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1.
A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K+ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K+ concentration produced a graded release of ATP from synaptosomes. Rb+ also released ATP but Na+, Li+ and choline did not. The response to elevated K+ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na+-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K+. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na+-channels was involved. Neither veratridine nor elevated K+ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K+ was diminished in a medium lacking CaCl+ or when EGTA was added to chelate Ca2+. In contrast, release by veratridine appeared to be augmented in Ca2+-free media or in the presence of EGTA. The K+-induced release of ATP, which is Ca2+ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca2+ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.  相似文献   

2.
Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na+, K+, Mg2+ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg2+ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na+ gave a higher charge contribution than did K+ or a combination of Na+ and K+. This implies that K+ gives a higher affinity for ATP than Na+ does and or that ATP mediates a higher affinity for Na+ than for K+.  相似文献   

3.
Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li+ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li+ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li+. Whole brain or regional contents of Na+ or K+ were unaffected by lithium treatment. Subcellular Li+ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li+ and K+ localization at 0–4°C. Li+ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li+ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K+ and Mg2+ and hence probably partial membrane depolarization. Addition of 1 mM Li+ in vitro also caused synaptosomal Mg2+ depletion. The results indicate that Li+ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li+ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li+.  相似文献   

4.
Whole homogenates of mouse brain and nerve-ending fractions of mouse and human brain were obtained at various age levels representative of maturity and old age. The mice were 3, 8 and 26–29 months old and the humans ranged in age from 19 to 84 y. Measurements of (Na++ K+)-ATPase in whole brain homogenate of mouse did not reveal any significant differences in relation to age. However, the ability of ethanol at various concentrations to inhibit membrane-bound synaptosomal (Na++ K+)-ATPase was significantly greater in older mice and humans. The data are interpreted as indicating a change in the property of synaptic membranes as a consequence of advancing age.  相似文献   

5.
Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent 22Na+ influx and sodium/proton (Na+/ H+) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K+ gradients in the presence of valinomycin or H+ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na+ into the vesicles was stimulated by a negative ΔΨ and had a Km for extrav-esicular Na+ of 34.8 ± 5.9 mol m3. The ΔΨ-dependent uptake of Na+ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K+/Na+ discrimination trait, and was also unaffected by growth in 50 mol m?3 NaCl. Inhibition of ΔΨ-dependent Na+ uptake by Ca2+ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na+-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of 14C-methylamine. The Na+/H+ antiport had a Km, for intravesicular Na+ of between 13 and 19 mol m?3. In the hexaploid, Na+/H+ antiport activity was greater when roots were grown in the presence of 50 mol m?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K+/Na+ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na+/K+ discrimination trait located on the 4D chromosome of wheat.  相似文献   

6.
A microsomal (Na++ K++ Mg2+)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na+ and K+ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na+ and K+. At low Na+ concentration (below 5 mM), the apparent Km decreases with increasing concentrations of K+ (1–20 mM). At 5 mM Na+, the K+ level does not change the apparent Km, while at Na+ levels above 10 mM, the apparent Km between enzyme and substrate increases with increasing concentration of K+. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na+ and K+. The Na+ binding is cooperative with different Km values and Hill coefficients (n) in the presence of low and high concentration of K+. At low Na+ level (< 5 mM). a negative cooperativity exists for Na+ (nNa < 1) which is more pronounced in the presence of high [K+]. When the concentration of Na+ is raised the negative cooperativity disappears and turns into a positive one (nNa > 1). Only K+ binding in the presence of low [Na+] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K+ (nK < 1 → nK > 1). In the presence of [Na+] > 10 mM, the changes in nk are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na+, K+ and MgATP. 4. In the presence of Na+ (< 10 mM), K+ is probably bound to two K sites, one of which translocates K+ through the membrane by an antiport Na+/K+ mechanism. This could be connected with an elevated K+ uptake in the presence of Na+ and could therefore explain some field properties of sugar beets.  相似文献   

7.
Sugar beet leaf homogenate contains Mg2+-stimulated ATPase activity with the highest specific activity in the 25,000–30,000 ×g-fraction. This fraction also has (Na++ K+)-activated ATPase activity. Both activities have two pH optima, one stable at pH 7.9 and one variable at lower pH. When optimal conditions of Na+ and K+ were tested with 64 combinations of these ions, at least two mountains of activity were revealed. The (Na++ K+)-ATPase had a high specificity for ATP. It had lost about 50% of its original activity after 56 days of storage at ?85°C. The activity drop was most pronounced at high ionic concentrations in the test medium. The (Na++ K+)-ATPase shows four peaks of activity when tested at constant ionic strength. The idea is put forward that the four peaks reflect two ATPases, one in the tonoplast and one in the plasmalemma, which undergo conformational changes in relation to the ionic milieu.  相似文献   

8.
Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.  相似文献   

9.
Effects of interrupted K+ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K+ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K+ supply. Root structure was greatly affected by the length of the period in K+ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K+ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K+ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K+. In comparison, both higher and lower internal/external K+ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca2+, Mg2+ and Na+ uptake. Uptake of Ca2+ and especially Mg2+ significantly increased after K+ interruption, while Na+ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K+ in the cells and maintain electroneutrality down to a certain minimal range of K+ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)?1] than in the root [20 to 30 μmol (g fresh weight)?1]. It is suggested that the critical K+ values are a measure of the minimal amount of K+ that must be present for physiological activity in the cells. At the critical levels, K+ (86Rb) influx and Ca2+ and Mg2+ concentrations were maximal. Below the critical K+ values, growth was reduced, and Ca2+ and Mg2+ could no longer substitute for K+ for electrostatic balance. In a short-term experiment, the ability of Ca2+ to compete with K+ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K+ status. The results indicate that K+, which was taken up actively and fastest at the external K+ concentration used (2.0 mM), partly determines the size of Ca2+ influx.  相似文献   

10.
Abstract: The effects of four K+-channel inhibitors on synaptosomal free Ca2+ concentrations and 86Rb+ fluxes are analysed. 4-Aminopyridine, α-dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca2+ concentration, although their potencies differ widely. In each case, the elevation in free Ca2+ concentration is reversed by the subsequent addition of tetrodotoxin. The transient 86Rb+ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4-aminopyridine and α-dendrotoxin. In contrast, when 4-aminopyridine or α-dendrotoxin is added to polarized synaptosomes, an enhanced86Rb+ flux is seen, both for uptake and for efflux with no change in the total 86Rb+/K+ content of the synaptosomes and with only a slight time-averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4-aminopyridine or α-dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine-stimulated 86Rb+ efflux, the elevation of free Ca2+ concentration, and the facilitation of glutamate exocytosis that are elicited by 4-aminopyridine or α-dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K+-channel inhibitors and that the enhanced 86Rb+ flux is a consequence of the activity of 4-aminopyridine- and α-dendrotoxin-insensitive K+ channels during these action potentials.  相似文献   

11.
Abstract— The swelling of intact, exposed primate cerebral cortex perfused in vioo under, isosmotic conditions was a linear function of the concentration of K+ in perfusate over the range 25–117 mM. The K+-dependent swelling was manifested throughout the depth of the cerebral cortex studied and was associated with an increased content of chloride in the swollen tissue, despite the constancy of the concentration of external chloride. The swelling of the cerebral cortex was a linear function of the temperature of the perfusate over the range 15–38°C, despite the constancy of the concentration of external K+. Moreover, the content of chloride in the swollen cerebral cortex was a linear function of the temperature of the overlying perfusate, despite the constancy of the external concentration of chloride. The changes in the contents of Na+ and K+ in the swollen cerebral cortex perfused with solutions containing constant concentrations of external Na+ and K+ but differing in temperature suggested that the fluid of swelling in the tissue was rich in both K+ and CI-, as had been shown previously in vitro. Perfusion of the exposed, intact cerebral cortex in uiuo with K+-rich fluids usually involved the reciprocal reduction of the concentrations of Na+ in the perfusate to maintain isotonicity. When comparable reductions in the concentration of external Na+ were achieved by replacement with choline (instead of K+), swelling of the perfused, exposed cortex was significantly less than that attributed to isotonic, K+-rich but Na+-poor fluids. These observations suggested that it was the elevated levels of K+ rather than lowered concentrations of Na+ that promoted the swelling of the perfused cerebral cortex. The apparent rate of influx of 36Cl from the perfusate into the underlying exposed and intact monkey cerebral cortex in vivo was a linear function of the concentration of K+ in perfusate over the range 25–117 mM and conformed to Michaelis-Menten kinetics when plotted according to Lineweaver and Burk. Moreover, the apparent influx of chloride from perfusate into swollen cerebral cortex was a linear function of the percentage swelling of cerebral cortex over the range 6–30 per cent. However, the apparent rate of influx of chloride from perfusate into unswollen cortex was not consistent with the linear correlation already described for swollen cerebral cortex. One reason for this discrepancy was the reduction in the size of the true (inulin) extracellular space associated with the K+-dependent swelling of cerebral cortex in vivo. The anatomical locus for this K+-dependent swelling of cerebral cortex was an expanded glial compartment, as demonstrated by electron-microscopy. The parenteral administration (50 mg/kg) or local perfusion (5 mM) of acetazolamide inhibited the K+-dependent swelling of cerebral cortex in vivo. Moreover, administration of acetazolamide inhibited the K+-dependent increase in content of C1- and the K+-dependent rate of influx of 36Cl into swollen cerebral cortex. We have discussed the possible enzymatic basis of these K+-dependent alterations in content of fluid and chloride and transport of chloride in mammalian cerebral cortex in viuo.  相似文献   

12.
Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na+ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na+ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K+ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I50) of Na+/H+ exchange and K+ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I50 for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na+/H+ exchange by these drugs.  相似文献   

13.
It has been shown that addition of phosphate to phosphate deficient yeast gives rise to an immediate increase in the rate of Na+ uptake and an immediate decrease in the rate of Rb+ uptake. In addition, phosphate uptake is enhanced specifically by Na ions presumably by a process with a very high affinity for phosphate with a Km of about 2 × 10−6M at pH 7.2, whereas the Km for phosphate uptake of the Na+ independent process amounts to 1.3 × 10−4M.  相似文献   

14.
The addition of LiCl stimulated the (Na++K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg. ATP concentrations. Apparent affinities for Li+ were estimated at the α-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the β-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent KM for Mg· ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the α-sites on the enzyme through which K+ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane NA+/K+ pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.  相似文献   

15.
Using compartmental analysis, unidirectional fluxes of K+ and Na+ and their intracellular compartmentation in excised barley (Hordeum distichon L. cv. Kocher-perle) root segments have been measured during a steady state in the presence or absence of ABA. Almost all flux rates were altered in the presence of external ABA, in particular the xylem transport R’ and the plasmalemma influx Øoc (see below) were strongly inhibited in the steady state. At the same time the presence of ABA induced a strong increase in the vacuolar K+ and Na+ content Qv and a decrease in the cytoplasmic one (Qc). Since the fluxes of an ion and its vacuolar or, in particular, cytoplasmic concentrations are interrelated, the ratios of fluxes originating from the cytoplasm and the cytoplasmic ion content were taken into account. On this basis ABA had the following effects: a) the secretion of K+ or Na+ to the xylem vessels was drastically inhibited; b) the plasmalemma K+ or Na+ efflux Øco was moderately stimulated and c) the tonoplast influx Øcv of Na+ was stimulated, while the tonoplast influx of K+ appeared to be unchanged (the decrease in Øcv being due to the decreased cytoplasmic K+ content). By a similar argument, also the apparent inhibition of the plasmalemma influx Øoc of K+ and Na+ in the steady state merely is an indirect effect of ABA. It only reflects the strong ABA-induced decrease in the xylem transport, that governs the magnitude of Øoc in the steady state. The results are discussed with reference to possible regulatory functions of ABA. In this respect it is suggested that – in particular under conditions of stress – ABA might regulate cellular metabolic processes by changing the cytoplasmic K+ level.  相似文献   

16.
The unidirectional influx of methionine into the brush border epithelium of chicken jejunum has been studied. Tissues leached of Na+ transport methionine from a medium devoid of Na+ with reduced apparent affinity (Kt) and maximal flux (Jmax). Addition of Na+ to the medium during a 1-min incubation with substrate, or during a 30-min preincubation, restored Kt but affected Jmax slightly. Theophylline was found to maintain Jmax in the absence of Na+. Essentially complete restoration of Kt and Jmax could be attained when theophylline-treated tissue was exposed to Na+ for 30 min. Influx from a Na+ medium was unaffected by theophylline pretreatment in Na+-containing buffer. Kt was increased without an effet upon Jmax when influx was studied from choline medium following preincubation in Na+.Modifiers of tissue cyclic AMP levels were investigated in conjunction with theophylline. Histamine and carbachol were found to inhibit theophylline-stimulated transport. Secretin was found to stimulate influx in Na+-leached tissue, but did not potentiate the theophylline effect. Amino acids in the incubation medium inhibited theophylline-stimulated influx, whereas preloaded lysine or methionine had no effect.The results are interpreted in terms of a model which envisions roles for cellular and external Na+ and for cyclic AMP in the activation and regulation of amino acid transport in intestine.  相似文献   

17.
Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na+ and K+ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na+ and K+ are suggested. 2) In the absence of K+, Na+ acts as an uncompetitive modifier raising the apparent Km and Vmax for MgATP. 3) In the absence of Na+, K+ activates non-competitively with respect to MgATP. Thus K+ increases Vmax but does not affect the apparent affinity constant. 4) K+ and Na+ double the rate constants. 5) In the presence of Na+ or K+, Mg2+ in excess acts as a weak inhibitor to Na+ and/or K+ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.  相似文献   

18.
Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.  相似文献   

19.
Abstract: The effects of K+ depolarization and of stimulation by veratridine on apparent cytosolic free Ca2+ ([Ca2+]cyt) and net Ca2+ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca2+]cyt was determined with fura-2, and Ca2+ accumulation was measured with tracer 45Ca. [Ca2+]cyt was ~ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]0, was increased from the normal 5 mM to 30 or 50 mM, 45Ca uptake and [Ca2+]cyt both increased within 1 s. Both increases were directly related to [Ca2+]0 for [Ca2+]0= 0.02–1.2 mM; however, the increase in 45Ca uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads ≤100 μmol/L of cell water, equivalent to the Ca2+ entry during a train of ≤60 impulses), the 45Ca uptake exceeded the increase in [Ca2+]cyt by a factor of nearly 1,000. This indicates that ~99.9% of the entering Ca2+ was buffered and/or sequestered within ~ 1 s. With larger Ca2+ loads, a larger fraction of the entering Ca2+ was buffered; ~99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca2+ entry and the increase in [Ca2+]cyt, the “calcium buffer ratio”β, was therefore ~ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca2+]cyt, which ranged between ~7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca2+]0= 0.2–0.6 mM), 45Ca influx and [Ca2+]cyt increased progressively for ~10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in 45Ca influx and [Ca2+]cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca2+]cyt and the veratridine-stimulated 45Ca within the terminals to decline, thereby demonstrating that the Ca2+ loading is reversible in the presence of extracellular Ca2+. These data show that synaptosomes are capable of buffering and metabolizing Ca2+ in a manner expected for intact neurons.  相似文献   

20.
Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between 22Na+ and 42K+ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na+-free medium or on 0.2 × sea water. 22Na+ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na+ m?3, particularly in Na+-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. 42K+ uptake decreased with increasing salinity in Na+-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na+ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of 42K+ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na+ uptake was suggested at low Na+ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.  相似文献   

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