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1.
In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca2+-channels, Na+–Ca2+ exchange and Ca2+-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca2+-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na+–K+ ATPase were decreased whereas the Mg2+-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na+–K+ ATPase activity, whereas Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg2+-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na+–Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca2+-transport processes is regulated by thyroid hormones and the modification of Ca2+-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.  相似文献   

2.
The aim of the study was to find out whether low phospholamban level in atria as compared with ventricles is associated with differences in sarcoplasmic reticular Ca2+-uptake and contractile performance. Relationship between phospholamban and -adrenergic stimulation in rat left atria and papillary muscles were examined by means of contractile measurements, sarcoplasmic reticular oxalate-supported Ca2+-uptake, and Western blotting of phosphorylated phospholamban. Phosphoprotein determination after -adrenergic stimulation demonstrated that the levels of Ser16 and Thr17 phosphorylated phospholamban in atria remained at about one-third of that in ventricles. However, comparison of sarcoplasmic reticular Ca2+-uptake in control and isoproterenol perfused preparations demonstrated that the effect of -adrenergic stimulation on sarcoplasmic reticular Ca2+-uptake was stronger in atrial preparations. Moreover, atria responded to isoproterenol with much larger increases in developed tension, contractility and relaxation rates than papillary muscles. Thus, despite lower level of phospholamban, the -adrenergic activation of sarcoplasmic reticular Ca2+-uptake and contractile indices are higher in atria.  相似文献   

3.
The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemia-reperfusion induced changes in heart function as well as Ca2+-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 M captopril or 100 M losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca2+-pump ATPase, Ca2+-uptake and Ca2+-release activities. Northern blot analysis revealed that mRNA levels for SR Ca2+-handling proteins such as Ca2+-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca2+-pump ATPase and Ca2+-uptake activities in the postischemic hearts but had no effect on changes in Ca2+-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca2+-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca2+-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca2+-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca2+-handling proteins.  相似文献   

4.
Ca2+ transients and the rate of Ca2+ release (dCaREL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca2+ indicators. Single pulse experiments with different returning potentials showed that Ca2+ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca2+ removal (dCaREM/dt) was studied by fitting the decaying phase of the Ca2+ transient (Melzer, Ríos & Schneider, 1986) and dCaREL/dt was calculated as the difference between dCa/dt and dCaREM/dt. The fast Ca2+ release decayed as a consequence of Ca2+ inactivation of Ca2+ release. Double pulse experiments showed inactivation of the fast Ca2+ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca2+ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca2+]i and the inhibited amount of Ca2+ release was 0.98. The [Ca2+]i for 50% inactivation of dCaREL/dt was 0.25 m, and the minimum number of sites occupied by Ca2+ to inactivate the Ca2+ release channel was 3.0. These data support Ca2+ binding and inactivation of SR Ca2+ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.  相似文献   

5.
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   

6.
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   

7.
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   

8.
Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca2+-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca2+-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca2+-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.  相似文献   

9.
Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twich muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased45Ca-binding.These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.  相似文献   

10.
Adult women have longer QT intervals compared with men of a similar age, indicating differences in the speed of repolarisation of the ventricles. We investigate the influences of gender on ventricular electrophysiology and intracellular Ca2+ regulation of the guinea pig heart. Comparing sexually mature animals, females exhibited a significantly longer APD. Peak L-type Ca2+ current (ICaL) was larger in females and when this current was inhibited with nifedipine the gender differences in APD were removed. APD differences also disappeared when the SR was depleted of Ca2+. Inactivation of ICaL during a clamp step is faster in females but slower during an action potential and SR Ca2+ content is larger. We suggest that gender differences in APD result from variation in the kinetics of ICaL stemming from alterations to Ca2+ release.  相似文献   

11.
The Ca2+-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca2+-uptake and Ca2+-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca2+-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca2+-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca2+-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca2+-ATPase. Regulation of the Ca2+-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca2+-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.  相似文献   

12.
Sarcoplasmic reticulum contains the internal Ca2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca2+ level is the same all throughout the SR; however, whether the Ca2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca2+ buffer capacity of the SR by comparing the reduction in SR Ca2+ levels with the corresponding increase in [Ca2+]i during activation of either IP3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca2+ influx, indicate the relevance of luminal SR Ca2+ buffer capacity in the [Ca2+]i response. To further study the importance of luminal SR Ca2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP3R-mediated Ca2+ release.  相似文献   

13.
Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca2+. Although MCC-135, an intracellular Ca2+-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca2+ uptake by SR and myofilament sensitivity to Ca2+. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca2+ uptake and myofilament sensitivity to Ca2+, respectively. In diabetic rats, SR Ca2+ uptake was decreased, which was related to decrease in protein level of SR Ca2+-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca2+ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca2+ was increased, without significant change in myofilament sensitivity to Ca2+ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca2+ in diabetic rats. These results suggest that MCC-135 enhances SR Ca2+ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca2+. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.  相似文献   

14.
Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca2+-pump activity and thereby may cause the occurrence of intracellular Ca2+ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca2+-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca2+ uptake and Ca2+-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca2+-stimulated ATPase activity and ATP-dependent Ca2+ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca2+-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca2+ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.  相似文献   

15.
Heart failure is common among the elderly and an alteration in myocardial Ca2+ transport is believed to be involved in its depressed contractile performance. Although ATP-dependent sarcoplasmic reticular (SR) Ca2+ transport has been reported to decrease in old hearts, virtually nothing appears to be known about the Ca2+ pump activity of SR in aging myocardium in the presence of calmodulin, one of its endogenous activators. In this study, the activity of the Ca2+ pump of aging cardiac SR was assessed in the presence of this endogenous stimulator. This assessment was therefore designed to give additional information about the status of this enzyme in old hearts. Male Sprague-Dawley rats were used and were divided into 3 groups: young (4–6 months old); middle-aged (15–17 months old) and old age (24–25 months old). Purified SR membranes were isolated from ventricular tissues. ATP-dependent Ca2+ accumulation by membrane vesicles of middle-aged and old hearts was significantly depressed in comparison to young hearts at all Ca2+ concentrations employed in the absence and presence of calmodulin. The activity of this Ca2+ transporter was similar in middle-aged and old hearts even in the presence of calmodulin. These results suggest that the activity of the Ca2+ pump in SR of aging hearts is depressed even in the presence of calmodulin.C. E. Heyliger is a Scholar of the British Columbia Heart Foundation.  相似文献   

16.
The conformational states of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca2+ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca2+ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca2+-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca2+ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca2+ gradient-mediated conformational changes in SR · Ca2+-ATPase.Abbreviations SR sarcoplasmic reticulum - HB hypocrellin B - Trp tryptophan - DMSO dimethysulfoxide - Hepes N-2-hydroxyethyl piperazine-N-ethanesulfonic acad - SR(50005) SR vesicles with 1000-fold transmembrane Ca2+ gradient - SR(5050) SR vesicles without Ca2+ gradient - Ksv(app) apparent Stern-Volmer constant - Ksvi Stern-Volmer constant of component i for dynamic quenching  相似文献   

17.
Ca microdomains in smooth muscle   总被引:1,自引:0,他引:1  
In smooth muscle, Ca2+ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca2+ to perform these multiple functions is the cell's ability to localize Ca2+ signals to certain regions by creating high local concentrations of Ca2+ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca2+ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca2+ store. A single Ca2+ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca2+] and the rapid rates of decline target Ca2+ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca2+ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca2+. In this review, the generation of microdomains arising from Ca2+ influx across the plasma membrane and the release of the ion from the SR Ca2+ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.  相似文献   

18.
Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca2+-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca2+-transporter showed high affinity for Ca2+ (KCa 2+ = 0.04 ± 0.01 M), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca2+-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5–1 mM), a high affinity Ca2+-dependent ATPase activity was observed (KCa 2+ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca2+-transport nor high affinity Ca2+-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg+)-ATPase activity, the presence of both a high affinity Ca2+-pump and high affinity Ca2+-dependent ATPase. However, the high affinity Ca2+-ATPase activity does not appear to be the biochemical expression of the Ca2+-pump.Abbreviations Ca2+-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate  相似文献   

19.
Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs+-conducting medium. We found that the pharmacology, Cs+ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10–7 to 10–3 M Ca2+, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca2+, respectively, the cardiac SR channel remained activated in the presence of high [Ca2+]. In correlation to these studies, [3H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca2+] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [3H]ryanodine binding of cardiac SR at millimolar Ca2+ in the absence of adenine nucleotides.When Ca2+-induced Ca2+ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca2+-induced inactivation at high [Ca2+] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca2+ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca2+-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.  相似文献   

20.
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   

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