The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake

Abstract: The Na⁺ and K⁺ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na⁺ (290 MM) and low internal K⁺ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na⁺, 96 mM; K⁺, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na⁺ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na⁺ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca²⁺]_cyt elevation is associated with Ca²⁺-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca²⁺ channels and their regulation remains limited in planta . A type of voltage-dependent Ca²⁺-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba²⁺ and Ca²⁺, and their activities can be inhibited by micromolar Gd³⁺. The unitary conductance and the reversal potential of the channels depend on the Ca²⁺ or Ba²⁺ gradients across the plasma membrane. The inward whole-cell Ca²⁺ (Ba²⁺) current, as well as the unitary current amplitude and NP_o of the single Ca²⁺ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

Cellular mechanisms of potassium transport in plants

Potassium (K⁺) is the most abundant ion in the plant cell and is required for a wide array of functions, ranging from the maintenance of electrical potential gradients across cell membranes, to the generation of turgor, to the activation of numerous enzymes. The majority of these functions depend more or less directly upon the activities and regulation of membrane-bound K⁺ transport proteins, operating over a wide range of K⁺ concentrations. Here, we review the physiological aspects of potassium transport systems in the plasma membrane, re-examining fundamental problems in the field such as the distinctions between high- and low-affinity transport systems, the interactions between K⁺ and other ions such as NH₄⁺ and Na⁺, the regulation of cellular K⁺ pools, the generation of electrical potentials and the problems involved in measurement of unidirectional K⁺ fluxes. We place these discussions in the context of recent discoveries in the molecular biology of K⁺ acquisition and produce an overview of gene families encoding K⁺ transporters.     

K+ and Na+ influxes in Nitella modified by pH and electric current

K⁺ and Na⁺ influxes into Nitella translucens Agardh in buffered artificial pond water have been measured in the pH range 5.7 to 8.1 with and without sub-threshold electric current. For pH levels below 7 the results confirm previous observations that neither ion is a major carrier of membrance current. At pH levels greater than 7 the effect of applied current is generally to depress cation influx. It is argued that this is evidence in favour of OH⁻ being the principal charge carrier under these conditions. Observations of the effects of OH⁻ gradients on K⁺ influx tend to support this hypothesis.     

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD-like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca²⁺ increases accompany all forms of SD, but persistently elevated Ca²⁺ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca²⁺ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca²⁺ influx via P/Q type channels is important for SD generated by localized application of high K⁺ solutions. In contrast, SD-like events recorded in in vitro ischemia models are not usually prevented by Ca²⁺ removal, but under some conditions, Zn²⁺ influx via L-type channels contributes to SD initiation. This review addresses different roles of Ca²⁺ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn²⁺ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.     

Ca(2+) and Mg(2+)/ATP independently trigger homotypic membrane fusion in gastric secretory membranes

Exocytic activation of gastric parietal cells represents a massive transformation. We studied a step in this process, homotypic fusion of H,K-ATPase-containing tubulovesicles, using R18 dequenching. Ca²⁺ and Mg²⁺/ATP each caused dramatic dequenching, reflecting a change in R18 distribution from 5% to 65–90% of the assay's membranes in 2.5 min. These stimuli also triggered fusion between tubulovesicles and liposomes. Independent confirmation that dequenching represented membrane fusion was established by separating tubulovesicle–liposome fusion products on density gradients. Only agents that trigger fusion allowed the transmembrane H,K-ATPase to move to low-density fractions along with R18. EC₅₀ for Ca²⁺-triggered fusion was 150 n m and for Mg²⁺/ATP-triggered fusion 1 m m , the latter having a Hill coefficient of 2.5. ATP-triggered fusion was specific for Mg²⁺/ATP, required ATP hydrolysis, and was insensitive to inhibition of NSF and/or H,K-ATPase. Fusion initiated by either trigger caused tubulovesicles to become resistant to subsequent challenge by either trigger. Ca²⁺-and Mg²⁺/ATP-triggered fusion required protein component(s) in tubulovesicles, though this was required in only one of the fusing membranes since tubulovesicles fused well with liposomes containing no proteins. Our data suggest that exocytosis in parietal cells is triggered by separate but interacting pathways and is regulated by self-inhibition.     

CATION MODULATION OF SYNAPTOSOMAL RESPIRATION

Abstract— Synaptosomes were prepared from the cerebral cortex of the adult rat by a rapid technique, involving the use of centrifugation in a Ficoll-sucrose discontinuous gradient. Adequate respiratory control ratios were obtained with glutamate and succinate plus rotenone. The addition of Na⁺ to the incubation medium stimulated synaptosomal, State-4 respiration, with a half-maximal response at 15 mM Na⁺. The stimulation by Na⁺ was inhibited by atractylate, oligomycin, ouabain or EDTA. A cooperative interaction between Na⁺ and low concentrations of Mg²⁺ was observed. A significant proportion (39 per cent) of the total Na-K ATPase (EC 3.6.1.4) activity in the discontinuous gradient was localized in the synaptosomal fraction. In the absence of exogenous Mg²⁺, Na⁺ induced a 64 per cent stimulation of the synaptosomal ATPase activity which was sensitive to ouabain. Such stimulation of ATP hydrolysis would account for the formation of increased amounts of ADP, with consequent recycling to ATP through adequately controlled oxidative phosphorylation. These observations demonstrate a significant role for transmembrane cationic gradients in the control of synaptosomal respiration and mitochondrial oxidative phosphorylation. The preparation exhibits moderate respiratory control and should prove useful in studies of integrated mitochondrial oxidative metabolism and neuronal membrane function.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

Characterization of the pyrophosphate-dependent proton transport in microsomal membranes from maize roots

Cleared maize ( Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP- and pyrophosphate (PP_i)-dependent proton-pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PP_i-dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H⁺-PPase was Mg²⁺-dependent and the K_m for PP_i (in the presence of 3 m M MgSO₄) was 28 μ M . The pump was electrogenic, K⁺-dependent and a permeant anion was necessary to dissipate the membrane potential (NO₃⁻= I⁻ >Br⁻ > Cl⁻). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H⁺-PPase was insensitive to vanadate, oligomycin and molybdate. -Diethylstilbestrol (DES) and N,N'-dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μ M .     

Salinity effects on oxygen consumption, gill Na+, K+-ATPase and ion regulation in juvenile coho salmon

The metabolic response of juvenile coho salmon Oncorhynchus kisutch to different salinities was examined, using whole-animal oxygen consumption rates and gill Na⁺, K⁺-ATPase activities as indicators of osmoregulatory energetics. Coho salmon smolts were acclimated to fresh water (FW), isosmotic salinity (ISO, 10‰) and sea water (SW, 28‰) and were sampled for up to 6 weeks for plasma levels of cortisol, glucose and ions (Na⁺, K⁺, Cl⁻), gill Na⁺, K⁺-ATPase activity and oxygen consumption rates. Following an initial adjustment period, plasma constituents in SW fish returned to near-FW values, indicating that the fish were acclimated to SW by day 21. Gill Na⁺, K⁺-ATPase activities on days 21 and 42 were lowest in ISO, higher in FW and highest in SW. This result is consistent with the idea that less energy would be required to maintain ion balance in an isosmotic environment, where the ionic gradients between extracellular fluid and water would be minimal. Oxygen consumption rates of swimming fish (1 body length s⁻¹), however, did not differ significantly between the three test salinities after 6 weeks. The results of this study suggest that the metabolic response of juvenile salmonids to changes in salinity is dependent on life-history stage (e.g. fry v . smolt), and that oxygen consumption rates do not necessarily reflect osmoregulatory costs.     

Passive uptake of K+(86Rb+) in sunflower roots at low external K+ concentrations

Passive fluxes of K⁺ (⁸⁶Rb) into roots of sunflower ( Helianthus annuus L. cv. Uniflorus) were determined at low K⁺ concentration (0.1 and 1.0 mM K⁺) in the ambient solution. Metabolic uptake of K⁺ was inhibited by 10⁻⁴M 2,4-dinitrophenol (DNP). K⁺ (⁸⁶Rb) fluxes were studied both continuously and by time differentiation of uptake. In high K⁺ roots passive uptake was directly proportional to the K⁺ concentration of the uptake solution, indicating free diffusion. This assumption was supported by the fact that passive Rb⁺ uptake was not affected by high K⁺ concentrations. In low K⁺ roots the passive uptake of K⁺ was higher than in high K⁺ roots. The increase was possibly due to carrier-mediated K⁺ transport. As K⁺ effluxes were quantitatively similar to influxes, it is suggested that passive K⁺ fluxes represent exchange diffusion without relation to net K⁺ transport.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Uptake of Cd2+ and Fe2+ by excised roots of Tamarix aphylla

The uptake of Cd²⁺ by excised roots of Tamarix aphylla (L.) Karst, was investigated using roots of hydroponically grown plants. The concentration isotherm of Cd²⁺ uptake approached saturation with a single phase hyperbola. The time course of Cd²⁺ absorption was generally hyperbolic, with an apparent linear section between 2 and 30 min. The temperature response varied among different temperature ranges: a Q₁₀ of approximately 1.9 was found between 10 and 20°C, but at higher and lower temperatures Q₁₀ values were only 1–1.3. It is concluded that Cd²⁺ uptake by the roots of T. aphylla at moderate temperatures is mediated by a metabolic process, combined with a passive influx component that becomes dominant at higher and lower temperatures. The distribution of the absorption sites for Cd²⁺ and for Fe²⁺ along the roots of T. aphylla was also investigated. Cadmium uptake showed no apparent pattern, whereas a distinct pattern of uptake was observed for Fe²⁺, with the highest rates at the root tip. Iron absorption was stimulated in the presence of nutrients, whereas that of Cd²⁺ was inhibited. Adsorption and absorption of Cd²⁺ were strongly inhibited by Ca²⁺ and by Mg²⁺, but were unaffected by Fe²⁺. Monovalent ions (Na⁺, K⁺, Li⁺) also reduced Cd²⁺ absorption, but to a lesser extent than Ca²⁺ and Mg²⁺. Uptake of Cd⁺ was reduced at lower pH of the medium. The importance of interfering cations for Cd²⁺ tolerance of T. aphylla is emphasized.     

Comparative studies of inorganic substances in the blood of fishes from the Scagerac Sea

The concentrations of the main plasma inorganic electrolytes Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^- and and PO₄^3- have been determined for different orders of marine fishes. For Na⁺ and Cl^- a typical decrease was found when passing from cyclostomes, holocephalans and elasmobranchs to teleosts. The concentrations of K⁺, Ca²⁺ and Mg²⁺ showed a similar trend except that there was a rise in the teleost group, which showed a large range of variation for these three ions. In the case of PO₄^3- no significant differences between groups were found.     

INHIBITION OF SYNAPTOSOMAL NORADRENALINE UPTAKE BY VERATRIDINE, GRAMICIDIN D AND VALINOMYCIN

Abstract— —The effects of brief exposures of rat brain synaptosomes to veratridine, gramicidin D and valinomycin on noradrenaline uptake were investigated. All three drugs inhibited the Na⁺-dependent component of noradrenaline uptake by synaptosomes. These effects were independent of extracellular Ca^{2 +} concentrations, indicating that the reductions were not due to the release of newly accumulated noradrenaline.
Gramicidin D reduced the V_max for noradrenaline uptake, whereas veratridine and valinomycin reduced the V^max and also increased the V^m for uptake.
Most of these findings can be explained on the basis of the effects that these drugs have on the inward-directed electrochemical gradients for Na⁺ across synaptosomal membranes, although, in the cases of veratridine and valinomycin, the elevated K_m's suggest that an impairment of noradrenaline binding to its carriers might also be involved.     

Aluminum enhancement of plant growth in acid rooting media. A case of reciprocal alleviation of toxicity by two toxic cations

The generally rhizotoxic ion Al³⁺ often enhances root growth at low concentrations. The hypothesis that Al³⁺ enhances growth by relieving H⁺ toxicity was tested with wheat seedlings ( Triticum aestivum L.). Growth enhancement by Al³⁺ only occurred under acidic conditions that reduced root elongation. Al³⁺ increased cell membrane electrical polarity and stimulated H⁺ extrusion. Previous investigations have shown that Al³⁺ decreases solute leakage at low _pH and that the alleviation of H⁺ toxicity by cations appears to be a general phenomenon with effectiveness dependent upon charge (C³⁺>C²⁺>C^l+). Alleviation of one cation toxicity by another toxic cation appears to be reciprocal so that Al³⁺ toxicity is relieved by H⁺. It has been argued previously that this latter phenomenon accounts for the apparent toxicity of ALOH²⁺ and Al(OH)⁺₂. Reduction of cell-surface electrical potential by the ameliorative cation may reduce the cell-surface activity of the toxic cation.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.