Intracellular chloride activity of leech neurones and glial cells in physiological,low chloride saline

Leech blood apparently contains considerably less chloride than generally used in physiological experi ments. Instead of 85–130 mM Cl^– used in experimental salines, leech blood contains around 40 mM Cl^– and up to 45 mM organic anions, in particular malate. We have reinvestigated the distribution of Cl^– across the cell membrane of identified glial cells and neurones in the central nervous system of the leech Hirudo medicinalis L., using double-barrelled Cl^–- and pH-selective micro electrodes, in a conventional leech saline, and in a saline with a low Cl^– concentration (40 mM), containing 40 mM malate. The interference of anions other than Cl^–to the response of the ion-selective microelectrodes was estimated in Cl^–-free salines (Cl^– replaced by malate and/or gluconate). The results show that the absolute intracellu lar Cl^– activities (aCl_i) in glial cells and neurones, but not the electrochemical gradients of Cl^– across the glial and the neuronal cell membranes, are altered in the low Cl^–, malate-based saline. In Retzius neurones, aCl_i is lower than expected from electrochemical equilibrium, while in pressure neurones and in neuropil glial cells, aCl_i is distributed close to its equilibrium in both salines, re spectively. The steady-state intracellular pH values in the glial cells and Retzius neurones are little affected (0.1 pH units) in the low Cl^–, malate-based saline.     

Effect of Single-Charged Anions of Different Nature on the DNA Molecule Conformation in Water–Salt Solutions

Methods of intrinsic viscosity () and beam flow birefringence were used to study the effects of some single-charged ions (F^–, Cl^–, Br^–, I^–, NO^– ₂, NO^– ₃, ClO^– ₄, SCN^–, CH₃COO^–) on the size and thermodynamic rigidity of a DNA molecule in aqueous solutions of sodium salts in a broad interval of ionic strength when temperature T is changed. It has been shown that the close interactions in a macromolecule and the resulting DNA persistent length a are independent of the type of the salt anion over the whole interval of . On the contrary, the specific volume of the DNA molecule in solution, proportional to the value, is quite sensitive to the anionic composition of the solvent, which is due to the effect of anions and their hydration on the long-range interactions in the macromolecule. The presence of polyatomic and halide anions is manifested differently in the value of DNA. Possible factors responsible for the observed effect and the role of structural alterations of water upon anion hydration are discussed.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Transinhibition and Voltage-gating in a Fungal Nitrate Transporter

We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894–9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H⁺-NO ₃ ^– symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I _{NO 3} ^– . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H⁺+NO ₃ ^– )_o, the product concentration inside, [P] = [H⁺] _i ² · [NO ₃ ^– _i, may rise substantially near the plasma membrane, violating the condition [P] [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K _1/2 20 µM for I _{NO 3} ^– at pH_o from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 ± 0.5 for inactivation, and transition probabilities of 0.3–1.3 s^–1 at V = 0. This gating mode impedes loss of cellular NO ₃ ^– during depolarization.     

Inhibition of the catalase reaction of Photosystem II by anions

A new binding site for anions which inhibit the water oxidizing complex (WOC) of Photosystem II in spinach has been identified. Anions which bind to this site inhibit the flash-induced S₂/S₀ catalase reaction (2H₂O₂2H₂O+O₂) of the WOC by displacing hydrogen peroxide. Using a mass spectrometer and gas permeable membrane to detect the ³²O₂ product, the yield and lifetime of the active state of the flash-induced catalase (to be referred to simply as flash-catalase) reaction were measured after forming the S₂ or S₀-states by a short flash. The increase in flash-catalase activity with H₂O₂ concentration exhibits a K_m=10–20 mM, and originates from an increase in the lifetime by 20-fold of the active state. The increased lifetime in the presence of peroxide is ascribed to formation of the long-lived S₀-state at the expense of the unstable S₂-state. The anion inhibition site differs from the chloride site involved in stimulating the photolytic water oxidation reaction (2H₂OO₂+4e^-+4H⁺). Whereas water oxidation requires Cl^- and is inhibited with increasing effectiveness by F^-CN^-N₃ ^-, the flash-catalase reaction is weakly inhibited by Cl^-, and with increasing effectiveness by F^-CN^-, N₃ ^-. Unlike water oxidation, chloride is unable to suppress or reverse inhibition of the flash-catalase reaction caused by these anions. The inhibitor effectiveness correlates with the pK_a of the conjugate acid, suggesting that the protonated species may be the active inhibitor. The reduced activity arises from a shortening of the lifetime of the flash-induced catalase active state by 3–10 fold owing to stronger anion binding in the flash-induced states, S₂ and S₀, than in the dark S-states, S₁ and S_-1. To account for the paradoxical result that higher anion concentrations are required to inhibit at lower H₂O₂ concentrations, where S₂ forms initially after the flash, than at higher H₂O₂ concentrations, where S₀ forms initially after the flash, stronger anion binding to the S₀-state than to the S₂-state is proposed. A kinetic model is given which accounts for these equilibria with anions and H₂O₂. The rate constant for the formation/release of O₂ by reduction of S₂ in the WOC is <0.4 s^-1.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - BTP bis [tris(hydroxymethyl)methylamino]-propane - CCCP carbonylcyanide m-chlorophenylhyrazone - DCBQ 2,5-dichlorobenzoquinone - DMBQ 2,3-dimethylbenzoquinone - WOC water oxidizing complex     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

An effect of corticosteroids on thymocytes not mediated by macromolecule synthesis

Rat thymocytes were incubated for 2 min at 37°C and the cells then broken by osmotic shock in 1.5 mM MgCl₂ and the nuclei harvested. Treatment with 50 nM dexamethasone for 2 min resulted in about one third of nuclei showing abnormalities in appearance, in shape and density. This was not prevented by prior incubation for 10 min with actinomycin D and cycloheximide, but was when nuclei were isolated in the presence of anions larger than F^– and Cl^–, including I^–, Br^–, SO = ₄ and citrate . Subsequent addition of Cl^– ion, however, resulted in development of abnormalities in steroid-treated nuclei. It is concluded that the steroid induces a mechanism resulting in influx of chloride ion leading to nuclear edema, which is not mediated by processes involving synthesis of macromolecules.     

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.     

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).     

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Molecular and crystal structure of Nα-(9-fluorenyl)- methoxycarbonyl-L-ornithine hydrochloride diethyl ether solvate

The solid-state conformation of the first N-protected ornithine derivative has been established by X-ray analysis. The hydrochloride of N-(9-fluorenyl)methoxycarbonyl-L-ornithine crystallises as diethyl ether solvate. The backbone (₀, , , ¹) torsion angles are (174.9°, –84.0°, 145.9°, –171.0°). The conformation of the urethane amide bond is trans. The ornithine aliphatic side chain adopts preferred fully extended conformation which is stabilised by the hydrogen bonding of the –NH₃ ⁺ group to the diethyl ether molecule, carboxyl group and Cl^- anions.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells

Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl^– (Cl _i ^– ) or cell volume of MDCK cells acutely exposed to normal (pH_norm=7.4) and alkaline (pH_alk=7.7) conditions. At pH_norm, OTA increased Cl _i ^– by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH _i . At pH_alk, application of OTA increased Cl _i ^– by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH _i by 0.11±0.03 (n= 8, P<0.05). The Cl^–HCO ₃ ^– exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH _i and Cl _i ^– . OTA did not affect cell volume under both pH_norm and pH_alk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl _i ^– without changing cell volume. The driving force of plasma membrane Cl^–/HCO ₃ ^– exchange dissipates, leading to a rise of pH _i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH _i and Cl _i ^– homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca²⁺ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger.     

Regulation of the anion channel of the chloroplast envelope from spinach

Several anions such as Cl^–, NO^– ₂, SO^2– ₄, and PO^3– ₄ are known to modulate the photosynthetic activity. Moreover, the chloroplast metabolism requires the exchange of both inorganic and organic (e.g., triose phosphate, dicarboxylic acid, ATP) anions between the cytoplasmand the stroma. A chloride channel form the chloroplast envelope was reconstituted in planar lipid bilayers. We show that the channel is active in conditions prevailing in the plant. The open probability increases with the ionic strength of the experimental solutions and is maximal at 0 mV. This suggests that the channel could play a role in the osmotic regulation of the chloroplast. Amino group reagents affect the channel activity in a way that demonstrated that lysine residues are important for channel gating but not for ATP binding. Together, our results provide new information on the functioning of this channel in the chloroplast envelope membranes. They indicate that the open probability of the channel is low (P _o 0.2) in vivo and that this channel can account for the chloride flux through the chloroplast envelope.     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

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.     

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.     

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     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)