External K+ Deficiency Inhibits Photosynthetic Activity Through Superoxide Anion Production in Protoplasts Isolated from the Thallus of Ulva pertusa

To investigate the effects of potassium on photosynthetic activity in the green alga Ulva pertusa, we enzymatically isolated protoplasts from thallus samples. Photochemical quenching showed that protoplasts were capable of electron transport by photosystem II (PS II) during illumination. This quenching was dependent on external pH, with a reduced electron transport rate at pH >6.8 and less ability to use HCO ₃ ^? under alkaline conditions. In the presence of external Na⁺, K⁺ enhanced PS II quantum yield, indicating a functional role for K⁺ during photosynthesis. That yield was enhanced in a [K⁺]-dependent manner, with maximum activity at 100 mM. However, potassium alone did not maintain photochemical activity, and its addition supported photosynthetic O₂ evolution only in the presence of Na⁺. A deficiency of K⁺ led to the production of superoxide anions. Because of that generation, activities of superoxide dismutase and ascorbate peroxidase, two key enzymes involved in scavenging reactive oxygen species in the water–water cycle, also increased during such stress. These results strongly suggest that a series of ROS-scavenging systems are initiated in Ulva chloroplasts in response to K⁺ deficiency and that enzyme activities might protect algal cell photosynthesis.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Decreased Production of the Superoxide Anion Radical in Neutrophils Exposed to a Near-Null Magnetic Field

Biophysics - This paper reports that pre-incubation of a neutrophil suspension in the presence of a near-null magnetic field produced using a system of magnetic shields (a residual constant...     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

External Copper Inhibits the Activity of the Large-Conductance Calcium- and Voltage-sensitive Potassium Channel from Skeletal Muscle

We have characterized the effect of external copper on the gating properties of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle, incorporated into artificial bilayers. The effect of Cu2+ was evaluated as changes in the gating kinetic properties of the channel after the addition of this ion. We found that, from concentrations of 20 microM and up, copper induced a concentration- and time-dependent decrease in channel open probability. The inhibition of channel activity by Cu2+ could not be reversed by washing or by addition of the copper chelator, bathocuproinedisulfonic acid. However, channel activity was appreciably restored by the sulfhydryl reducing agent dithiothreitol. The effect of copper was specific since other transition metal divalent cations such as Ni2+, Zn2+ or Cd2+ did not affect BK(Ca) channel activity in the same concentration range. These results suggest that external Cu2+-induced inhibition of channel activity was due to direct or indirect oxidation of key amino-acid sulfhydryl groups that might have a role in channel gating.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

烟草根皮层原生质体质膜钾通道的特性研究

采用膜片钳技术对烟草根皮层原生质体质膜上的钾通道进行全细胞记录,从而深入研究烟草K^＋的吸收机制和调控机理。结果表明,内向钾通道在膜电压低于-40mV时,可以被K^＋激活。内向电流可以被钾通道的专一抑制剂TEA^＋抑制。动力学分析表明内向钾电流产生的K^＋表观解离常数（Km）≈15．2mmol／L,类似于低亲和性钾通道。该通道具有依赖于胞外K^＋浓度的特性,对胞外NH4^＋、Ca^2＋、Mg^2＋浓度变化反应敏感,内向K^＋电流可被不同程度地抑制。     

Stimulating Effects of Mercuric-and Silver Ions on the Superoxide Anion Production in Human Polymorphonuclear Leukocytes

In a survey of a number of heavy metal ions for effects on the oxidative metabolism (respiratory burst) of human polymorphonuclear leukocytes (neutrophils) we have found that mercury(II) and silver ions in micromolar concentration significantly increase the production of superoxide anions in cells, initiated by formyl-methionyl-leucylphenylalanine (fMLP). The stimulation of radical formation induced by a certain ion concentration varied considerably in cells isolated from different blood donors, from a moderate increase to a very large (up to 400% of control values). When the soluble stimulator phorbol myristate acetate (PMA) or the particulate stimulator Zymosan were used to initiate the cell respiratory burst, no additional stimulating effects by the metal ions on superoxide anion formation were observed. This fact might indicate that the effect of the metal ions on the fMLP-dependent initiation of cell activity is a mechanism coupled to the interaction between the chemotactic peptide and its corresponding receptor molecules on the cell surface.

By increasing the concentration of silver ions during pre-incubation of resting neutrophils, a spontaneous activation of the cells could be recorded at a concentration exceeding 5 μM. However, the silver ion concentration at which such spontaneous initiation of the respiratory burst occurred varied significantly between blood samples from different donors with a concentration range of 5 to 15 μM. This effect could not be shown for mercuric ions due to the toxicity of the metal above 5 μM. Blood samples from some donors contained neutrophils that could be activated by either mercuric- or silver ions at concentration as low at 1 μM.

The spontaneous activation of neutrophils with elevated concentrations of silver ions is kinetically similar to the PMA-induced. The onset of superoxide anion formation is preceeded by a lag period whose length varies in time with the concentration of agent applied to the cells. It is a known fact that once the neutrophils have been activated with fMLP it is not possible to reactivate the cells by a second supplementation of fMLP. However, after cessation of the fMLP-induced activation, addition of PMA or silver ions gives rise to renewed production of superoxide anions.

We propose two different mechanisms of action of silver ions on oxidative metabolism of neutrophils. At a low concentration the metal ions are thought to interact with an activating agent and a corresponding cell surface receptor molecule, while at elevated ion concentrations, we postulate an action like that of phorbol-esters on neutrophils, (i.e., an interaction between activating agent and the enzyme protein kinase C of the cells).     

Evidence for the Involvement of Loosely Bound Plastosemiquinones in Superoxide Anion Radical Production in Photosystem II

Recent evidence has indicated the presence of novel plastoquinone-binding sites, Q_C and Q_D, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O₂^•−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O₂^•−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the Q_B site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the Q_D site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b₅₅₉ (cyt b₅₅₉). Based on these results, we propose that O₂^•− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the Q_B site and one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_C site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_D site seems to be ambiguous. In spite of the fact that the existence of Q_C and Q_D sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.     

External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving approximately 37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located approximately 13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.     

Responses by Coleoptiles of Intact Rice Seedlings to Anoxia: K+ Net Uptake from the External Solution and Translocation from the Caryopses

This study evaluated the effects of anoxia on K⁺ uptake andtranslocation in 3–4-d-old, intact, rice seedlings (Oryzasativa L. cv. Calrose). Rates of net K⁺ uptake from the mediumover 24 h by coleoptiles of anoxic seedlings were inhibitedby 83–91 %, when compared with rates in aerated seedlings.Similar uptake rates, and degree of inhibition due to anoxia,were found for Rb⁺ when supplied over 1·5–2 h,starting 22 h after imposing anoxia. The Rb⁺ uptake indicatedthat intact coleoptiles take up ions directly from the externalsolution. Monovalent cation (K⁺ and Rb⁺) net uptake from thesolution was inhibited by anoxia to the same degree for thecoleoptiles of intact seedlings and for coleoptiles excised,‘aged’, and supplied with exogenous glucose. Transportof endogenous K⁺ from caryopses to coleoptiles was inhibitedless by anoxia than net K⁺ uptake from the solution, the inhibitionbeing 55 % rather than 87 %. Despite these inhibitions,osmotic pressures of sap (_sap) expressed from coleoptiles ofseedlings exposed to 48 h of anoxia, with or without exogenousK⁺, were 0·66 ± 0·03 MPa; however,the contributions of K⁺ to _sap were 23 and 16 %, respectively.After 24 h of anoxia, the K⁺ concentrations in the basal10 mm of the coleoptiles of seedlings with or without exogenousK⁺, were similar to those in aerated seedlings with exogenousK⁺. In contrast, K⁺ concentrations had decreased in aeratedseedlings without exogenous K⁺, presumably due to ‘dilution’by growth; fresh weight gains of the coleoptile being 3·6-to 4·7-fold greater in aerated than in anoxic seedlings.Deposition rates of K⁺ along the axes of the coleoptiles werecalculated for the anoxic seedlings only, for which we assessedthe elongation zone to be only the basal 4 mm. K⁺ depositionin the basal 6 mm was similar for seedlings with or withoutexogenous K⁺, at 0·6–0·87 µmolg^–1 f. wt h^–1. Deposition rates in zones above6 mm from the base were greater for seedlings with, thanwithout, exogenous K⁺; the latter were sometimes negative. Weconclude that for the coleoptiles of rice seedlings, anoxiainhibits net K⁺ uptake from the external solution to a muchlarger extent than K⁺ translocation from the caryopses. Furthermore,K⁺ concentrations in the elongation zone of the coleoptilesof anoxic seedlings were maintained to a remarkable degree,contributing to maintenance of _sap in cells of these elongatingtissues.     

Tentoxin Inhibits Both Photophosphorylation in Thylakoids and the ATPase Activity of Isolated Coupling Factor F1 from the Cyanobacterium Anacystis nidulans

Tentoxin strongly inhibited the ATPase activity of isolatedcoupling factor 1 (AF₁) from the cyanobacterium Anacystis nidulans,with 50% inhibition occurring at 0.3 µM. When thylakoidsfrom A. nidulans were preincubated with 0.3 µM tentoxinfor 30 min, photophosphorylation was inhibited by 50%. Measurementsof fluorescence from 9-aminoacridine indicated that tentoxininhibited the utilization of the proton gradient by ATP formationin thylakoids. These results indicate that tentoxin is a strongenergy-transfer inhibitor of photophosphorylation in A. nidulans.Tentoxin decreased the level of ATP in intact cells both inthe light and in darkness, its effects being much stronger inthe dark. Tentoxin at 50 µM strongly inhibited the growthof the cells. ³Present address: Corporate Research and Development Laboratory,Tonen Co. 1-3-1 Nishi-tsurugaoka, Ohi-machi, Saitama, 354 Japan ⁴Present address: Technology and Engineering Laboratories, AjinomotoCo., Inc. Suzuki-cho 1, Kawasaki, 210 Japan     

Superoxide Production and the Activity of Extracellular Peroxidase in Plant Tissues under Stress Conditions

The production of reactive oxygen species on the plant-cell surface is considered. Along with the plasmalemmal redox systems, cell-wall peroxidase is involved in the production of superoxide and hydrogen peroxide. Under stress conditions, some soluble peroxidase isoforms are easily secreted into the apoplast. Various membranotropic compounds, salicylic acid in particular, can also induce this process. Mobile peroxidase forms are supposed to induce the plant defense response.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).     

3-Methylcrotonylglycine Disrupts Mitochondrial Energy Homeostasis and Inhibits Synaptic Na+,K+-ATPase Activity in Brain of Young Rats

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO₂ production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I–IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na⁺,K⁺-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO₂ production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na⁺,K⁺-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na⁺,K⁺-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na⁺,K⁺-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.