Inhibition of Malate Synthesis by Vanadate: Implications for the Cytosolic Alkalinization Induced by Vanadate Concentrations Partially Inhibiting the Plasmalemma H+ Pump

The effects of Na-orthovanadate, at concentrations only partiallyinhibiting net H⁺ extrusion, were determined on vacuolar andcytosolic pH by the weak base and weak acid distribution atequilibrium. Treatment with vanadate induces in Elodea densaleaves and in Arabidopsis thaliana seedlings a moderate acidificationof both cell sap and vacuole. Conversely, it induces an alkalinizationof cytosol, this effect being in apparent contrast with a conditionof reduced activity of the H⁺-transporting plasmalemma ATPase,which should be associated with a cytosolic acidification. InArabidopsis seedlings treated with vanadate, the increase inpH of both cytosol and external medium is associated with adecrease in cell sap buffer capacity, more evident for highervanadate concentrations, and particularly marked in the pH rangebetween 3·5 and 5·5. In these conditions, themalate content is strongly reduced, its decrease almost completelyaccounting for the decrease in cell sap buffer capacity. Anin vitro analysis of the vanadate effect on phosphoenolpyruvatecarboxylase indicates that the decrease in malate content seemssubstantially due to an inhibiting effect of vanadate on thisenzyme. These results stress that the in vivo use of vanadateas an inhibitor of the plasmalemma H⁺-ATPase must be taken withcaution; in particular, for studying the correlations betweenchanges in net H⁺ extrusion and changes in cytosolic pH andrelated processes. Key words: Vanadate, malate, cytosolic pH, Elodea densa, Arabidopsis thaliana     

The Effects of Light Irradiation on the Orientation of Microtubules in Seedlings of Avena sativa L. and Pisum sativum L.

The effects of light irradiation on the arrangement of corticalmicrotubules (MTs) were examined in etiolated A vena mesocotylsand coleoptiles, and in Pisum epicotyls. Elongation of A venamesocotyls ceased as a result of irradiation with white lightwithin 1 h. The predominant orientation of MTs became more longitudinalwithin 1 h in epidermal cells and changed from transverse tooblique, after the elongation ceased, in parenchymal cells.Irradiation with red and with blue light also caused cessationof cell elongation and the same changes in the orientation ofMTs. Elongation of Avena coleoptiles ceased as a result of irradiationwith white light within 24 h. The predominant orientation ofMTs became more longitudinal in epidermal cells and changedfrom transverse to oblique in parenchymal cells. The changein orientation of MTs in epidermal cells preceded that in parenchymalcells. In Pisum epicotyls, elongation ceased as a result ofirradiation with white light within 1 h. Although the orientationsof MTs in epidermal cells did not show any remarkable change,those in parenchymal cells changed from transverse to obliqueafter cell elongation ceased. The change in orientation of MTs and the cessation of cell elongationof A vena mesocotyls induced by white-light irradiation wereboth significantly retarded by treatment with IAA. This resultsuggests that IAA is involved in maintaining the transverseorientation of MTs in Avena mesocotyls. (Received February 22, 1989; Accepted August 2, 1989)     

The Influence of the Cell Membrane on some Toxicity Effects of Growth Substances in Nitella

When cells of Nitella are placed in a solution of some plantgrowth substances there is a profound increase in the membraneresistance as measured by means of an internal silver/silverchloride micro-electrode. This impedance effect is accompaniedby some marked visible changes within the cell cytoplasm, thesechanges being dependent upon the concentration of undissociatedgrowth substance in the vicinity of the membrane, and upon thepH of the external environment. A detailed study is made of these visible changes and, takinginto account the negatively charged membrane, one possible interpretationof the results is that only undissociated molecules of the growthsubstance may enter the cytoplasm of Nitella from the environment,the diffusion obeying a simple Fick Law relationship. The substances are used in sufficient quantity to kill the Nitellacell, death following the visible effects, and they appear tobe unique in that their toxic effect produces a system of highmembrane impedance.     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

The Fine Structure of Bamboo Fibres: II. REFRACTIVE INDICES AND WALL DENSITY

In the fibres of Dendrocalamus strictus and D. longispathusit has been shown that the refractive indices of the outer walllamella, parallel and perpendicular to cell length, are correlatednot only with cell length but also with the density of the wholewall. The indications nevertheless are still that the variationin refractive indices with cell length are due mainly to changesin the orientation of the cellulose microfibrils. There is,however, a subsidiary effect of factors associated with density.These factors are briefly discussed     

The Structure and Orientation of Guard Cells in Plants Showing Stomatal Responses to Changing Vapour Pressure Difference

Transmission electron micrographs revealed that a substantialpart of the guard cell wall of both Quercus robur L. and Populusnigra ‘italica’ L. was either free of cuticle orcovered with a greatly reduced cuticular layer. In Quercus thestructure of the guard cell was such that the area of limitedcuticular development would be exposed to the evaporating powerof the atmosphere even when the stomata were closed. Lanthanumstaining confirmed that this area might be an important siteof evaporation. A similar evaporation site was identified inthe guard cell wall of Pinus sylvestris L. Light micrographsrevealed that this area could also be exposed on the outsideof the leaf when the stomata were closed. It appears that guardcell orientation with respect to the epidermal plane dependsupon epidermal turgor. Changes in orientation of the guard cellcoupled with the exact location of the cuticle-free area inthe guard cell wall may explain the nature of the stomatal responseof individual species to changing VPD and the effect of othervariables, e.g. water deficit, on this response. Quercus robur L, oak, Populus nigra L, poplar, stomata, guard cells, cuticle, evaporation, vapour pressure difference     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

Transient light-induced changes in ion channel and proton pump activities in the plasma membrane of tobacco mesophyll protoplasts

Rapid, transient changes of the membrane potential upon lighttransitions are generally observed in microelectrode studies.In a patch-clamp study similar responses to light transitionswere found in current clamp. Corresponding with the changesof membrane potential, light-induced current changes in voltageclamp were observed. This paper evaluates the involvement ofoutward rectifying conductances and plasma membrane bound H⁺-ATpases(proton pump) to these light responses in mesophyll protoplastsof Nicotiana tabacum L. The contribution of K⁺-channels to theseresponses, could be minimized by variation of the holding potentialor addition of the K⁺-channel blocker verapamil. It was concludedthat light transitions modulate both proton pump and K⁺-channelactivity. Effects of light on membrane current were not observedin root cells and chlorophyll-deficient cells, suggesting thatthe response requires photosynthetic activity. However, blockersof photosystems I and II did not affect current changes. Key words: Light, patch-clamp, plasma membrane, tobacco, whole cell     

Effects of Fusicoccin in 'Isolated' Guard Cells of Commelina communis L

The effects of 3 ? 10^–2 mol m^–3 FC on rubidium fluxesand contents in isolated guard cells of Commelina communis L.have been studied using ⁸⁶RbCl. Fusicoccin causes a marked stimulationof influx and an immediate, apparently irreversible, decreasein efflux of ⁸⁶Rb. The effect on influx is usually more importantin determining the new net flux of Rb. Observed fluxes differmarkedly from those predicted by the Goldman-Hodgkin-Katz equation,suggesting that FC does not act solely via an effect upon theplasmalemma potential. Fusicoccin appears to have a more directeffect upon the ion movements associated with changes in stomatalaperture than either ABA or transfer to the dark. Observed changesin Rb content cannot account fully for the osmotic changes associatedwith aperture increase. Key words: Fusicoccin, Guard cells, Ion fluxes, Commelina communis     

Implications of the feeding current structure of Euchaeta rimana, a carnivorous pelagic copepod, on the spatial orientation of their prey

Many marine planktonic organisms create water currents to entrainand capture food items. Rheotactic prey entrained within thesefeeding currents often exibit escape reactions. If the directionof escape is away from the feeding current, the prey may successfullydeter predation. If the escape is towards the center of thefeeding current, the prey will be re-entrained towards its predatorand remain at risk of predation. The direction of escape isdependent on (i) the ability of the prey to escape in a directiondifferent than its pre-escape orientation and (ii) the orientationcaused by the interaction of the prey's body with the movingfluid. In this study, the change in orientation of Acartia hudsonicanauplii as a result of entrainment within the feeding currentof Euchaeta rimana, a planktonic predatory copepod, was examined,When escaping in still water, A.hudsonica nauplii were ableto vary their pre-escape direction by only 10. This allowsonly a limited ability to escape in a direction different thantheir pre-escape orientation. Analyses of the feeding currentof E.rimana show the flow speed to be most rapid in the centralregion with an exponential decrease in speed distally. In contrast,flow vorticity is minimal in the center of the feeding currentand maximal at 1.75 mm along the antennae. As a result, thedegree of rotation of the prey towards the center of the feedingcurrent shows a strong dependency on the prey's location withinthe feeding current. The feeding current of E.rimana rotatedthe prey 14 when near the center of the flow field and up to160 when located more distal in the feeding current Since theprey's escape abilities cannot compensate for the rotation dueto the flow, this mechanism will maintain the escaping preywithin the feeding current of their predator. Therefore, thefeeding current facilitates predatory copepods in capturingprey by (i) increasing the amount of water which passes overtheir sensors and through their feeding appendages and (ii)controlling the spatial orientation of their prey prior to escape.     

Light-Triggered Changes of Membrane Potential in Cells of Anthoceros punctatus and their Relation to Activation of Chloroplast ATPase

Post-illumination transients of the membrane potential wererevealed in cells of Anthoceros punctatus upon short (1–2s) irradiation with photosynthetically active light. An initialfast depolarization of the cell by 20–30 mV after a lagperiod of 2 s and a subsequent slow repolarization were recordedwith micro-electrodes positioned both in the chloroplast andin transparent parts of the cell. The potential changes of similarkinetics were also observed upon continuous illumination. Post-illuminationpotential changes were abolished by 3-(3, 4-dichlorophenyl)-l,l-dimethylurea and dicyclohexylcarbodiimide and were stimulatedby the addition of NH₄C1. It is assumed that the light-triggeredpotential change across the plasmalemma of A. punctatus aredetermined by the light activation of chloroplast ATPase. Thisassumption is further supported by observations of post-illuminationtransient increase of the chlorophyll fluorescence in preparationsof A. punctatus. Key words: Cell membrane potential, Chloroplast, Photosynthesis     

Effects of Light and Inhibitors on Polylamellation and Shift of Microfibril Orientation in Boergesenia Cell wall

New round cells of Boergesenia forbesii, which develop fromprotoplasts, form crossed polylamellate cell walls. The frequencyof the shift in microfibril orientation in generating wall layerswas affected not by the photoperiod, but by the total lengthof the illumination period. The dependency of the shift uponlight intensity and quality, and its inhibition by 3-(3,4-dichlorophenyl)-l,l-dimethylureaindicated that the shift in fibril orientation was primarilydependent upon photosynthesis. KCN and NaN₃ strongly inhibitedwall thickening but the frequency of the shift only a little.Therefore, the system controlling microfibril orientation functionsindependently of wall synthesis. Both colchicine and cytochalasinB did not affect microfibril orientation as well as wall synthesisin Boergesenia cells. (Received August 1, 1981; Accepted December 14, 1981)     

An LQT mutant minK alters KvLQT1 trafficking

Cardiac I_Ks, the slowly activated delayed-rectifier K⁺ current, is produced by the protein complex composed of - and -subunits: KvLQT1 and minK. Mutations of genes encoding KvLQT1 and minK are responsible for the hereditary long QT syndrome (loci LQT1 and LQT5, respectively). MinK-L51H fails to traffic to the cell surface, thereby failing to produce effective I_Ks. We examined the effects that minK-L51H and an endoplasmic reticulum (ER)-targeted minK (minK-ER) exerted over the electrophysiology and biosynthesis of coexpressed KvLQT1. Both minK-L51H and minK-ER were sequestered primarily in the ER as confirmed by lack of plasma membrane expression. Glycosylation and immunofluorescence patterns of minK-L51H were qualitatively different for minK-ER, suggesting differences in trafficking. Cotransfection with the minK mutants resulted in reduced surface expression of KvLQT1 as assayed by whole cell voltage clamp and immunofluorescence. MinK-L51H reduced current amplitude by 91% compared with wild-type (WT) minK/KvLQT1, and the residual current was identical to KvLQT1 without minK. The phenotype of minK-L51H on I_Ks was not dominant because coexpressed WT minK rescued the current and surface expression. Collectively, our data suggest that ER quality control prevents minK-L51H/KvLQT1 complexes from trafficking to the plasma membrane, resulting in decreased I_Ks. This is the first demonstration that a minK LQT mutation is capable of conferring trafficking defects onto its associated -subunit. potassium channel; hereditary arrhythmia; electrophysiology; protein interaction     

Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn²⁺) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn²⁺ with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl₂) to PAEC. We observed that Zn²⁺-exposed PAECs exhibited a dose-dependent increase in superoxide (O₂^–·) generation that was localized to the mitochondria. Furthermore, we found Zn²⁺-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn²⁺-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn²⁺-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn²⁺-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn²⁺ homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn²⁺ levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress. reactive nitrogen species; apoptosis mitochondrial dysfunction     

The Relationship Between the Distribution of Periclinal Cell Divisions in the Shoot Apex and Leaf Initiation

Periclinal cell divisions in vegetative shoot apices of Pisumand Silene were recorded from serial thin sections by mappingall the periclinal cell walls formed less than one cell cyclepreviously. The distribution of periclinal divisions in theapical domes corresponded to the distributions subsequentlyoccurring in the apices when the young leaf primordia were forming.In Pisum, periclinal divisions were almost entirely absent fromthe I₁ region of the apical dome for half a plastochron justafter the formation of a leaf primordium and appeared, simultaneouslyover the whole of the next potential leaf site, about half aplastochron before the primordium formed. In Silene periclinaldivisions seemed to always present in the apical dome at thepotential leaf sites and also round the sides of the dome wherethe ensheathing leaf bases were to form. Periclinal divisionstherefore anticipated the formation of leaf primordia by occuring,in Pisum about one cell cycle and in Silene two or more cellcycles, before the change in the direction of growth or deformationof the surface associated with primordial initiation. Pisum, Silene, planes of cell division, orientation of cell walls, leaf primordia, shoot apical meristem, plastochron     

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1

Volatile anesthetics have been shown to activate various two-pore (2P) domain K⁺ (K_2P) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K⁺ channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K_2P channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K⁺ current in rat ventricular myocytes (which strongly express TASK-1) and selected human K_2P channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K⁺ (K_ir) current (I_K1) and acid-sensitive outward K⁺ current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(–)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K_2P channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations. volatile anesthetics; etomidate; propofol; ion channels     

Effects of soil resistance to root penetration on leaf expansion in wheat (Triticum aestivum L.): composition, number and size of epidermal cells in mature blades

Wheat seedlings {Triticum aestivum L.) were grown on soils withcontrasted resistances to root penetration (measured as penetrometerresistance, R_s. High R_s reduced the rates of leaf appearanceand expansion. Although the duration of expansion was increased,mature leaves were smaller. Underlying changes in leaf anatomywere investigated on cleared mature leaves, focusing on theepidermes. Three leaves were analysed: leaves 1 and 3 whichstarted their development in the embryo, and leaf 5 which wasinitiated on the seedling, after imposition of contrasted soilconditions. In all leaves, high R_s, caused a reduction in maturecell sizes, lengths and widths, and a shift in the relativeproportions of functionally different cell types, with a decreasein the relative proportions of stomata and associated cell types(interstomatal and sister cells) and an increase in the proportionsof unspecialized elongated epidermal cells and of trichomes.In leaves 3 and 5 the number of cellular files across the bladewas also reduced, while in leaf 1 it was similar at the twoR_s. These differences between leaves are attributed to differencesin their developmental stage when root stress was first perceived.Remarkably, R_s had no effect (leaf 1) or relatively small effects(leaves 3 and 5) on the total number of cells per file, suggestingthat this parameter is either largely insensitive to variationin root environment, or is programmed at the outset before stresswas perceived at the apex. Key words: Wheat, anatomy, mature epidermis, root impedance     

Arachidonic acid reversibly enhances N-type calcium current at an extracellular site

We examined the effects of arachidonic acid(AA) on whole cell Ca²⁺ channel activity in rat superiorcervical ganglion neurons. Our companion paper (Liu L, Barrett CF, andRittenhouse AR. Am J Physiol Cell Physiol 280:C1293-C1305, 2001) demonstrates that AA induces severaleffects, including enhancement of current amplitude at negativevoltages, and increased activation kinetics. This study examines themechanisms underlying these effects. First, enhancement is rapidlyreversible by bath application of BSA. Second, enhancement appears tooccur extracellularly, since intracellular albumin was without effecton enhancement, and bath-applied arachidonoyl coenzyme A, anamphiphilic AA analog that cannot cross the cell membrane, mimickedenhancement. In addition, enhancement is voltage dependent, in thatcurrents were enhanced to the greatest degree at 10 mV, whereasvirtually no enhancement occurred positive of +30 mV. We alsodemonstrate that AA-induced increases in activation kinetics arecorrelated with enhancement of current amplitude. An observed increasein the voltage sensitivity may underlie these effects. Finally, themajority of enhancement is mediated through N-type current, thusproviding the first demonstration that this current type can beenhanced by AA.

     

Growth and the Current Pattern Around Internodal Cells of Nitella flexilis L.

The ionic currents that traverse the internodal cells of thegreen alga Nitella flexilis L. have been measured with an extracellularvibrating probe. In adult interriodes illuminated with whitelight a pattern of self-generated currents exists along thecell, featuring alternating zones of inward and outward current.At inward current zones current densities of up to 25 µAcm^–2 were measured, at outward current zones the maximaldensity recorded was 7.5 µA cm^–2. The zones withinward current had an average length of 1.0 mm, and those withoutward current averaged 1.5 mm. When the light was turned offthe overall current density decreased drastically and the patternchanged. Currents first appear in growing internodal cells thatare about 1.0 mm long. With increasing length a current patterndevelops, with the zones of inward and outward current at firstshifting laterally along the surface. Only in adult cells dothe zones become stationary and form a typical current pattern.In addition to the current pattern different chloroplast volumescan be observed along the internode. In outward current zonesthe volumes of chloroplasts are 3.2 times those of inward currentzones. The natural current pattern observed in Nitella internodescauses loops of electric current that extend outward from thecell surface into the bathing medium. We speculate that thesecurrent loops might provide a mechanism of electrophoretic iontransport through the medium. Such a mechanism could increasethe supply of for the internodal cells in their natural stagnant water habitat. Key words: Nitella flexilis, Internode growth, Current pattern, Vibrating probe     

Initial Anatomical Changes Associated with Tuber Formation on Single-node Potato (Solanum tuberosum L.) Cuttings: A Re-evaluation

Cell division and cell expansion during early stages of tuberdevelopment were studied using developing axillary buds on single-leafcuttings from potato (Solanum tuberosum L.). Cuttings takenfrom plants induced to form tubers, by short day (SD) treatment,were compared with cuttings from non-induced (long day, LD)plants. In the apical zone of the buds, cell division occurredfrom the first day after cutting, in both LD and SD cuttings.The planes of these divisions were transverse, associated withelongation of the buds. At day 5, a new orientation of celldivision was observed in the subapical zone of SD cuttings only.These divisions were longitudinal, associated with radial growth.Cell expansion occurred in both SD and LD cuttings, and wasnot uniquely related to the onset of tuber formation. Copyright1999 Annals of Botany Company Solanum tuberosum L., potato, tuber formation, cell division, cell expansion.