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1.
The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.  相似文献   

2.
Investigation of Ca2+ transport by calcium pump of the cell plasma membrane of the gastric glands isolated from guinea pigs and its inhibition by metal cations has been performed. The mainly competitive type of Ca2+ translocation inhibition by the calcium pump by metals cations (0.025-1.00 mM) was determined. Potency of inhibition increases in such an order (I50, mM): Ba2+ (0.336) < Sr2+ (0.251) < Mn2+ (0.099) < Co2+ (0.029) < Cd2+ (0.016). It was shown by one-factor dispersion analysis that potency of inhibition depends on ionic radii and hydration enthalpy of metal cations and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 83.73-85.95). Dependence of the inhibition constants (I50) on ionic radii is most adequately described by the parabolic equation, such a dependence on hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that selective Ca2+ translocation by the calcium pump and its inhibition by metal cations is determined by the interaction between energy of their interaction with cation-binding sites of the transport system and energy of hydration. Energetics of such interactions depends on the steric factors. The physicochemical model of the Ca2+ selective translocation by calcium pump and its inhibition by metal cations has been proposed.  相似文献   

3.
C.A Pasternak 《Cell calcium》1986,7(5-6):387-397
An increase in non-specific permeability across the plasma membrane of cells bathed in physiological media leads to an increase in intracellular Ca2+. The extent of the increase depends on the extent of the membrane lesion; the duration of the increase depends on the metabolic capability of cells to pump Ca2+ out. A slight, transient increase in cytosolic Ca2+ can be beneficial because it activates repair mechanisms; a large, sustained, increase is toxic because it impairs normal cell function. Extracellular Ca2+ prevents increases in non-specific permeability: only when the protective action of extracellular Ca2+ is overcome by pore-forming agents does intracellular Ca2+ rise to toxic levels. The beneficial actions of Ca2+, superimposed on its damaging effect, account for some of the paradoxical effects of Ca2+ that have been described in the literature.  相似文献   

4.
It has been found that Sr2+, La3+ Mn2+ (10-50 microM) inhibit Ca2+ transport into mitochondria in a competitive manner. Cd2+ ions show the mixed type inhibition of this transport. The inhibitory constants (Ki, microM) of the metals cations effect on Ca2+ transport increases in such a sequence: La3+ (2,11), Cd2+ (10,36), Mn2+ (49,29), Sr2+ (66,43). The metals cations inhibitory effect has an insignificant dependence on their ionic radii. But it is good correlated with the series of metals cations, based on the stability constants of their complexes with acetate (r = -0.96), aspartic (r = -0.91) and glutaminic acids and their hydratation enthalpy (r = -0.78). These data reveal that hydratation of metals cations and their interaction with carboxyles of Ca(2+)-uniporter plays an important role in the process of Ca2+ transport into mitochondrial matrix space and its inhibition by the metals cations. The mixed type inhibition of mitochondrial Ca2+ uptake by Cd2+ seems to be caused by the partial de-energization of mitochondria owing to Cd2+ interaction with SH-containing respiratory chain components and pore-forming ligands of mitochondrial membrane.  相似文献   

5.
The element Cd is considered to have no biological function and is highly toxic to humans and animals. Toxic effects of this metal upon cell membrane structure and function have been shown. On the other hand, Ca is an essential element in a wide variety of cellular activities. The present study was initiated to research whether the interaction between Ca and Cd could affect D-galactose absorption across the rabbit jejunum in vitro. In media with Ca2+, when CdCl2 was present at 0.5 or 1 mM, Cd was found to significantly reduce the sugar absorption. In Ca2+-free media, where CaCl2, was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by Cd, but when CaCl2 was replaced isotonically with MgCl2, the inhibition is observed. Verapamil at 10−6 M (blocking mainly Ca2+ transport) did not modify the inhibitory effect of cadmium on D-galactose transport. When 10−6 M of A 23187 (Ca2+ specific ionophore) was added in media with/without Ca2+; CdCl2 produced no change in D-galactose transport. These results suggest that Ca and Cd could have affinity for the same chemical groups of enterocyte membrane, which would be related with the intestinal absorption of D-galactose.  相似文献   

6.
Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.  相似文献   

7.
The data are presented concerning the role of cAMP-dependent phosphorylation of sarcoplasmic reticulum and sarcolemma proteins in the transport of Ca2+ through the cardiac skeletal and smooth muscle membranes. Phosphorylation of membrane proteins by soluble and membrane-bound cAMP-dependent protein kinases is shown to have a regulating effect on Mn2+-, Ca2+-ATPase activity and to change membrane permeability for Ca2+. The molecular mechanisms and the character of the interaction between the transport systems and the phosphorylating protein substrates have not been yet established.  相似文献   

8.
A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.  相似文献   

9.
The influence of Cd2+ on the function and structure of liver mitochondria of rats and lamprey (Lampetra fluviatilis L.) has been studied in vitro. It is shown that Cd2+ can penetrate into the mitochondrial matrix due to Ca2+-transport mechanism. Being stored in the mitochondria, Cd2+ inhibits respiration and an energy dependent transport of penetrating cations (Cs+-valinomycin), and disturbs passive permeability of the inner mitochondrial membrane for monovalent cations and H+. The effect of Cd2+ on the lamprey liver mitochondria is more pronounced than in the case of rats.  相似文献   

10.
The anti-calmodulin drugs calmidazolium (CMZ) and trifluoperazine (TFP) were shown to have a number of effects on 45Ca transport by plasmalemmal vesicles from gastric smooth muscle. Although these compounds produced the expected dose-dependent inhibition of the plasmalemmal ATP-dependent Ca2+ transport system, they also evoked a Ca2+ release comparable to that observed in the presence of the Ca2+ ionophore, ionomycin. This increased transmembrane Ca2+ flux was so large that it accounted for much of the apparent decrease in 45Ca uptake produced by these agents. Thus, direct effects of CMZ and TFP on ATP-dependent 45Ca uptake could only be reliably assessed for brief (less than or equal to 30 seconds) drug exposures. The explanation for the observed effects of CMZ and TFP on membrane Ca2+ permeability is unclear. The increased transmembrane Ca2+ flux may reflect nonspecific effects on membrane permeability or it may reflect a specific interaction of the anticalmodulin drugs with a Ca2+ release channel or with the Ca2+ transport ATPase. In any case, these results suggest the need for caution in the design and interpretation of studies using both CMZ and TFP as anticalmodulin agents.  相似文献   

11.
Sidedness of the effect of K+ on Ca transport by the sarcoplasmic reticulum Ca pump reconstituted into soybean phospholipid vesicles was investigated. The reconstituted vesicles which sustained a high rate of Ca transport even in the absence of Ca-precipitating anions exhibited low passive permeabilities to 42K+, 86Rb+, or 45Ca2+. Evidence was presented that K+ activated the Ca pump on the external surface of the vesicles and that it was not taken up by the vesicles during the pump activity. In the presence of high externally added K+, the reconstituted vesicles preloaded with K+ exhibited a significantly higher Ca transport activity than the vesicles preloaded with Tris+ but not the ones preloaded with Li+. Ca transport by the K+-loaded vesicles was accompanied by a small amount of K+ efflux, which corresponded to about 20% of the amount of Ca+ taken up. Since the intravesicular K+ did not affect the turnover of the ADP-insensitive component (E2P) of the phosphoenzyme intermediate formed during the pump cycle, it was concluded that the intravesicular K+ stimulated the Ca pump activity indirectly by compensating the charge imbalance caused by the electrogenic Ca2+ movement. These results thus indicate that K+ activates the Ca pump only on the cytoplasmic side of the sarcoplasmic reticulum membrane, but it is not obligately transported across the membrane under conditions where K+ fully activates the Ca pump.  相似文献   

12.
The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.  相似文献   

13.
Dependence of the red blood cell calcium pump on the membrane potential   总被引:4,自引:0,他引:4  
(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.  相似文献   

14.
Two major Ca2+ transport mechanisms co-function in a preparation of synaptosomal plasma membrane vesicles: an (ATP + Mg2+)-dependent Ca2+ pump, and a reversible Na+-Ca2+ exchanger (Gill, D. L., Grollman, E.F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192). An accurate comparative analysis of the kinetics of the two Ca2+ transporters under free Ca2+ conditions precisely buffered with EGTA, reveals that both mechanisms have high affinity for Ca2+. The ATP-dependent Ca2+ pump displays simple saturation kinetics with a Km for Ca2+ of 0.11 microM and a Vmax of 2.2 nmol/min/mg of protein. In contrast, the Na+-Ca2+ exchanger has a complex dependence on free Ca2+, the activity continuing to saturate over a wide range of free Ca2+ concentrations from 0.03 microM to 3 mM. The curvilinear Eadie-Hofstee analysis reveals a distinct high affinity component for the exchanger with a Km for Ca2+ of approximately 0.5 microM, and a lower affinity component not accurately resolvable into a discrete Km value. 2 mM amiloride blocks Na+-Ca2+ exchange-mediated Ca2+ uptake by 90% over a wide range of free Ca2+ (0.3-3000 microM), suggesting a similar noncompetitive inhibition of both low and high affinity Ca2+ sites. Ca2+ accumulated in vesicles via either the Ca2+ pump or Na+-Ca2+ exchanger is rapidly (in less than 1 min) released by 0.1% saponin (w/v), although a minor component (8-10%) of Ca2+ pump activity is resistant to saponin addition. The IC50 for the effect of saponin is the same (0.01%, w/v) for both Ca2+ transport mechanisms. The ATP-dependent Ca2+ pump is shown to be highly sensitive to vanadate inhibition (Ki = 0.5 microM). The high saponin sensitivity of both Ca2+ transporters and the potent effect of vanadate on Ca2+ pumping, together with previous Na+ channel and Na+ pump flux studies in the same membrane vesicles (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990), all strongly suggest that both of the high affinity Ca2+ transporters function in the plasma membrane where they are of major functional importance to the regulation of intrasynaptic free Ca2+ levels.  相似文献   

15.
The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.  相似文献   

16.
Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+:Mg2+-ATPase pump protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 +/- 0.19) X 10(-3) and (1.25 +/- 0.18) X 10(-3). Damage to vesicle function was assayed by measurements of increases in passive Ca2+ permeability. Rates of passive Ca2+ movement into the SR lumen were increased by dye photosensitization in proportion to radiation absorbed. Active Ca2+ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 microM and 1.16 microM, respectively. The photochemical lability of the Ca2+ pump protein and alterations in passive and active Ca2+ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.  相似文献   

17.
A Chu  P Volpe  B Costello  S Fleischer 《Biochemistry》1986,25(25):8315-8324
Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

18.
The Ca2+ pump of rat heart sarcolemma has been studied via its ATP-dependent Ca2+ transport and (Ca2+ + Mg2+)-dependent ATPase activities. Direct incubation of the sarcolemmal vesicles with micromolar concentration of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) results in the reduction of Ca2+ uptake by 34 +/- 10% and ATP hydrolysis by 55 +/- 7%. Similar inhibition of the sarcolemmal Ca2+ pump is also observed with micromolar concentration of inositol trisphosphate (IP3), while GDP or inositol tetrakisphosphate (IP4) has no effect. Based on the evidence that these sarcolemmal vesicles are capable of generating IP3 upon stimulation by GTP gamma S, and that no additive effect is observed when both agents are incubated together with the membranes, it is concluded that the effect of GTP gamma S on the Ca2+ pump is mediated by IP3. The results here show for the first time that plasma membrane Ca2+ pump has a role in the primary Ca2+ signaling.  相似文献   

19.
To evaluate the effects of freezing and thawing on Ca2+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me2SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca2+ transport. However, freezing and thawing increase the membrane permeability to sucrose.  相似文献   

20.
The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.  相似文献   

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