Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats

The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.     

Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes

Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.     

Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.     

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), spring (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p less than or equal to 0.05). In the 51 day E group ATPase activity (0.378 +/- 0.009 mumol Pi X mg-1 X min-1) was greater than at 10 and 21 days (0.307 +/- 0.006 and 0.323 +/- 0.008 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). No change occurred in the S group from 10 to 21 and 51 days (p greater than or equal to 0.05). Both the 21 and 51 day S (0.318 +/- 0.011 and 0.399 +/- 0.010 mumol Pi X mg-1 X min-1) and E (0.323 +/- 0.008 and 0.378 +/- 0.009 mumol Pi X mg-1 X min-1) groups had higher activity compared to the C group (0.193 +/- 0.029 and 0.172 +/- 0.031 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). Maturation (10--51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca2+ binding (p less than or equal to 0.05) while Ca2+ uptake ability did not change (p greater than or equal to 0.05). SR yield, Ca2+ binding and uptake were not altered with S training (p greater than or equal to 0.05). The E training resulted in greater Ca2+ uptake at 51 days compared to C and S (p less than or equal to 0.05), with no change in Ca2+ binding (p greater than or equal to 0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.     

Kinetics of ATP-dependent Ca2+ uptake by permeabilized rat enterocytes. Effects of inositol 1,4,5-trisphosphate

Isolated rat enterocytes were permeabilized by saponin treatment. 45Ca2+ was accumulated by these cells when provided with ATP in a medium containing Ca2+ ligands. The use of oxalate, vanadate and mitochondrial inhibitors indicated that both non-mitochondrial and mitochondrial pools are involved. Kinetic analysis of non-mitochondrial Ca2+ uptake revealed a Km of 0.1 microM Ca2+ and a Vmax of 0.4 nmol Ca2+/mg protein X min for this Ca2+-pumping ATPase activity. Mitochondria started to take up Ca2+ between 0.2 and 0.3 microM free Ca2+ reaching maximal rates around 2 microM. At 1 microM free Ca2+ mitochondria accumulated 20 times more Ca2+ than the non-mitochondrial pool. Inositol 1,4,5-trisphosphate released 40% of the Ca2+ content of the non-mitochondrial pool. Half-maximal release was observed at 0.5 and 1.5 microM IP3 in duodenal and ileal cells respectively. These findings support the possibility that the phosphatidyl inositide metabolism plays a role in regulation of electrolyte transport in enterocytes.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.     

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.     

The heavy metal ions Ag+ and Hg2+ trigger calcium release from cardiac sarcoplasmic reticulum

Heavy metal ions have been shown to induce Ca2+ release from skeletal sarcoplasmic reticulum (SR) by binding to free sulfhydryl groups on a Ca2+ channel protein and are now examined in cardiac SR. Ag+ and Hg2+ (at 10-25 microM) induced Ca2+ release from isolated canine cardiac SR vesicles whereas Ni2+, Cd2+, and Cu2+ had no effect at up to 200 microM. Ag(+)-induced Ca2+ release was measured in the presence of modulators of SR Ca2+ release was compared to Ca2(+)-induced Ca2+ release and was found to have the following characteristics. (i) Ag(+)-induced Ca2+ release was dependent on free [Mg2+], such that rates of efflux from actively loaded SR vesicles increased by 40% in 0.2 to 1.0 mM Mg2+ and decreased by 50% from 1.0 to 10.0 mM Mg2+. (ii) Ruthenium red (2-20 microM) and tetracaine (0.2-1.0 mM), known inhibitors of SR Ca2+ release, inhibited Ag(+)-induced Ca2+ release. (iii) Adenine nucleotides such as cAMP (0.25-2.0 mM) enhanced Ca2(+)-induced Ca2+ release, and stimulated Ag(+)-induced Ca2+ release. (iv) Low Ag+ to SR protein ratios (5-50 nmol Ag+/mg protein) stimulated Ca2(+)-dependent ATPase activity in Triton X-100-uncoupled SR vesicles. (v) At higher ratios of Ag+ to SR proteins (50-250 nmol Ag+/mg protein), the rate of Ca2+ efflux declined and Ca2(+)-dependent ATPase activity decreased gradually, up to a maximum of 50% inhibition. (vi) Ag+ stimulated Ca2+ efflux from passively loaded SR vesicles (i.e., in the absence of ATP and functional Ca2+ pumps), indicating a site of action distinct from the SR Ca2+ pump. Thus, at low Ag+ to SR protein ratios, Ag+ is very selective for the Ca2+ release channel. At higher ratios, this selectivity declines as Ag+ also inhibits the activity of Ca2+,Mg2(+)-ATPase pumps. Ag+ most likely binds to one or more sulfhydryl sites "on" or "adjacent" to the physiological Ca2+ release channel in cardiac SR to induce Ca2+ release.     

Myofibril MgATPase activities and energy metabolism in cardiomyopathic mice with diastolic dysfunction

To study the genomic physiology of cardiac myofibril proteins in the heart, we have successfully created a cardiac troponin I (cTnI; a myofibril protein) gene knockout mouse model using gene targeting techniques. The phenotype of the cTnI gene knockout mouse is a cardiomyopathy with diastolic dysfunction resulting in sudden death in neonates. In the present studies, energy metabolism was analyzed in myocardial cells from cTnI-null hearts. Myofibril MgATPase activities were determined in myocardial cells from either wild-type or cTnI mutant mouse hearts. Furthermore, the quantity and quality of the mitochondria in wild-type and cTnI mutant animals were counted and analyzed. Our results demonstrate that damaged relaxation and increased Ca²⁺-independent force production in cTnI-null hearts is in part related to the increased myofibril MgATPase activities accompanied by an increase in mitochondria quantity and mitochondrial ATPase activities. These data indicate that cardiomyopathies with diastolic dysfunction are different from cardiomyopathies caused by systolic dysfunction. The former involves the damage of cardiac relaxation due to increased MgATPase activities and increased Ca²⁺-independent force production inside of myofilaments, while the latter involves the damage of systolic contraction due to decreased MgATPase activities and decreased force production.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification

The (Ca2+ + Mg2+) ATPase of dog heart sarcolemma (Caroni, P., and Carafoli, E. (1980) Nature 283, 765-767) has been characterized. The enzyme possesses an apparent Km (Ca2+) of 0.3 +/- 02 microM, a Vmax of Ca2+ transport of 31 nmol of Ca2+/mg of protein/min, and an apparent Km (ATP) of 30 microM. It is only slightly influenced by monovalent cations and is highly sensitive to orthovanadate (Ki = 0.5 +/- 0.1 microM). The high vanadate sensitivity has been used to distinguish the sarcolemmal and the contaminating sarcoplasmic reticulum Ca2+-dependent ATPase in heart microsomal fractions. Calmodulin has been shown to be present in heart sarcolemma. Its depletion results in the transition of the Ca2+-pumping ATPase to a low Ca2+ affinity; readdition of calmodulin reverses this effect. The Na+/Ca2+ exchange system was not affected by calmodulin. The results of calmodulin extraction can be duplicated by using the calmodulin antagonist trifluoperazine. The calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column. One major (Mr = 150,000) and 3 minor protein bands could be eluted from the column with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The major protein band (72%) has Ca2+-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+-dependent reaction.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Impairment of cardiac contractility and sarcoplasmic reticulum Ca2+ ATPase activity by hypochlorous acid: reversal by dithiothreitol.

Isolated rat hearts perfused with 100 microM hypochlorous acid (HOCl), a powerful oxidant produced by activated neutrophils, exhibited progressive impairment of contractile performance suggestive of a cytosolic Ca2+ overload (increased left ventricular end-diastolic pressure, increased aortic root perfusion pressure, and depressed pulse pressure). Sarcoplasmic reticulum (SR) enriched microsomal preparations isolated from HOCl-perfused hearts showed a significant decline, when compared with control hearts, in both Ca2+ ATPase activity (123 +/- 40 vs. 473 +/- 46 nmol Pi.mg-1 protein.min-1) and Ca2+ uptake (12 +/- 5 vs. 46 +/- 4 nmol Ca2+.mg-1 protein.min-1). The sulfhydryl content in Ca2+ ATPase and other proteins, as determined by [14C]iodoacetamide binding, was also progressively depleted in HOCl-perfused hearts. Perfusion of the HOCl-treated hearts with dithiothreitol (DTT), a disulfide reducing agent, resulted in a time-dependent attenuation, and eventual partial reversal, of the dysfunction in both contractility and SR Ca2+ ATPase activity. Protein thiol levels were concomitantly restored to near control values. The data indicate that HOCl-induced contractile dysfunction in heart is related to the inactivation of the SR Ca2+ ATPase as a result of thiol oxidation and suggest that DTT is capable of reversing this dysfunction in situ by reducing the oxidized sulfhydryls in the Ca2+ ATPase.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.