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1.
In resting muscle, cytoplasmic Ca2+ concentration is maintained at a low level by active Ca2+ transport mediated by the Ca2+ ATPase from sarcoplasmic reticulum. The region of the protein that contains the catalytic site faces the cytoplasmic side of the membrane, while the transmembrane helices form a channel-like structure that allows Ca2+ translocation across the membrane. When the coupling between the catalytic and transport domains is lost, the ATPase mediates Ca2+ efflux as a Ca2+ channel. The Ca2+ efflux through the ATPase channel is activated by different hydrophobic drugs and is arrested by ligands and substrates of the ATPase at physiological pH. At acid pH, the inhibitory effect of cations is no longer observed. It is concluded that the Ca2+ efflux through the ATPase may be sufficiently fast to support physiological Ca2+ oscillations in skeletal muscle, that occur mainly in conditions of intracellular acidosis.  相似文献   

2.
The regulatory role of Ca2+-stimulated adenosine 5-triphosphatase (Ca2+-ATPase) in Ca2+ transport system of rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+-ATPase activity was calculated by subtracting Mg2+-ATPase activity from (Ca2+–Mg2+)-ATPase activity. The release of Ca2+ from the Ca2+-loaded nuclei was evoked progressively after Ca2+ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca2+ from the Ca2+-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca2+ uptake and induced a promotion of Ca2+ release from the Ca2+-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca2+-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca2+ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca2+ channels, did not prevent the NAD+ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca2+ release, suggesting that Ca2+ channels do not involve on Ca2+ release from the nuclei. These results indicates that an inhibition of nuclear Ca2+-ATPase activity causes the decrease in nuclear Ca2+ uptake and the release of Ca2+ from the Ca2+-loaded nuclei. The present finding suggests that Ca2+-ATPase plays a critical role in the regulatory mechanism of Ca2+ uptake and release in rat liver nuclei.  相似文献   

3.
In order to identify defects in Na+-Ca2+ exchange and Ca2+-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na+-dependent Ca2+ uptake, Na+-induced Ca2+ release, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na+-induced Ca2+-release. Similar results were obtained in Ca2+-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca2+ following a 5 min perfusion with Ca2+-free medium. Although a 2 min reperfusion of the Ca2+-free perfused hearts depressed sarcolemmal Ca2+-pump activities without any changes in Na+-induced Ca2+-release, Na+-dependent Ca2+ uptake was increased. These results indicate that alterations in the sarcolemmal Ca2+-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca2+ overload.  相似文献   

4.
The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.  相似文献   

5.
Relevant Ca2+ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca2+-mobilizing agents. Vasopressin produced a cytoplasmic Ca2+ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca2+ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca2+. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca2+ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca2+ pool was not. The vasopressin-induced cytoplasmic Ca2+ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca2+ pool, was sensed as a mitochondrial Ca2+ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca2+ peak was affected by cyclosporin A when the Ca2+ signal was induced by irreversible discharge of the intracellular Ca2+ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca2+ signals generated in the reticular store.  相似文献   

6.
Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca2+ concentration is necessary and sufficient for this process. The predominant source of Ca2+ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca2+ to the cytosol. The ER store is (re)filled by the store-specific Ca2+-ATPase. Ultimately, the depleted ER is replenished by Ca2+ which enters from the extracellular space to the cytosol via store-operated Ca2+ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca2+ channels and plasma membrane Na+/Ca2+ exchangers are additional means for cytosolic Ca2+ entry. Cytosolic Ca2+ levels can be modulated by mitochondria, which can take up cytosolic Ca2+ via the Ca2+ uniporter and release Ca2+ into cytosol via the mitochondrial Na+/Ca2+ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca2+ sources generates cytosolic Ca2+ dynamics that can drive Ca2+-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.  相似文献   

7.
Summary 45Ca fluxes and free-cytosolic Ca2+ ([Ca2+] i ) measurements were used to study the effect of Ca2+-mobilizing hormones on plasma membrane Ca2+ permeability and the plasma membrane Ca2+ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca2+ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca2+. In the present study, we show that activation of this pathway increases the plasma membrane Ca2+ permeability by approximately sevenfold. Despite that, the cells reduce [Ca2+]i back to near resting levels. To compensate for the increased plasma membrane Ca2+ permeability, a plasma membrane Ca2+ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca2+ pump. Activation of the plasma membrane Ca2+ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca2+ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca+ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca2+ pump.  相似文献   

8.
Ca2+ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca2+ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca2+ ATPase, the enzyme that participates in the vectorial translocation of Ca2+ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca2+ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.  相似文献   

9.
The cytoplasmic Ca2+ concentration ([Ca2+]cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca2+ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca2+]cyt. We measured changes of [Ca2+]cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca2+] from 1.3 mM to 5 mM induced an incrase in [Ca2+]cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd3+, 10 μM La3+ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca2+ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca2+) resulted in a decrease of [Ca2+]cyt from 105±10 nM to 88.5±10 nM with La3+, from 103±12 to 89±12 nM with Gd3+ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca2+ entry beside its function in Ca2+ signaling contributes to the regulation of resting [Ca2+]cyt.  相似文献   

10.
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   

11.
Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca2+ transport, but the Mg2+-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+. Additional experiments show that the Ca2+ sensitivity of the Ca2+-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca2+-ATPase activity. Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.  相似文献   

12.
We reported previously that a Ca2+-ATPase in rat testes and goat spermatozoa could be activated by Ca2+ alone without Mg2+, though it has a lot of similarities with the well known Ca2+, Mg2+-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca2+ and at low temperature. Increase of the concentration of Ca2+ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca2+, Mg2+-ATPase. The findings strengthened our previous report that Mg2+-independent Ca2+-ATPase is involved in Ca2+ transport and Ca2+ uptake like Ca2+, Mg2+-ATPase.  相似文献   

13.
Sergio de la Fuente 《BBA》2010,1797(10):1727-1735
We have investigated the kinetics of mitochondrial Ca2+ influx and efflux and their dependence on cytosolic [Ca2+] and [Na+] using low-Ca2+-affinity aequorin. The rate of Ca2+ release from mitochondria increased linearly with mitochondrial [Ca2+] ([Ca2+]M). Na+-dependent Ca2+ release was predominant al low [Ca2+]M but saturated at [Ca2+]M around 400 μM, while Na+-independent Ca2+ release was very slow at [Ca2+]M below 200 μM, and then increased at higher [Ca2+]M, perhaps through the opening of a new pathway. Half-maximal activation of Na+-dependent Ca2+ release occurred at 5-10 mM [Na+], within the physiological range of cytosolic [Na+]. Ca2+ entry rates were comparable in size to Ca2+ exit rates at cytosolic [Ca2+] ([Ca2+]c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca2+]c. As a consequence, the presence of [Na+] considerably reduced the rate of [Ca2+]M increase at [Ca2+]c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca2+]c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca2+]M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca2+ buffering, and comparison of our results with data on total mitochondrial Ca2+ fluxes indicate that the mitochondrial Ca2+ bound/Ca2+ free ratio is around 10- to 100-fold for most of the observed [Ca2+]M range and suggest that massive phosphate precipitation can only occur when [Ca2+]M reaches the millimolar range.  相似文献   

14.
To investigate Ca2+ uptake by Ca2+-depleted bovine chromaffin cells we depleted these cells of Ca2+ by incubating them in Ca2+-free buffer, then measured changes in cytoplasmic Ca2+ concentration ([Ca2+ 1)45Ca2+ uptake, and Mn2+ uptake in response to added Ca2+ or MN2+. In depleted cells, the increase in [Ca2+]i after Ca2+ addition, and the Mn2+ and45Ca2+ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca2+ pools in depleted cells increased after Ca2+ addition. The times for [Ca2+]i rise and Mn2+ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca2+ stores. In Ca2+-depleted cells and cells which had been loaded with BAPTA,45Ca2+ uptake was much higher than in control cells. These results suggest that extracellular Ca2+ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn2+ influx depended on the level of filling of the Ca2+ stores, suggesting that some signalling takes place between the intracellular stores and Ca2+ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca2+]i cytosolic Ca2+ concentration - IP3 inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy  相似文献   

15.
Activation of the human red cell calcium ATPase by calcium pretreatment   总被引:1,自引:0,他引:1  
Some kinetic parameters of the human red cell Ca2+-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V max of about 0.4 μmol Pi/mg × hr and a K s of 0.3 μm Ca2+ were found. When Mg2+ (10 mm) or Ca2+(10 μm) were also added during preincubation, V maxbut not Kwas altered. Ca2+ was more effective than Mg2+, thus increasing V max to about 1.3 μmol Pi/mg × hr. The presence of both Ca2+ and Mg2+ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V max. Conversely, addition of ATP (2 mm) with either Ca2+ or Ca2+ plus Mg2+increased Vmax without affecting K. Preincubation with Ca2+ for periods longer than 30 min further increased Vmaxand reduced Kto levels as low as found with calmodulin treatment. The Ca2+ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from each other. Moreover, immunodetection of Ca2+-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca2+ plus ATP in the presence of Mg2+ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca2+ effect. Ca2+ preincubation may stimulate the Ca2+-ATPase activity by stabilizing or promoting the E1 conformation.  相似文献   

16.
Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca2+ i homeostasis and altered two Ca2+ release mechanisms in these cells (IP3 and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca2+ pump. One of the primary Ca2+ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP3 and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca2+ homeostasis in VSMC chronically exposed to atherogenic oxLDL.  相似文献   

17.
Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca2+-pump ATPase or sarcolemmal Ca2+-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca2+-pump ATPase or plasma membrane Ca2+-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.  相似文献   

18.
Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca2+ binding and accumulating activities as well as (Mg2+ + Ca2+)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca2+ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca2+ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca2+ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca2+ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca2+ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca2+ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca2+ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca2+ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca2+ accumulation (V (nmol Ca2+/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca2+ required for half-maximal velocities did not differ significantly with age (K0.5 for Ca2+ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca2+ binding also indicated that the velocity of Ca2+ binding but not the concentration of Ca2+ required for half-maximal binding was altered due to aging. At identical Ca2+ concentrations, the combined Ca2+ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca2+ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca2+ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg2+ + Ca2+)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca2+ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca2+ transport. Further, the age-related increment in the Ca2+ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca2+ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca2+ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.  相似文献   

19.
Summary Intracellular calcium [Ca2+] i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca2+] i transient followed by a steady-state increase as the characteristic [Ca2+] i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca2+] i signal in single cells. The distribution of [Ca2+] i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca2+] i present in the subplasmalemmal space and in one cell pole. [Ca2+] i gradients within these regions were not constant but showed temporal changes in the form of [Ca2+] i oscillations and spatial changes in the form of [Ca2+] i waves. [Ca2+] i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca2+] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca2+], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca2+] i waves was also independent of influx of extracellular Ca2+. [Ca2+] i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca2+] i induced by different agonists were encoded into changes of baseline [Ca2+] i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca2+] i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca2+] i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca2+] i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar9,Met(O2)11]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.  相似文献   

20.
A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (1 M) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca2+-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca2+-stimulated ATP hydrolysis was characterized by a reduction inV max for Ca2+. Levorphanol pretreatment reduced the Hill coefficient (HN) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca2+ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca2+/Mg2+ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca2+-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca2+-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV max for both Ca2+ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca2+-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca2+/Mg2+ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca2+ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca2+ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca2+ following inhibition of Ca2+/Mg2+ ATPase may underlie some of the pharmacological effects of opiate drugs.  相似文献   

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