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1.
The modulation of the Ca2+- (or Pb2+-)activated K+ permeability in human erythrocytes by vanadate, menadione and chloro-substituted menadione analogs was investigated by measurements of K+ fluxes and single-channel currents. Vanadate and menadione stimulate the K+ permeability by increasing the probability of channel openings; the menadione analogs, on the other hand, inhibit the K+ permeability by increasing the probability of channel closings. The compounds used in these experiments also interact with oxidoreductases; it is demonstrated that menadione analogs in contrast to menadione strongly inhibit the membrane-bound dehydrogenase in the erythrocytes. Concentrations of Pb2+ above 10 μmol/l, but not of Ca2+, inhibit the enzyme activity as well as the K+ permeability. The parallel effects on dehydrogenase activity and the K+ channels suggest a direct relationship between these two systems in the membrane of erythrocytes. 相似文献
2.
Modulation of Ca2+-activable K+ permeability was compared with modulation of a membrane-bound oxidoreductase activity in human erythrocytes. Changes in the K+ permeability were monitored by flux measurements and single-channel recordings. The enzyme activity was detected by measuring reduction of ferricyanide. Pb2+, Atebrin and menadione had parallel effects on the channel protein and the enzyme. In contrast, propranolol stimulates K+ permeability, but is without effect on enzyme activity. The results demonstrate that the K+ channel and the enzyme are distinct membrane proteins but that the enzyme activity may influence channel gating. 相似文献
3.
To study the effect of propranolol on the Ca2+- or Pb2+-activated K+ permeability in human erythrocytes, K+ effluxes were compared with single-channel currents. The results demonstrate that propranolol has a twofold effect: (1) it renders the channel protein more sensitive to Ca2+ or Pb2+; and (2) it simultaneously inhibits channel activity and slightly reduces single-channel conductance. The number of active channels is not affected. 相似文献
4.
Mechanical deformation of normal ATP-replete human erythrocytes increased their permeability to Ca2+ sufficiently to turn on the Ca(2+)-activated K+ channel (the Gardos channel). When Ca2+ is absent, mechanical deformation of normal erythrocytes induces an equivalent increase the permeability of both Na+ and K+, In the presence of 0.1 to 1 mM Ca2+, a further increase in the K+ efflux rate was seen. There was no increase in Na+ flux above that induced by deformation itself. The involvement of the Ca(2+)-activated H channel was verified by showing the specific inhibitors of the channel, quinine and charybdotoxin, prevent the Ca(2+)-induced increase in K+ efflux. These results are consistent with a model of sickle cell dehydration proposed by Bookchin et al. ((1987) Prog. Clin. Biol. Res. 240, 193-200). The estimated rate of Ca2+ entry under these conditions (37 degrees C, 1000 dyne/cm2, and laminar shear) was about 1 mmol/loc per h. 相似文献
5.
Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions. 相似文献
6.
Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes 总被引:2,自引:0,他引:2
Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
ATP-sensitive potassium channel regulates astrocytic gap junction permeability by a Ca2+-independent mechanism 总被引:3,自引:0,他引:3
Using the scrape-loading technique in cultured astrocytes, we show that sulfonylureas such as tolbutamide and glybenzcyclamide, which inhibit the ATP-sensitive K+ channel, prevent the inhibition of gap junction permeability caused by several structurally unrelated uncouplers such as oleic acid, arachidonic acid, endothelin-1, octanol, and alpha-glycyrrhetinic acid. When the intracellular level of Ca2+ was diminished, all the uncouplers tested were still able to inhibit gap junction communication, indicating that their inhibitory effect was not mediated by Ca2+. In addition, tolbutamide and glybenzcyclamide prevented the inhibitory effect of these uncouplers in Ca(2+)-depleted astrocytes, suggesting that the inhibition of the ATP-sensitive K+ channel increases gap junction permeability through a Ca(2+)-independent mechanism. The activation of the ATP-sensitive K+ channel caused by potassium channel openers such as diazoxide and pinacidil led to the inhibition of gap junction communication and overcame the effect of sulfonylureas. These results suggest that the ATP-sensitive K+ channel regulates gap junctional permeability. 相似文献
8.
Two tests were performed to assess the relationship between the Ca2+-activated K+ channel and the Ca2+-pumping ATPase in human erythrocytes. Antibodies against the purified ATPase inhibited the ATPase in resealed erythrocytes, but had no effect on the K+ channel (as assessed by Rb+ efflux). Reconstituted liposomes containing the purified active Ca2+-pumping ATPase showed no Ca2+-activated Rb+ influx. Both of these results suggest that some molecule other than the Ca2+-ATPase is responsible for the K+ channel. 相似文献
9.
Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats 总被引:2,自引:0,他引:2
R L Shoemaker R T Worrell 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1991,196(3):325-332
We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously. 相似文献
10.
Properties of the Ca-activated K conductance of human red cells as revealed by the patch-clamp technique 总被引:1,自引:0,他引:1
Application of Ca2+ to the inner surface of red-cell membranes activates unitary currents that can be measured in cell-attached and cell-free membrane patches. Ca2+ can be replaced by Pb2+ to activate the single channels. In addition to internal Ca2+ external K+ has to be present. The channels are preferentially permeable to K+ with a selectivity ratio PK:PNa of about 15:1 as estimated from measurement of reversal potentials. The dependence of channel activity on Ca2+ is compatible with the conception that the binding of two Ca2+ is necessary to open a single channel. Both the channel activity and the single-channel conductance exhibit inward rectification. External and internal Na+ inhibit the K+ currents. The reported results suggest that the unitary current events are responsible for the Ca2+-dependent K+ permeability known from measurement on cell suspensions. Therefore, comparison of the two techniques allows calculation of the number of K+ channels per red cell, which on average is about 10. 相似文献
11.
Plasma membrane nadh dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species 总被引:1,自引:0,他引:1
C Miner S López-Burillo J García-Sancho B Herreros 《Biochimica et biophysica acta》1983,727(2):266-272
Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity. 相似文献
12.
Quinones have been shown to possess antineoplastic activity; however, their effects on ionic currents remain unclear. The effects of 2-mercaptophenyl-1,4-naphthoquinone (2-MPNQ), menadione (MD) and 1,4-naphthoquinone (1,4 NQ) on cell proliferation and ionic currents in pituitary GH3 lactotrophs were investigated in this study. 2-MPNQ was more potent than menadione or 1,4-naphthoquinone in inhibiting the growth of GH3 cells. 2-MPNQ decreased cell proliferation in a concentration-dependent manner with an IC50 value of 3 microM. In whole-cell recording experiments, 2-MPNQ reversibly caused an inhibition of Ca2+-activated K+ current (I(K(Ca)) in a concentration-dependent manner. The IC50 value for 2-MPNQ-induced inhibition of I(K(Ca)) was 7 microM. In the inside-out configuration of single channel recording, 2-MPNQ (30 microM) applied intracellularly suppressed the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels but did not modify single channel conductance. Menadione (30 microM) had no effect on the channel activity, whereas 1,4-naphthoquinone (30 microM) suppressed it by about 26%. Both 2-MPNQ and thimerosal suppressed the dithiothreitol-stimulated channel activity. 2-MPNQ also blocked voltage-dependent K+ currents, but it produced a slight reduction of L-type Ca2+ inward current. However, unlike E-4031, 2-MPNQ (30 microM) did not suppress inwardly rectifying K+ current present in GH3 cells. Under the current clamp configuration, the presence of 2-MPNQ (30 microM) depolarized the cells, and increased the frequency and duration of spontaneous action potentials. The 2-MPNQ-mediated inhibition of K+ currents would affect hormone secretion and cell excitability. The blockade of these ionic channels by 2-MPNQ may partly explain its inhibitory effect on the proliferation of GH3 cells. 相似文献
13.
Accumulation of Ca2+ induced by cytotoxic levels of menadione in the isolated, perfused rat liver 总被引:1,自引:0,他引:1
Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity. 相似文献
14.
Menadione is a synthetic derivative of the natural vitamins K with antiinflammatory activity among its potentially significant clinical properties. We have found this agent to stimulate the production of superoxide anion (O2-) in human polymorphonuclear leukocytes (PMN) and dimethylsulfoxide-differentiated HL-60 cells in a time-, cell number-, and drug concentration-dependent manner. Conversely, menadione attenuates both O2- production and lysozyme release in cells stimulated by phorbol myristate acetate (PMA), fMet-Leu-Phe, or Ca2+ ionophore. 4-Acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2-2'disulfonic acid stilbene, agents which inhibit transmembrane O2-) flux, do not alter menadione's effects on superoxide dismutase (SOD) inhibitable cytochrome c reduction in resting or PMA-stimulated PMN. Likewise, quinone reductase inhibitors, warfarin and dicumarol, known to attenuate vitamin K-dependent responses and enhance quinone-mediated oxidative stress, have no effect upon menadione-stimulated O2- production. Furthermore, menadione-induced suppression of stimulus-mediated lysozyme release is not reversed by cotreatment with oxygen metabolite scavenging enzymes SOD and catalase. Nevertheless, under conditions of restricted oxygen supply, the suppressive effect of menadione on stimulant-induced lysozyme release is greatly diminished. Thus, although pharmacological manipulation suggests otherwise, there appears to exist at least a component of the inhibitory activity of menadione that is oxygen dependent, and may be oxidative stress-related. 相似文献
15.
Inhibition of hepatocyte plasma membrane Ca2+-ATPase activity by menadione metabolism and its restoration by thiols 总被引:4,自引:0,他引:4
Incubation of isolated rat hepatocytes with cytotoxic concentrations of menadione resulted in inhibition of plasma membrane Ca2+-ATPase activity. This could be restored by subsequent treatment with either dithiothreitol or reduced glutathione, suggesting that the inhibition by menadione was due to oxidation of sulfhydryl groups critical for Ca2+-ATPase activity. 相似文献
16.
H A Pershadsingh R D Gale D M Delfert J M McDonald 《Biochemical and biophysical research communications》1986,135(3):934-941
Increased membrane permeability (conductance) that is specific for K+ and directly activated by Ca2+ ions, has been identified in isolated adipocyte plasma membranes using the K+ analogue, 86Rb+. Activation of these K+ conductance pathways (channels) by free Ca2+ was concentration dependent with a half-maximal effect occurring at 32 +/- 4 nM free Ca2+ (n = 7). Addition of calmodulin further enhanced the Ca2+ activating effect on 86Rb+ uptake (K+ channel activity). Ca2+-dependent 86Rb+ uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K+ channels that are activated by Ca2+ and amplified by calmodulin. 相似文献
17.
Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes 总被引:2,自引:0,他引:2
R Grygorczyk 《Biochimica et biophysica acta》1987,902(2):159-168
The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed. 相似文献
18.
Lead Enters Bovine Adrenal Medullary Cells Through Calcium Channels 总被引:17,自引:1,他引:16
Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca. 相似文献
19.
The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components. 相似文献
20.
Calcium and voltage dependence of single Ca2+-activated K+ channels from cultured hippocampal neurons of rat 总被引:3,自引:0,他引:3
F Franciolini 《Biochimica et biophysica acta》1988,943(3):419-427
Calcium and voltage dependence of the Ca2+-activated K+ channel, K(Ca), was studied at the single-channel level in cultured hippocampal neurons from rat. The K(Ca) channel has approx. 220 pS conductance in symmetrical 150 mM K+, and is gated both by voltage and by Ca2+ ions. For a fixed Ca2+ concentration at the inner membrane surface, [Ca]i, channel open probability, Po, increases e-fold for 14 mV positive change in membrane potential. At a fixed membrane potential (0 mV), channel activity is first observed at [Ca]i = 10(-6) M, and increases with Ca2+ concentration approximating an absorption isotherm with power 1.4. The [Ca]i required to half activate (Po = 0.5) the channel is 4.10(-6) M. When compared to other preparations, the K(Ca) channel from hippocampal neurons reported here shows the lowest Ca2+ sensitivity and the highest voltage sensitivity. These findings are interpreted in evolutionary terms. 相似文献