Cl^– starvation and NH ₄ ⁺ in terms involving only changes in Cl^– concentration and cytoplasmic pH. Conversely, the combined effects of Cl^– starvation and pH jump cannot be described by the model if the effect of the pH jump is the consequence of increased cytoplasmic pH. The simple interpretation of experiments on whole cells involving manipulation of (the electrochemical potential difference for protons across the plasma membrane) is questioned in the light of these and previous findings that secondary factors can determine the response of Cl^– transport in Chara.Abbreviations CPW Chara pond water - [Cl^–]_c cytoplasmic Cl^– concentration - pH_c cytoplasmic pH - pH_o external pH - transmembrane electrochemical gradient of protons - a membrane electrical potential difference     

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.     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

The energetics of Cl− active transport inChara

Summary The rate of Cl^– influx in intactChara was inhibited whenever the ATP concentration was reduced by application of metabolic inhibitors. In perfused cells, however, a net influx of Cl^– against its electrochemical gradient could be observed in the absence of ATP. Addition of ATP to the perfusion medium slightly stimulated Cl^– influx in one experiment but had no effect in another. Addition of ADP, NADH or metabolic inhibitors did not alter the influx rate. Consideration of the potential energy gradients across theChara plasmalemma in the perfused state leads to the conclusion that Cl^– influx occurs by cotransport with H⁺ or OH^–.     

Mechanism of Cl− transport at the plasma membrane ofChara corallina: II. Transinhibition and the determination of H+/Cl− binding order from a reaction kinetic model

Summary Internal Cl^– and low internal pH are strong inhibitors of Cl^– influx at the plasma membrane ofChara. The present investigation seeks to understand the mechanism by which this is achieved. Since both Cl^– and H⁺ are transported by the same system, one possible mechanism is simply through a change in the electrochemical gradients of these ions. However, it is found that transport is more sensitive to theinternal concentrations of the two ions than to their respective gradients. It is demonstrated that Cl^– influx, which shows Michaelis-Menten kinetics with respect to external concentration, is affected only in itsV _max by internal Cl^– and pH; the apparentK _m of the transport system for external Cl^– is unchanged. In addition, it is found that there is an apparent interaction between internal Cl^– and pH in their effects on Cl^– influx, both in intact cells and those that have been perfused internally. A kinetic model is proposed which can account quantitatively for all these observations simply through the effects of substrate concentration on the apparent rate constants of a recycling carrier. The model predicts (i) strictly ordered binding of Cl^– and H⁺ to the carrier at both internal and external surfaces, with Cl^– first on and first off (ii) movement of charge through the membrane on the loaded, rather than the unloaded, carrier. The present model is expected to account for similar kinetic observations from a variety of other cotransport systems.     

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.     

Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Summary Ouabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K⁺(Rb⁺)Cl^– fluxes were measured in low-K⁺ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K⁺ effluxes and Rb⁺ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K⁺Cl^– flux, metabolic depletion did not reduce K⁺Cl^– flux of normal or swollen cells. The anion preference of OR K⁺ efflux in NEM-treated cells was Br^–>Cl^–>HCO ₃ ^– =F^–I^–=NO ₃ ^– =CNS^–, and hence consistent with a reported Br^–>Cl^–>NO ₃ ^– sequence of the volume-dependent K⁺Cl^– transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb⁺Cl^– fluxes suggesting their immunologic identity, a conclusion also supported by anti-L₁ absorption studies. Since pretreatment with anti-L₁ prevented the activation of Rb⁺ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb⁺Cl^– influx, the NEM reactive SH groups must be located apart from the L₁ antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K⁺Cl^– transport path(s) regulated by a protein with two functional subunits or domains; a chemically (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl^– transport stimulated throughC _s by NEM orV _s by cell swelling.     

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.     

Cellular and whole-plant chloride dynamics in barley: insights into chloride–nitrogen interactions and salinity responses

The first analysis of chloride fluxes and compartmentation in a non-excised plant system is presented, examining ten ecologically pertinent conditions. The short-lived radiotracer couple ³⁸Cl/³⁹Cl was used as a Cl^– tracer in intact barley (Hordeum vulgare L. cv. Klondike) seedlings, which were cultured and investigated under four external [Cl^–], from abundant (0.1 mM) to potentially toxic (100 mM). Chloride–nitrogen interactions were investigated by varying N source (NO₃ ^– or NH₄ ⁺) and strength (0.1 or 10 mM), in order to examine, at the subcellular compartmentation level, the antagonism, previously documented at the influx level, between Cl^– and NO₃ ^–, and the potential role of Cl^– as a counterion for NH₄ ⁺ under conditions in which cytosolic [NH₄ ⁺] is excessive. Cytosolic [Cl^–] increased with external [Cl^–] from 6 mM to 360 mM. Cl^– influx, fluxes to vacuole and shoot, and, in particular, efflux to the external medium, also increased along this gradient. Efflux reached 90% of influx at the highest external [Cl^–]. Half-times of cytosolic Cl^– exchange decreased between high-affinity and low-affinity influx conditions. The relationship between cytosolic [Cl^–] and shoot flux indicated the presence of a saturable low-affinity transport system (SLATS) responsible for xylem loading of Cl^–. N source strongly influenced Cl^– flux to the vacuole, and moderately influenced Cl^– influx and shoot flux, whereas efflux and half-time were insensitive to N source. Cytosolic pool sizes were not strongly or consistently influenced by N source, indicating the low potential for Cl^– to act as a counterion to hyperaccumulating NH₄ ⁺. We discuss our results in relation to salinity responses in cereals.Abbreviations [Cl^–]_cyt cytosolic chloride concentration - [Cl^–]_o external chloride concentration     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Current-voltage relationships and voltage sensitivity of the Cl− pump inHalicystis parvula

Summary The current-voltage (I–V) relationships for internally perfused and nonperfused cells ofHalicystis parvula were determined. In both types of cells theI–V curve shows a conspicuous region of negative slope, beginning at vacuole potentials around –30 mV and continuing to values of +20 to +40mV. The negative slope in perfused cells is abolished by the metabolic inhibitors, darkness and low temperature. In order to determine the origin of this negative slope, we measured the voltage sensitivity of the unidirectional fluxes of Cl^–, Na⁺ and K⁺ in perfused cells. The results show that the Cl^– influx, which is mediated primarily by a Cl^– pump, increases as the vacuole potential is clamped at increasingly morenegative values up to –50 mV, while the other fluxes measured changed in the directions predicted by the change in electrical driving force. The voltage sensitivity of the Cl^– pump quantitatively accounts for the negative slope of theI–V curve. Also, we observed a large transient outward current of 10–20-sec duration following an abrupt depolarization by voltage clamping. This transient current was reduced or abolished by low temperature, which suggests that it may be due to the voltage-sensitive Cl^– pump. Finally, we found an inverse relationship between the transprotoplasm resistance (R _m ) and thePD under standard conditions, which suggests that the activity of the electrogenic Cl^– pump lowerR _m , i.e., it is a conductive pump.     

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

Summary Internodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl^– and K⁺ in the excitation of the plasmalemma was studied.[Cl^–] _i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl^–. The lowered [Cl^–] _i was about 0.01mm. Cells with this low [Cl^–] _i generated action potential and showed anN-shapedV–I curve under voltage clamped depolarization like Cl^–-rich cells containing 13 or 29mm Cl^–.E _m at the peak of the action potential was constant at [Cl^–] _i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl^– during excitation is discussed.At [Cl^–] _i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K⁺ concentration to 100mm, NO ₃ ^– , F^–, SO ₄ ^2– , acetate, and propionate inhibited the generation of action potentials like Cl^–, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K⁺ concentration. It decreased as [K⁺] _i (K-methane sulfonate) increased. Increase in [Na⁺] _i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K⁺. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol. 30:249).     

Branchial Cl transport, anion-stimulated ATPase and acid-base balance inAnguilla anguilla adapted to freshwater: Effects of hyperoxia

Summary Freshwater eel gills are notorious for their limited ability to pump chloride. As a result there is a considerable discrepancy between the Na⁺ and Cl^– plasma levels, and plasma HCO₃ ^– and blood pH are relatively high in this species.When eels are kept in tanks aerated with pure oxygen, significant alterations in blood acid-base balance, an increase in plasma pCO₂ and a decrease in blood pH, are observed. In fish studied after 3 weeks hyperoxia, the decrease in blood pH is compensated by an increase in plasma HCO₃ ^–. Such fish exhibit a Cl^– influx 5 times higher than that observed in normoxic fish. This Cl^– influx is readily inhibited by addition of SCN^– to the external medium.An anion-stimulated ATPase activated by HCO₃ ^– and by Cl^– and inhibited by SCN^– was recently described in membrane fractions of the gills ofCarassius auratus, a fish noted for its high Cl^– pumping rate. This enzyme is also found in the gills of the eel. While the maximal rates of enzyme activation by HCO₃ ^– and by Cl^– are similar inCarassius andAnguilla, the affinity of the enzyme for Cl^– is 25 times higher inCarassius. In the microsomal fraction of the hyperoxic eel gills, the maximal anionstimulated ATPase activity remains unchanged but HCO₃ ^– affinity decreases by 50%, while Cl^– affinity increases 5 times. Thus some characteristics of this ATPase seem to be closely related to the Cl^– pump activity exhibited by the gill in fresh water.     

Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation      

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

Summary The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofV _m increase while theK _m for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO₃ ^–, CO₃ ^– and CH₃COO^– resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl^–-dependent.     

Is there a Cl−−OH− exchange (Cl−−H+ cotransport) mechanism in the brush-border membrane of the intestine of the fresh water trout (Salmo gairdneri,R.)?

Summary Experiments were performed to determine the presence of a Cl^––OH^– exchange (Cl^––H⁺ cotransport) in the brush-border membranes isolated from the intestinal epithelium of freshwater trout. Determinations of alkaline phosphatase activities have shown that vesicle suspensions had an enrichment factor of about 17 in this enzyme indicating a high degree of purification of the brush-border membrane preparation. Cl^– uptake by vesicles in the presence of a proton gradient occurs against a concentration gradient with an overshoot ratio of about 2 and is inhibited by SITS. Several lines of evidence suggest that the mechanism involved is electrical in nature: (i) Cl^– uptake is increased when the proton gradient is increased, but there is a linear relationship between the Cl^– uptake and the Nernst potential of protons. (ii) Cl^– uptake is increased when a proton ionophore is added at low concentration and inhibited at high concentration, suggesting that a proton conductance is involved in the Cl^– uptake. (iii) there is a linear relationship between the initial speed of the uptake of increasing Cl^– concentrations and the Cl^– concentration. (iv) Cl^– uptake can be modulated by different potassium gradients with or without valinomycin. It is concluded that the enterocyte of the freshwater trout is not equipped with a Cl^––OH^– exchange and the Cl^– uptake by vesicles is realized by a Cl^– conductance.     

The effects of nitrite on chloride regulation in the crayfish Pacifastacus leniusculus Dana (Crustacea: Decapoda)

Summary Nitrite in the external freshwater medium was found to be toxic to Pacifastacus leniusculus Dana (48 h LC₅₀0.7 mM NO ₂ ^– ). It produced significant changes in haemolymph ionic concentration and acid-base status. Exposure to 1.0 mM NO ₂ ^– resulted in a rapid, active accumulation of nitrite in the haemolymph (to 25 mM NO ₂ ^– after 24 h) and caused the partial inhibition of Cl^– uptake. Some reduction in Cl^– efflux rate was seen. In 1.0 mM NO ₂ ^– a rapid depletion of haemolymph [Cl^–] was observed (50 mM decrease in 27 h). Nitrite competitively inhibited active Cl^– uptake (K_m increased from 0.42 to 1.22 mM; K_i=0.45 mM). To achieve Cl^– balance in this medium, depleted crayfish would require a two-fold increase in external [Cl^–]. A lesser decrease in haemolymph [Na⁺] was found while osmotic pressure was relatively unaffected. Haemolymph [HCO ₃ ^– ] showed a significant increase and was accompanied, unexpectedly, by an acidosis. Possible sources of the excess HCO ₃ ^– , perhaps by inhibition of normal Cl^–/HCO ₃ ^– branchial exchange or release from CaCO₃ stores, are discussed. Haemolymph clearance of NO ₂ ^– was slower than uptake as was the restoration of [Cl^–] on recovery in nitrite-free medium.Abbreviations AFWM artificial freshwater medium - BOD biochemical oxygen demand - J _out ^Cl chloride efflux - J _in ^Cl chloride influx - J _in ^Cl chloride influx - J _net ^base net base flux - J _net ^base net base flux - J _in(p) ^Cl passive chloride influx - J _out efflux - LC ₅₀ median lethal concentration - NEDE N-1-Naphthylethylenediamine - SEM standard error of mean - TEP transepithelial potential difference - V _in ^Cl active chloride uptake