Nacl flux in the flounder (Pseudopleuronectes americanus) intestine: Effects of ph and transport inhibitors

• 1.1. The effects of extracellular pH on Na⁺ and Cl⁻ absorption were studied in vitro in the small intestine of the winter flounder, Pseudopleuronectes americanus.
• 2.2. Reductions in bathing solution pH inhibited J_ms^na (mucosal-to-serosal flux) and J_net^na (net flux) (r = 0.90) and J_net^Cl (r = 0.92) [due to an increase in J_sm^Cl, (serosal-to-mucosal)] and decreased short circuit current (Isc).
• 3.3. Luminal bumetanide (0.1 mM) and amiloride (1 mM) inhibited Na⁺ and Cl⁻ absorption by reducing J_ms.
• 4.4. Luminal barium (5mM) and luminal copper (100 μM) decreased J_ms^Cl and increased J_sm^Cl.
• 5.5. We conclude that reductions in extracellular pH inhibit a luminal membrane NaCl absorptive process (Na⁺-K⁺-2Cl⁻) and stimulate an electrogenic Cl⁻ secretory process.

     

The Nature of the In Vivo Sodium and Chloride Uptake Mechanisms through the Epithelium of the Chilean Frog Calyptocephalella gayi (Dum. et Bibr., 1841) : Exchanges of hydrogen against sodium and of bicarbonate against chloride

The Chilean frog, Calyptocephallela gayi, placed in dilute NaCl solutions may pump Na⁺ and Cl^- at very different rates depending on the kind of bath solutions in which it was preadapted. Furthermore, Na⁺ and Cl^- may be absorbed from solutions in which the accompanying coion, such as sulfate and choline, respectively, is impermeant. In all these cases it is obligatory to postulate the existence of two ionic exchange mechanisms, Cl^- and Na⁺, being exchanged against endogenous anions and cations, respectively. It has been determined that Na⁺ is exchanged against endogenous H⁺ and that Cl^- is exchanged against HCO₃^-. In animals pumping Na⁺ and Cl^- from dilute NaCl solutions Na⁺ or Cl^- uptake may be selectively inhibited, while the flux of the accompanying ion remains unchanged. This is considered to be an additional proof that both Na⁺ and Cl^- fluxes are always independent. The role of the ionic exchange mechanisms in the direct regulation of the Na⁺ and Cl^- levels in the internal medium is discussed as well as their relationship in the regulation of the acid-base equilibrium; other physioecological considerations have been treated.     

Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Editorial

The voltage dependence of the rat renal type II Na⁺/P_i cotransporter (NaP_i-2) was investigated by expressing NaP_i-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (P_i) induced inward currents (I_p) in the presence of 96 mM Na⁺ over the potential range −140 ≤ V ≤ +40 mV. With P_i as the variable substrate, the apparent affinity constant (K _m ^Pi) was strongly dependent on Na⁺, increasing sixfold for a twofold reduction in external Na⁺. K _m ^Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na⁺. The Hill coefficient was close to unity and the predicted maximum I_p (I_pmax) was 40% smaller at 50 mM Na⁺. With Na⁺ as the variable substrate, K _m ^Na was weakly dependent on both P_i and voltage, the Hill coefficient was close to 3 and I_pmax was independent of P_i at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na⁺-dependent manner, indicating the existence of uncoupled Na⁺ slippage. Voltage steps induced pre–steady state relaxations typical for Na⁺-coupled cotransporters. NaP_i-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na⁺, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V_0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e⁻. V_0.5 became more negative as Na⁺ was reduced. P_i suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V_0.5 to depolarizing potentials and suppressed relaxations in the absence of Na⁺, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na⁺ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na⁺ binding step.     

EFFECTS OF HEXYLRESORCINOL ON VALONIA

The effects on Valonia of guaiacol and hexylresorcinol are similar but the latter is more effective. Both substances lower or abolish the potassium effect; i.e., the ability of the cell to distinguish electrically between Na⁺ and K⁺. Both substances change the order of mobilities so that v _Cl > u _Na becomes u _Na > v _Cl or u _Na = v _Cl.     

Sodium,potassium, chloride and proline concentrations of chloroplasts isolated from a halophyte,Mesembryanthemum crystallinum L.

Concentrations of four major solutes (Na⁺, K⁺, Cl^-, proline) were determined in isolated, intact chloroplasts from the halophyte Mesembryanthemum crystallinum L. following long-term exposure of plants to three levels of NaCl salinity in the rooting medium. Chloroplasts were obtained by gentle rupture of leaf protoplasts. There was either no or only small leakage of inorganic ions from the chloroplasts to the medium during three rapidly performed washing steps involving precipitation and re-suspension of chloroplast pellets. Increasing NaCl salinity of the rooting medium resulted in a rise of Na⁺ und Cl^- in the total leaf sap, up to approximately 500 and 400 mM, respectively, for plants grown at 400 mM NaCl. However, chloroplast levels of Na⁺ und Cl^- did not exceed 160–230 and 40–60 mM, respectively, based upon a chloroplast osmotic volume of 20–30 l per mg chlorophyll. At 20 mM NaCl in the rooting medium, the Na⁺/K⁺ ratio of the chloroplasts was about 1; at 400 mM NaCl the ratio was about 5. Growth at 400 mM NaCl led to markedly increased concentrations of proline in the leaf sap (8 mM) compared with the leaf sap of plants grown in culture solution without added NaCl (proline 0.25 mM). Although proline was fivefold more concentrated in the chloroplasts than in the total leaf sap of plants treated with 400 mM NaCl, the overall contribution of proline to the osmotic adjustment of chloroplasts was small. The capacity to limit chloroplast Cl^- concentrations under conditions of high external salinity was in contrast to an apparent affinity of chloroplasts for Cl^- under conditions of low Cl^- availability.Abbreviation Chl chlorophyll     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Activation of Slo2.1 channels by niflumic acid

Slo2.1 channels conduct an outwardly rectifying K⁺ current when activated by high [Na⁺]_i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na⁺. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC₅₀ = 2.1 mM) or flufenamic acid (EC₅₀ = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K⁺]_e, the conductance–voltage (G-V) relationship had a V_1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V_1/2 to more negative potentials (EC₅₀ = 2.1 mM) and increased the minimum value of G/G_max (EC₅₀ = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V_1/2 of the G-V relationship was shifted to more positive potentials when [K⁺]_e was elevated from 1 to 300 mM (EC₅₀ = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K⁺]_e dependency (EC₅₀ = 23.5 mM). Conductance was also [Na⁺]_e dependent. Outward currents were reduced when Na⁺ was replaced with choline or mannitol, but unaffected by substitution with Rb⁺ or Li⁺. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K⁺]_e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K⁺]_e and [Na⁺]_e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na⁺.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V _e , which attends net Cl^– absorption,J _Cl ^net , and to assess how ADH might augment bothJ _Cl ^met andV _e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R _c /R _bl , and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10^–4 m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V _a . Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10^–4 m luminal furosemide reducedV _e andJ _Cl ^net to near zero values and hyperpolarized bothV _a andV _bl , the voltage across basolateral membranes; however, the depolarization ofV _bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV _a , Thus ADH-dependent increases inV _b were referable to greater depolarizations ofV _bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV _a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV _bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl^– current. Thus an increase in intracellular Cl^– activity may have contributed to the depolarization ofV _bl during net Cl^– absorption, and the intracellular Cl^– activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl^– cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl^– cotransport units in apical plasma membranes.     

Kinetic study on the effects of intracellular K++ and Na++ on Na++, K++, Cl+− cotransport of HeLa cells by Rb++ influx determination

Summary The effects of intracellular K⁺ and Na⁺ (K⁺ c, Na⁺ c) on the Na⁺,K⁺,Cl^+– cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb⁺ influx (JRb) at various intracellular concentrations of K⁺ and Na⁺ ([K⁺]c, [Na⁺]c). When [K⁺]c was increased and [Na⁺]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb⁺ or Na⁺ concentration ([Rb⁺]e, [Na⁺]e) was stimulated. However, the apparent K _0.5 for Rb⁺ e or Na⁺ e remained unchanged and the ratio of the apparent K ^+0.5 for K⁺ c and the apparent K _i for Na⁺ c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K _0.5 for Rb⁺ e. These results show that K⁺ c stimulates and Na⁺ c inhibits JRb, without change in the affinities of the pathway for Rb⁺ e and Na⁺ e. The affinity for K⁺ c is slightly lower than that for Na⁺ c. Hypertonicity enhances JRb without any change in the affinity for Rb⁺ e. We derived a kinetic equation for JRb with respect to K⁺ c and Na⁺ c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb⁺ e binds to the external K⁺ binding site of the pathway after the binding of K⁺ c to the internal regulatory site.We thank Mr. T. Masuya for technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 03202136) from the Japanese Ministry of Education, Science and Culture.     

On the Mechanism of Sodium Extrusion across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium efflux (J_out^Na) across the irrigated trout gill was rapid in sea water (SW), but only about 25 % as large in fresh water (FW). The difference correlated with a change in the potential difference across the gill (TEP). The latter was about +10 mV (blood positive) in SW, but –40 mV in FW. Both flux and electrical data indicated that gills in this fish are permeable to a variety of cations including Na⁺, K⁺, Mg²⁺, choline, and Tris. They are less permeable to anions; P_Na:P_K:P_Cl was estimated to be 1:10:0.3, and P_Cl > P_gluconate. The TEP was shown to be a diffusion potential determined by these permeabilities and the extant ionic gradients in SW, FW as well as in other media. J_out^Na appeared to be diffusive in all of the experiments undertaken. Exchange diffusion need not be posited, and the question of whether there is an active component remains open.     

渗透胁迫和离子毒害对转Bt基因棉幼苗生长及离子分布的影响

研究了渗透和盐胁迫处理对转Bt基因抗虫棉(Gossypium hirsutum) 99B种子的萌发和幼苗生长的影响,以及幼苗不同器官离子吸收和分配的差异。结果表明:渗透和盐胁迫均对转Bt基因抗虫棉幼苗的生长有抑制作用,其中PEG的抑制作用最强,而3种盐的抑制程度以CaCl₂>NaCl>Na₂SO₄,且在Na⁺含量相同时,Cl^-的毒害大于SO₄^2-。渗透胁迫下使根、茎和叶中的Na⁺和Cl^-含量提高,K⁺、Ca²⁺、SO₄^2-含量和K⁺/Na⁺、Ca²⁺/Na⁺和SO₄^2-/Cl^-比值降低,且地上部的变化幅度大于地下部的,其中以PEG的影响最为显著,其次是CaCl₂,Na₂SO₄处理最弱。这些说明,转Bt基因抗虫棉99B的耐盐性较弱。     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Intracellular Cl? Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J _Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K _m values (∼120 mM). However, the maximal J _Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J _Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.     

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K−Cl cotransport

Pathways of K⁺ movement across the erythrocyte membrane of frog Rana temporaria were studied using ⁸⁶Rb as a tracer. The K⁺ influx was significantly blocked by 0.1 mmol·l^-1 ouabain (by 30%) and 1 mmol·l^-1 furosemide (by 56%) in the red cells incubated in saline at physiological K⁺ concentration (2.7 mmol·l^-1). Ouabain and furosemide had an additive effect on K⁺ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K⁺ influx saturated as f(K⁺)_e with apparent K _m values for external K _e ⁺ concentration of 0.96±0.11 and 4.6±0.5 mmol·l^-1 and V _max of 0.89±0.04 and 2.8±0.4 mmol·l cells^-1·h^-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of K _e ⁺ medium concentration. Total K⁺ influx was significantly reduced when frog erythrocytes were incubated in NO _- ³ medium. Furosemide did not affect K⁺ transport in frog red cells in NO ₃ ^- media. At the same K _e ⁺ concentration the ouabain-furosemide-insensitive K⁺ influx in Cl^- medium was significantly greater than that in NO _- ³ medium. We found no inhibitory effect of 1 mmol·l^-1 furosemide on Na⁺ influx in frog red cells in Cl^- medium. K⁺ loss from the frog erythrocytes in a K⁺-free medium was significantly reduced (mean 58%) after replacement of Cl^- with NO _- ³ . Furosemide (0.5 mmol·l^-1) did not produce any significant reduction in the K⁺ loss in both media. The Cl^--dependent component of K⁺ loss from frog red cells was 5.7±1.2 mmol·l^-1·h^-1. These results indicate that about two-thirds of the total K⁺ influx in frog erythrocytes is mediated by a K–Cl cotransport which is only partially blocked by furosemide.Abbreviations DMSO dimethyl sulphoxide - K _e ⁺ external concentration of K⁺ - K _m apparent Michaelis constant for external - K⁺ K _e ⁺ at V _max/2 - RBC red blood cell(s) - V _max maximal velocity of the unidirectional K⁺ influx - TRIS tris(hydroxymethyl)aminomethane     

The Actual Ionic Nature of the Leak Current through the Na/Glucose Cotransporter SGLT1

Expression of the Na⁺/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a “Na⁺ leak” and represents some 5-10% of the maximal Na⁺/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (V_R) of ≈−15 mV as observed for wt SGLT1. V_R showed only a modest negative shift when extracellular Na⁺ concentration ([Na⁺]_o) was lowered and it was completely insensitive to changes in extracellular Cl⁻. When extracellular pH (pH_o) was decreased from 7.5 to 6.5 and 5.5, V_R shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pH_o but other ions must be involved at pH_o 7.5. In the presence of 15 mM [Na⁺]_o (pH_o = 7.5), addition of 75 mM of either Na⁺, Li⁺, Cs⁺, or K⁺ generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter.     

Salt-induced NO<Subscript>3</Subscript><Superscript>-</Superscript> uptake inhibition in cowpea roots is dependent on the ionic composition of the salt and its osmotic effect

Salinity remarkably inhibits NO₃ ^- uptake but the mechanisms are not well understood. This study was addressed to elucidate the role of ionic and osmotic components of salinity on NO₃ ^- influx and efflux employing classic kinetics involving a low affinity transport system (LATS) and a high affinity transport system (HATS). In the presence of KCl, NaCl, and Na₂SO₄ at 100 mM concentrations, in both LATS and HATS, Michaelis constant (K_m) was similar for the three salts and maximum rate (V_max) decreased as follows: KCl > NaCl > Na₂SO₄, compared to control indicating a non-competitive interaction with NO₃ ^-. Unexpectedly, iso-osmotic solutions (osmotic potential Ψ_π = -0.450) of polyethylene glycol (PEG, 17.84 %, v/v) and mannitol (100 mM) remarkably increased K_m in both the LATS and the HATS, but V_max did not change indicating a competitive inhibition. Under the PEG and mannitol treatments, K_m and V_max were higher than under the salt treatments. The salts increased slightly NO₃ ^- efflux in the following order KCl > NaCl > Na₂SO₄. In contrast, mannitol strongly stimulated and the PEG inhibited NO₃ ^- efflux. The obtained data reveal that salinity effects were not dependent on the anion type (Cl^- versus SO₄ ^2-) indicating a non-competitive inhibition mechanism between Cl^- and NO₃ ^-. In contrast, the cation types (K⁺ versus Na⁺) had a pronounced effect. The osmotic component is important to net NO₃ ^- uptake affecting remarkably the influx in both LATS and HATS components of cowpea roots.     

Ionic exchanges in isolated and open-circuited toad skin

Summary Net influxes of Na and Cl and effluxes of K and H (J _Na,J _Cl,J _K andJ _H) and volume flowJ _v across isolated open-circuited toad skins were measured using rotating chambers and a small volume of external solution, the ion fluxes being determined by chemical analysis of the external solution, in the range of 0.2 to 5.0mm external Na concentration. In this concentration range, with skin potential varying with (Na) _e ,J _Na is a linear function of the Na electrochemical potential difference across the skin, as expected on irreversible thermodynamic grounds. TheL _Na coefficient calculated asJ _Na/ _Na is equal to 5.5×10^–12 mole² joule^–1 cm^–2 min^–1, which is similar to values obtained for the same species in the short-circuited state and in higher ranges of (Na) _e . A positive correlation is observed betweenJ _Na andJ _K whenJ _Na varied with (Na) _e and also whenJ _Na varies in randomly selected skins. Antidiuretic hormone stimulatesJ _Na,J _K andJ _v in the range of 0.2 to 5.0mm (Na) _e and lowers the Na concentration in the equivalent solution absorbed by the skin (calculated asJ _Na/J _v ). Substitution of external Cl by SO₄ has no effect onJ _Na,J _K andJ _H and also in the skin potential in the range of (Na) _e studied. Substitution of external Na by K abolishesJ _Cl and reverses the skin polarity, the external solution now being positive to the internal one. Na removal from the external solution also reducesJ _K almost to zero.J _H is significantly reduced in this condition; however, a basal secretion still persists with (Na) _e equal to zero. The results of these experiments can be tentatively interpreted in terms of electrical coupling between ion fluxes, since only the procedures that result in alterations of skin potential are followed by changes in the rates of ion transport. The existence of other coupling mechanisms cannot be ruled out.     

THE KINETICS OF PENETRATION : I. EQUATIONS FOR THE ENTRANCE OF ELECTROLYTES

When the only solute present is a weak acid, HA, which penetrates as molecules only into a living cell according to a curve of the first order and eventually reaches a true equilibrium we may regard the rate of increase of molecules inside as See PDF for Equation where P_M is the permeability of the protoplasm to molecules, M_o, denotes the external and M_i the internal concentration of molecules, A_i denotes the internal concentration of the anion A^- and See PDF for Equation (It is assumed that the activity coefficients equal 1.) Putting P_MF_M = V_M, the apparent velocity constant of the process, we have See PDF for Equation where e denotes the concentration at equilibrium. Then See PDF for Equation where t is time. The corresponding equation when ions alone enter is See PDF for Equation. where K is the dissociation constant of HA, P_A is the permeability of the protoplasm to the ion pair H⁺ + A^-, and A_ie denotes the internal concentration of A_i at equilibrium. Putting P_AKF_M = V_A, the apparent velocity constant of the process, we have See PDF for Equation and See PDF for Equation When both ions and molecules of HA enter together we have See PDF for Equation where S_i = M_i + A_i and S_ie is the value of S_i at equilibrium. Then See PDF for Equation V_M, V_A, and V_MA depend on F_M and hence on the internal pH value but are independent of the external pH value except as it affects the internal pH value. When the ion pair Na⁺ + A^- penetrates and Na_i = BA_i, we have See PDF for Equation and See PDF for Equation where P _NaA is the permeability of the protoplasm to the ion pair Na⁺ + A^-, Na_o and Na_i are the external and internal concentrations of Na⁺, See PDF for Equation, and V _Na is the apparent velocity constant of the process. Equations are also given for the penetration of: (1) molecules of HA and the ion pair Na⁺ + A^-, (2) the ion pairs H⁺ + A^- and Na⁺ + A^-, (3) molecules of HA and the ion pairs Na⁺ + A^- and H⁺ + A^-. (4) The penetration of molecules of HA together with those of a weak base ZOH. (5) Exchange of ions of the same sign. When a weak electrolyte HA is the only solute present we cannot decide whether molecules alone or molecules and ions enter by comparing the velocity constants at different pH values, since in both cases they will behave alike, remaining constant if F_M is constant and falling off with increase of external pH value if F_M falls off. But if a salt (e.g., NaA) is the only substance penetrating the velocity constant will increase with increase of external pH value: if molecules of HA and the ions of a salt NaA. penetrate together the velocity constant may increase or decrease while the internal pH value rises. The initial rate See PDF for Equation (i.e., the rate when M_i = 0 and A_i = 0) falls off with increase of external pH value if HA alone is present and penetrates as molecules or as ions (or in both forms). But if a salt (e.g., NaA) penetrates the initial rate may in some cases decrease and then increase as the external pH value increases. At equilibrium the value of M_i equals that of M_o (no matter whether molecules alone penetrate, or ions alone, or both together). If the total external concentration (S_o = M_o + A_o) be kept constant a decrease in the external pH value will increase the value of M_o and make a corresponding increase in the rate of entrance and in the value at equilibrium no matter whether molecules alone penetrate, or ions alone, or both together. What is here said of weak acids holds with suitable modifications for weak bases and for amphoteric electrolytes and may also be applied to strong electrolytes.