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Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na+/K+-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pHi), intracellular Ca2+ ([Ca2+]i) and Na+/K+-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca2+ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca2+]i (200%). A decrease in pHi (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca2+. Inhibition of Na+/H2+ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca2+ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K+/H+ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca2+ influx via Na+/Ca2+ exchange, which under hyposmotic conditions activates the K+ and Ca2+ mitochondrial exchange systems (K+/H+ and Ca2+/H+).Deceased  相似文献   

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We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 μm, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.  相似文献   

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Summary The effects of cAMP, ATP and GTP on the Ca2+-dependent K+ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca2+-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K+ ghosts were incubated in an isotonic Na+ medium, the rate constant of Ca2+-dependent K+ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg2+ was added together with ATP, and it was abolished by raising free Ca2+ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K+ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K+ loss when the ghost free-Ca2+ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg2+ and it, was not overcome by raising internal Ca2+. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K+ loss. Although this effect was observed in the absence of added Mg2+ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg2+. The K+ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF2 (10mm), or MgF2 (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-32P)ATP under various conditions affecting K+ channel activity, was in direct correspondence to their effect on K+ efflux. The results suggest that the K+ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.  相似文献   

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When red cells are starved or incubated in the presence of metabolic poisons, with or without substrates, a large increase in K+ permeability is observed which depends on the presence of Ca2+ in the medium. The production or removal of a metabolite which controls the K+ permeability has been proposed to explain these effects. In the present experiments, a parallelism is found to exist between the rate of ATP depletion and the increase in Ca2+ uptake and K+ loss when red cells are depleted by different methods. The results support the view that the intracellular concentration of ATP may be the main factor on which the rate of Ca2+ uptake and the subsequent increase in K+ permeability depend.  相似文献   

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Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na++K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na++K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 m Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.  相似文献   

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1. 1. It has previously been demonstrated that an increase in extracellular Ca2+ concentration induces a transient increase in K+ permeability and associated hyperpolarization of the red cell membrane of the giant salamander, Amphiuma means. This phenomenon is analogous to the Ca2+-induced KCl loss observed in ATP-depleted human red cells and red cell ghosts.
2. 2. Histamine, which enhances the Ca2+-induced K+ loss from depleted human red cells, is without effect on this Ca2+-induced hyperpolarization of Amphiuma red cells.
3. 3. Promethazine (10 μM) and mepyramine (1 mM), which inhibit the Ca2+-induced K+ loss in depleted human red cells, also block the Ca2+-related hyperpolarization of Amphiuma erythrocytes.
4. 4. Chlorpromazine (25 μM), despite being a weak antihistamine, is equally effective in blocking the Ca2+-induced hyperpolarization of Amphiuma red cells.
5. 5. Ionophore A23187 causes a large and sustained Ca2+/K+-dependent hyperpolarization even in the presence of normal (1.8 mM) concentrations of Ca2+. This hyperpolarization is relatively insensitive to chlorpromazine and promethazine.
6. 6. The inhibition of the Ca2+-induced hyperpolarization of the Amphiuma red cell membrane by chlorpromazine and promethazine may be related to their properties as local anaesthetics.
Abbreviations: MOPS, morpholinopropane sulphonate  相似文献   

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Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are:K m =0.11 m Ca2+ andV max=81±4 nmol Pi/min·mg protein at 37°C. ATP-dependent Ca2+ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca2+/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75mm Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5mm an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na+–K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 m Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells.  相似文献   

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The ionic requirements for K+-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca2+ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K+-induced efflux. The Ca2+-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K+. In addition, verapamil did not antagonize 50 mM K+-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K+-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K+ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K+-evoked release, replacing Na+ with choline abolished the taurine efflux seen in response to K+ stimulation. Together, these findings suggest that neuronal N- and L-type Ca2+- and voltage-dependent Na+-channels are not involved in the influx of Ca2+ which appears to be necessary for K+-evoked taurine efflux, and that in addition to Ca2+, extracellular Na+ is also required.  相似文献   

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Summary We have studied the all or none cell response of Ca2+-dependent K+ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity=1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca2+ of the individual K+ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6–8 times the measured rate of uptake of external45Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca2+-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells. The above results suggest that the two all or none cell responses studied here can be explained by heterogeneity of agonist distribution among cells. Since pump-leak fluxes exist in both cases, differences of agonist distribution could be generated by heterogeneity of pumping among cells. This interpretation turns interest from K channels to Ca pumps to explain the heterogeneous behavior of red cells in response to a uniform stimulus.  相似文献   

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The increase in Ca2+ permeability by addition of ionophore A23187 in the presence of external Ca2+ did not alter the bumetanide-sensitive Na+/K+ effluxes in human red blood cells. An inhibition of this pathway by cellular Ca2+ could be observed only under conditions in which the cellular ATP content was drastically depleted.  相似文献   

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  • 1.1. Two components of Ca2+-Mg2+-ATPase are observed in kidneys of G. mirabilis. The high-affinity component has a K0.5Ca of 0.23μM; the low-affinity activity K0.5Ca is 90–110μM. The high-affinity activity requires Mg2+, displays Michaelis-Menten kinetics, has peak activity at 1.2 μM Ca2+, and is insensitive to ouabain and Na+ azide.
  • 2.2. In subcellular fractions, the high-affinity component segregates with Na+-K+-ATPase and is localized predominantly in BLM. The low-affinity component is broadly distributed among membranous organelles, including brush border, and may be equivalent to alkaline phosphatase.
  • 3.3. Specific activity of the high-affinity Ca2+-Mg2+-ATPase is modestly increased following adaptation of fish to FW, but total renal high-affinity activity is greatest in the hypertrophied kidneys of FW-adapted fish and is least in kidneys of fish adapted to 200% SW.
  • 4.4. High-affinity Ca2+-Mg2+-ATPase may be associated with active Ca2+ transport or with regulation of intracellular Ca2+ concentration of tubular cells.
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  • 1.1. The expected higher gill (Na++K+)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
  • 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg2+, 50 mM Na+, 2.5 mM K+, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg2+, 100 mM Na+, 10 mM K+, pH 7.5 in BW.
  • 3.3. The change of the enzyme activation kinetics by Mg2+, ATP, Na+ and K+ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
  • 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.
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20.
Z. Ping  I. Yabe  S. Muto 《Protoplasma》1992,171(1-2):7-18
Summary K+, Cl, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K+, K+ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K+ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K+ channels showed a significant discrimination against Na+ and Cl. The Cl channel opened at positive vacuolar membrane potentials for cytoplasmic Cl influx had a high conductance of 110pS in symmetrical 100mM Cl. When K+ and Cl channels were excluded from opening, no traces were found of Ca2+ channel activity for vacuolar Ca2+ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca2+ channel which allowed influx of cytoplasmic Ca2+ into the vacuole when the Ca2+ concentration on the cytoplasmic side was high. When Ca2+ was substituted by Ba2+, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba2+Sr2+Ca2+Mg2+=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca2+ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd2+, but was insensitive to La3+, Gd3+, Ni2+, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K+, Cl, and Ca2+ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.  相似文献   

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