The sodium cycle. II. Na-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na⁺ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na⁺-motive respiratory chain of V. alginolyticus. Na⁺ loaded cells incubated in a K⁺ or Li⁺ medium fail to synthesize ATP in response to lactate addition. The addition of Na⁺ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na⁺ level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na⁺ in the Na⁺-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔPNa, i.e., [Na⁺]_in > [Na⁺]_out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K⁺-loaded cells incubated in a high Na⁺ medium, i.e., when ΔpNa of the proper direction ([Na⁺]_in < [Na⁺]_out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K⁺ medium (ΔpNa is low) is decreased by CCCP even without monensin. Artificial formation of ΔpNa by adding 0.25 M NaCl to the K⁺-loaded cells (Na⁺ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na⁺ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na⁺ instead of H⁺ as the coupling ion.     

Extracellular Na removal enhances granule secretion in platelets 3-evidence that Na/H exchange is inhibitory to secretion induced by some agonists

The effect of extracellular Na⁺ ([Na⁺]_e) removal on agonist-induced granule secretion in platelets in relation to [ph]_i and [Ca²⁺]_i changes was investigated. Substitution of [Na⁺]_e with choline⁺ of K⁺ resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC₈. Increases in [Ca²⁺]_i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH]_i increases induced by thrombin, U46619, PMA and diC₈ were abolished and a drop in [pH]_i (0.05–0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH]_i and greatly increased secretion with all the agonists, particularly in the absence of [Na⁺]_e, clear evidence that [pH]_i rises due to Na⁺/H⁺ exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH₄Cl, which restored the increase in [pH]_i in the absence of [Na⁺]_e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin-induced secretion was observed when Na⁺ was replaced with Li⁺, which allowed a normal increase in [pH]_i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH]_i rise and potentiated thrombin-induced secretion. With collagen and U46619, the results suggest that removal of [Na⁺]_e per se rather than inhibition of Na⁺/H⁺ exchange results in enhanced secretion. It is concluded that [Na⁺]_e per se and [pH]_i elevations via Na⁺/H⁺ exchange both have important inhibitory roles in the control of platelet granule secretion.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Glycinebetaine-induced modulation of antioxidant enzymes activities and ion accumulation in two wheat cultivars differing in salt tolerance

Modulation of water relations, activities of antioxidant enzymes and ion accumulation was assessed in the plants of two wheat cultivars S-24 (salt tolerant) and MH-97 (moderately salt sensitive) subjected to saline conditions and glycinebetaine (GB) applied foliarly. Different levels of GB, i.e., 0 (unsprayed), 50 and 100 mM (in 0.10% Tween-20 solution) were applied to the wheat plants at the vegetative growth stage. Leaf water potential, leaf osmotic potential and turgor potential were decreased due to salt stress. Salt stress increased the Na⁺ and Cl⁻ accumulation coupled with a decrease in K⁺ and Ca²⁺ in the leaves and roots of both cultivars thereby decreasing tissue K⁺/Na⁺ and Ca²⁺/Na⁺ ratios. Furthermore, salt stress decreased the activities of superoxide dismutase (SOD), whereas it increased the activities of catalase (CAT) and peroxidase (POD) in both wheat cultivars. However, accumulation of GB in the leaves of both wheat cultivars was consistently increased with an increase in concentration of exogenous GB application under both non-saline and saline conditions. Accumulation of Na⁺ was decreased with an increase in K⁺ accumulation upon a consistent increase in GB accumulation under salt stress conditions thereby resulting in better K⁺/Na⁺ and Ca²⁺/Na⁺ ratios in the leaves and roots. High accumulation of GB and K⁺ mainly contributed to osmotic adjustment, which is one of the factors known to be responsible for improving growth and yield under salt stress. The activities of all antioxidant enzymes, SOD, CAT and POD were enhanced by GB application in cv. MH-97 under saline conditions, whereas all these except SOD were reduced in cv. S-24. It is likely that both applied GB and intrinsic SOD scavenged ROS in the tolerant cultivar thereby resulting into low activities of CAT and POD enzymes under salt stress. In conclusion, the adverse effects of salt stress on wheat can be alleviated by the exogenous application of 100 mM GB by modulating activities of antioxidant enzymes and changes in water relations and ion homeostasis. Furthermore, effectiveness of GB application on regulation of activities of antioxidant enzymes was found to be cultivar-specific.     

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide

In addition to the (Na⁺+K⁺)ATPase another P-ATPase, the ouabain-insensitive Na⁺-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg²⁺, MgATP and furosemide on the Na⁺-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na⁺-ATPase at pH 7.0 are: (a) K_0.5 for Na⁺=8.9±2.2 mM; (b) K_0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg²⁺: one stimulatory (K_0.5=0.20±0.06 mM) and other inhibitory (I_0.5=1.1±0.4 mM); and (d) I_0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na⁺ (K_0.5=19.5±0.4) and MgATP (K_0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg²⁺ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na⁺ (K_0.5=18.7±1.5 mM) and furosemide (I_0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg²⁺. The data presented in this paper indicate that the modulation of the Na⁺-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg²⁺ and MgATP complex may play an important role in the Na⁺-ATPase physiological regulatory mechanisms.     

Changes in action potentials and intracellular ionic homeostasis in a ventricular cell model related to a persistent sodium current in SCN5A mutations underlying LQT3

In LQT3 patients, SCN5A mutations induce ultraslow inactivation of a small fraction of the hNav1.5 current, i.e. persistent Na⁺ current (I_pNa). We explored the time course of effects of such a change on the intracellular ionic homeostasis in a model of guinea-pig cardiac ventricular cell [Pasek, M., Simurda, J., Orchard, C.H., Christé, G., 2007b. A model of the guinea-pig ventricular cardiomyocyte incorporating a transverse–axial tubular system. Prog. Biophys. Mol. Biol., this issue]. Sudden addition of I_pNa prevented action potential (AP) repolarization when its conductance (g_pNa) exceeded 0.12% of the maximal conductance of fast I_Na (g_Na). With g_pNa at 0.1% g_Na, the AP duration at 90% repolarization (APD₉₀) was initially lengthened to 2.6-fold that in control. Under regular stimulation at 1 Hz it shortened progressively to 1.37-fold control APD₉₀, and intracellular [Na⁺]_i increased by 6% with a time constant of 106 s. Further increasing g_pNa to 0.2% g_Na caused an immediate increase in APD₉₀ to 5.7-fold that in control, which decreased to 2.2-fold that in control in 30 s stimulation at 1 Hz. At this time diastolic [Na⁺]_i and [Ca²⁺]_i were, respectively, 34% and 52% higher than in control and spontaneous erratic SR Ca release occurred.

In the presence of I_pNa causing 46% lengthening of APD₉₀, the model cell displayed arrhythmogenic behaviour when external [K⁺] was lowered to 5 mM from an initial value at 5.4 mM. By contrast, when K⁺ currents I_Kr and I_Ks were lowered in the model cell to produce the same lengthening of APD₉₀, no proarrhythmic behaviour was observed, even when external [K⁺] was lowered to 2.5 mM.     

Responses of apical ion fluxes to NaCl stress in Elaeagnus angustifolia seedlings

Aims Elaeagnus angustifolia is one of the most salt-tolerant species. The objective of this study was to understand the mechanisms of ion transporation in E. angustifolia exposed to different salt concentrations through manipulations of K⁺/Na⁺ homeostasis.
Methods Seedlings of two variants of the species, Yinchuan provenance (YC, salt-sensitive type) and the Alaer provenance (ALE, salt-tolerant type), were treated with three different NaCl application modes, and the ion fluxes in the apical regions were measured using non-invasive micro-test technology (NMT). In mode 1, Na⁺ and K⁺ fluxes were measured after 150 mmol·L^-1 NaCl stress lasted for 24 h. In mode 2, K⁺ and H⁺ fluxes were quantified with a transient stimulation of NaCl solution. In mode 3, Amiloride (Na⁺/H⁺ antiporters inhibitor) and tetraethylammonium (TEA, K⁺ channel inhibitor) was used to treat apical regions of E. angustifolia seedlings after NaCl stress for 24 h, respectively.
Important findings Under NaCl stress for 24 h, net effluxes of Na⁺ and K⁺ were increased significantly. The net Na⁺ effluxes of YC provenance seedlings (720 pmol·cm^-2•s^-1) were lower than that of ALE provenance (912 pmol·cm^-2·s^-1), but the net K⁺ efflux was higher in YC provenance. Under the instantaneous NaCl stimulation, net K⁺ efflux was remarkably increased, with the net K⁺ efflux of YC provenance always higher than that of ALE provenance. Interestingly, H⁺ at the apical regions was found from influx to efflux, with the net H⁺ efflux of ALE provenance greater than that of the YC provenance. Under the NaCl and NaCl + Amiloride treatment, the net Na⁺ efflux of ALE provenance seedlings was higher than that of YC provenance, while the net K⁺ efflux was less in ALE provenance seedlings. On the other hand, the differences in net Na⁺ and K⁺ effluxes were insignificant between the two provenances under the control group and NaCl + TEA treatment. In conclusion, NaCl stress caused Na⁺ accumulation and K⁺ outflows of E. angustifolia seedlings; The E. angustifolia seedlings utilize Na⁺/H⁺ antiporters to reduce Na⁺ accumulation by excretion; and the maintenance of K⁺/Na⁺ homeostasis in salt-tolerant E. angustifolia provenance seedlings roots accounted for a greater Na⁺ extrusion and a lower K⁺ efflux under NaCl stress. Results from this study provide a theoretical basis for further exploring salt-tolerant E. angustifolia germplasm resource.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

The (Na + K)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl2

Na⁺-dependent ADP/ATP exchange activity, of a (Na⁺ + K⁺)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of ¹⁴C into ATP during incubations with unlabeled ATP and [¹⁴C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl₂ concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg²⁺, Mg · ATP, and Mg · ADP present, with a range of MgCl₂, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg²⁺, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg²⁺, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg²⁺, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.     

The effect of ionic strength on melting of DNA modified by platinum(II) complexes

Thermal denaturation of calf thymus DNA modified by antitumor cis-diamminedichloroplatinum(II) (cis-DDP) and by two related Pt(II) compounds which had been shown to be clinically inefective, viz. trans-diamminedichloroplatinum(II) (trans-DDP) or monodentate diethylenetriaminechloroplatinum(II) chloride {[Pt(dien)Cl)]Cl}, was studied by monitoring changes of absorbance at 260 nm. The melting of DNA platinated to different levels was investigated in neutral media containing varying concentrations of Na⁺. It has been shown that the ionic strength has a strong influence on the character and magnitude of changes in the melting temperature of DNA (T_m) induced by the platination. The modification of DNA by either platinum complex used in this work results in an increase of T_m if DNA melting is measured in media containing low Na⁺ concentrations (ca. 1 mM). This effect is reversed at higher Na⁺ concentrations. The concentration of Na⁺ at which this reversal occurs is, however, markedly lower for DNA modified by cis-DDP than for DNA modified by the other two platinum complexes. These results have been iterpreted to mean that at least three factors affect the thermal stability of DNA modified by the platinum(II) complexes: stabilization effects of the positive charge on the platinum moiety and of interstrand cross-links, and a destabilization effect of conformational distortions in DNA. Thus, in order to compare and interpret the melting behavior of DNA modified by different compounds, a great attention has to be paid to the composition of the medium in which the melting experiments are carried out.     

Effect of cesium on the volume of the helix-coil transition of dA.dT polymers and their ligand complexes

The pressure dependence of the helix–coil transition of poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of NaCl and CsCl at concentrations between 10 and 200 mM is reported and used to calculate the accompanying volume change. We also investigated the binding parameters and volume change of ethidium bromide binding with poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of these two salts. The volume change of helix–coil transition of poly(dA)∙poly(dT) in Cs⁺-containing solutions differs by less than 1 cm³ mol^− 1 from the value measured when Na⁺ is the counter-ion. We propose that this insensitivity towards salt type arises if the counter-ions are essentially fully hydrated around DNA and the DNA conformation is not significantly altered by salt types. Circular dichroism spectroscopy showed that the previously observed large volumetric disparity for the helix–coil transition of poly[d(A-T)]·poly[d(A-T)] in solutions containing Na⁺ and Cs⁺ is likely result of a Cs⁺-induced conformation change that is specific for poly[d(A-T)]·poly[d(A-T)]. This cation-specific conformation difference is mostly absent for poly(dA)∙poly(dT) and EB bound poly[d(A-T)]·poly[d(A-T)].     

Physiological responses to salt stress in young umbu plants

Soil salinity affects plant growth and development due to harmful ion effects and water stress caused by reduced osmotic potential in the soil solution. In order to evaluate the effects of salt stress in young umbu plants, research was performed in green house conditions at the Laboratory of Plant Physiology at Federal Rural University of Pernambuco, Brazil. Growth, stomatal behaviour, water relations, and both inorganic and organic solutes were studied aiming for a better understanding of the responses of umbu plants to increasing salinity. Plants were grown in washed sand with Hoagland and Arnon nutrient solution with 0, 25, 50, 75, and 100 mM NaCl. Growth, leaf water potential, transpiration, and diffusive resistance were evaluated. Na⁺, K⁺, Cl⁻, soluble carbohydrates, and free amino acid contents were measured in several plant organs. Most variables were affected with salinity above 50 mM NaCl showing decreases in: number of leaves, plant height, stems diameter, and dry masses, and increases in root-to-shoot ratio. Reductions in ψ_pd were observed in plants grown under 75 and 100 mM NaCl. All salt levels above zero increased Na⁺ and Cl⁻ contents in leaves. However, K⁺ content was not affected. Na⁺ and Cl⁻ in stems and roots reached saturation in treatments above 50 mM NaCl. Organic solute accumulation in response to salt stress was not observed in umbu plants. These results suggest that umbu plants tolerate salt levels up to 50 mM NaCl without showing significant physio-morphological alterations.     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Possible interaction of [H]glutamate binding sites with anion channels in rat neural tissues

The effect of various ions on [³H] -glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1–100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2°C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30°C. Ammonium chloride (1–100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10⁻³ M) as well as by inhibitors of anion transport including ethacrynic acid (10⁻³ M) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (10⁻⁴−10⁻³ M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of -aspartic acid resulted in the complete abolition of the Na⁺-dependent binding while sparing the Cl⁻-dependent binding. Scatchard analysis revealed that Cl⁻ ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na⁺ ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10⁻⁵−10⁻³ M) inhibited the Cl⁻-dependent binding of [³H]Glu to a significantly greater extent than the inhibition on Na⁺-dependent binding. acid and kainic acid exerted no preventive action on the basal, Cl⁻-dependent and Na⁺-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [³H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [³H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na⁺ ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures.

These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.     

盐胁迫下菊芋根系脱落酸对钠离子转运和光系统Ⅱ的影响

通过根系施加脱落酸(ABA)合成抑制剂钨酸钠,研究盐胁迫(150 mmol·L^-1 NaCl)下菊芋根系ABA信号对根系Na⁺转运、叶片Na⁺积累和光系统Ⅱ(PSⅡ)的影响。结果表明:钨酸钠抑制盐胁迫下根系ABA合成,降低根系Na⁺外排,提高根系Na⁺向叶片的转运系数。盐胁迫增加叶片Na⁺含量,没有影响叶片膜脂过氧化、PSⅡ反应中心蛋白合成和PSⅡ最大光化学效率(F_v/F_m)。根系ABA合成受抑制,显著增加盐胁迫下叶片Na⁺积累,加剧叶片膜脂过氧化,损伤PSⅡ反应中心蛋白,显著降低F_v/F_m,诱发PSⅡ光抑制。总之,盐胁迫下菊芋根系ABA信号诱导根系Na⁺外排,抑制Na⁺向地上部转运,有利于减少叶片Na⁺积累,防御PSⅡ氧化损伤。     

A significant fraction of calcium transients in intact Guinea Pig ventricular myocytes is mediated by Na-Ca exchange

Ca²⁺ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca²⁺ changes across the cell, suggesting the presence of significant spatial Ca²⁺ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca²⁺ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca²⁺]_i elicited by membrane depolarization. The overall spatial distribution of [Ca²⁺]_i changes appeared unchanged. Ca²⁺ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the exchange mechanism. Conversely, when the reversal potential of the exchange was shifted to negative potentials by lowering [Na⁺]₀ or by increasing [Na⁺]_i by treatment with 20 μM monensin, the amplitude of these Ca²⁺ transients increased. Ca²⁺ transients elicited by membrane depolarization and largely mediated by reverse operation of Na⁺-Ca²⁺ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca²⁺ influx through the exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.     

Modification of isolated subunit c of the F1F0-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

Subunit c of the F₁F₀-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β- -maltoside with [¹⁴C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na⁺ subunit c was partially protected from labeling with [¹⁴C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [¹⁴C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na⁺ ions. The isolated c-subunit therefore apparently retains in part the Na⁺ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.     

Evidence that adrenaline activates key oxidative enzymes in rat liver by increasing intramitochondrial [Ca]

The effects of intramitochondrial Ca²⁺ on the activities of the Ca²⁺-sensitive intramitochondrial enzymes, (i) pyruvate dehydrogenase (PDH) phosphate phosphatase, and (ii) oxoglutarate dehydrogenase (OGDH), were investigated in intact rat liver mitochondria by measuring (i) the amount of active PDH (PDHa) and (ii) the rate of decarboxylation of -[1-¹⁴C]oxoglutarate (at non-saturating [oxoglutarate]), at different concentrations of extramitochondrial Ca²⁺. In the presence of Na²⁺ and Mg²⁺, both PDH and OGDH could be activated by increases in extramitochondrial [Ca²⁺] within the expected physiological range (0.05–5 μM). When liver mitochondria were prepared from rats treated with adrenaline, and then incubated in Na-free media containing EGTA, both PDH and OGDH activities were found to be enhanced. Evidence is presented that the activation of these enzymes by adrenaline is brought about by a mechanism involving increases in intramitochondrial [Ca²⁺].     

NaCl胁迫对4种豆科树种幼苗生长和K+、Na+含量的影响

以合欢、刺槐、国槐和皂荚4种豆科树种盆栽实生幼苗为试验材料,研究了NaCl胁迫下4个树种幼苗的生长、耐盐临界浓度和Na⁺、K⁺含量的变化,并对其耐盐性进行了比较.结果表明：NaCl胁迫抑制了4个树种幼苗的生长,苗木的干物质积累量减小、根冠比增大,尤其对合欢和皂荚的影响较大;以相对干质量降至对照组50%时的NaCl浓度作为生长临界NaCl浓度(C₅₀)指标,4个树种的耐盐强弱顺序为：刺槐(5.0‰)＞国槐(4.5‰)＞皂荚(3.9‰)＞合欢(3.0‰);随NaCl浓度的增加,各树种幼苗根、茎、叶中Na⁺含量逐渐增加,K⁺含量先增加后减小（合欢根除外）,而K⁺/Na⁺差异较大.相同浓度NaCl胁迫下,幼苗器官的Na⁺分布为根＞茎＞叶,K⁺因树种和NaCl浓度不同而各异,以叶片中较多,K⁺/Na⁺为叶＞茎＞根.NaCl胁迫下,刺槐的K⁺含量和K⁺/Na⁺较高,地上部分Na⁺含量较低,幼苗干物质量大,耐盐性较强;而合欢的K⁺/Na⁺较小,高浓度NaCl胁迫下地上部分的Na⁺含量较高,幼苗干物质量小,耐盐性较差.苗木地上部分对K⁺的积累和根部对Na⁺的滞留是影响豆科树种耐盐性能的主要因素.