Energy expenditure by Ba(2+) contracture in rat ventricular slices derives from cross-bridge cycling.
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To clarify the energy-expenditure mechanism during Ba(2+) contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O(2) consumption (VO(2)) of quiescent slices in Ca(2+)-free Tyrode solution and VO(2) during Ba(2+) contracture by substituting Ca(2+) with Ba(2+). We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba(2+) contracture VO(2). The Ca(2+)-free VO(2) corresponds to that of basal metabolism (2.32 +/- 0.53 ml O(2). min(-1). 100 g LV(-1)). Ba(2+) increased the VO(2) in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic VO(2). Blockade of the sarcoplasmic reticulum (SR) Ca(2+) pump by CPA (10 micromol/l) did not at all decrease the Ba(2+)-activated VO(2). BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba(2+)activated VO(2) almost to basal metabolic VO(2). These energetic results revealed that the Ba(2+)-activated VO(2) was used for the cross-bridge cycling but not for the Ca(2+) handling by the SR Ca(2+) pump. (+info)
Chronic and acute exercise do not alter Ca2+ regulatory systems and ectonucleotidase activities in rat heart.
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The purpose of this investigation was to examine the effects of chronic and acute exercise on the main components involved in excitation-contraction coupling and relaxation in rat heart. Sixty male Wistar rats were divided into a sedentary (S) and three 12-wk treadmill-trained groups (T-1, moderate intensity; T-2, high intensity; T-3, interval running). After 12-wk, 15 rats from the S group and 15 rats from the T-2 group were subjected to a single treadmill-exercise session until exhaustion before being killed at 0, 24, or 48 h (acute exercise). The remaining animals were killed 48 h after the last standard exercise session (chronic exercise). The efficacy of the training programs was confirmed by an increase in treadmill endurance time and in skeletal muscle citrate synthase activity. None of the exercise programs modified heart weight or cardiac oxidative capacity. [(3)H]PN200-110 and [(3)H]ryanodine binding to cardiac homogenates indicated that the density of L-type and sarcoplasmic reticulum (SR) Ca(2+) channels was the same in S and trained rats. The SR Ca(2+)-ATPase activity was also unmodified. Finally, the activities of the ectoenzymes Mg(2+)-ATPase and 5'-nucleotidase, which are involved in degradation of extracellular nucleotides, were not affected by either of the running programs. After the acute exercise session, no changes were detected in either of the tested parameters in heart homogenates of S and T-2 animals. We conclude that neither treadmill-exercise training for 12 wk nor exhaustive exercise alters the density of Ca(2+) channels involved in excitation-contraction coupling or the SR Ca(2+)-ATPase and the ectonucleotidase activities in rat heart. (+info)
Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.
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The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression. (+info)
Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.
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The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides. (+info)
Effect of beta-blockers on free radical-induced cardiac contractile dysfunction.
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BACKGROUND: We examined the effects of hydroxyl radicals (OH.) on human myocardial contractility and on sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and the effects of the beta-receptor antagonists metoprolol, carvedilol, and its metabolite BM-910228. METHODS AND RESULTS: Isometric force of contraction was determined in isolated human myocardium. H(2)O(2) 1 mmol/L and Fe(3+)-nitrilotriacetic acid (Fe(3+)-NTA) 0.1 mmol/L used for generation of OH. induced a decrease in basal force of contraction and an increase in diastolic tension in atrial and left ventricular myocardial preparations. After challenge with OH., the maximum positive inotropic response to Ca(2+) 1.8 to 15 mmol/L was decreased by 60% and by 39%, respectively. The effects of OH. could be blocked by catalase. Carvedilol and its metabolite BM-910228 attenuated the OH.-induced impairment of the inotropic response to Ca(2+) in atrial myocardial preparations. Metoprolol had no significant effect. The stimulation frequency (0.5 to 3.0 Hz)-dependent increase in force of contraction and decrease in diastolic tension were abolished after exposure of atrial trabeculae to OH. In parallel, SERCA activity was decreased by OH. concentration-dependently, as determined in myocardial membrane preparations. BM-910228 partially restored the force-frequency relationship and preserved SERCA activity. CONCLUSIONS: OH. radicals induce an impairment of contraction and relaxation and an attenuation of the force-frequency relationship in human myocardium accompanied by an inhibition of SERCA. Carvedilol and BM-910228 partly prevented OH.-induced contractile dysfunction. These observations could explain the improvement of ejection fraction in heart failure trials with carvedilol without a restoration of beta-adrenergic receptor density. (+info)
The behaviors of Ca(2+)-ATPase embedded in interdigitated bilayer.
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We investigated the behavior of a membrane protein, Ca(2+)-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca(2+)-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca(2+)-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions. (+info)
Phosphoenzyme conversion of the sarcoplasmic reticulum Ca(2+)-ATPase. Molecular interpretation of infrared difference spectra.
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Time-resolved Fourier transform infrared difference spectra of the phosphoenzyme conversion and Ca(2+) release reaction (Ca(2)E(1)-P --> E(2)-P) of the sarcoplasmic reticulum Ca(2+)-ATPase were recorded at pH 7 and 1 degrees C in H(2)O and (2)H(2)O. In the amide I spectral region, the spectra indicate backbone conformational changes preserving conformational changes of the preceding phosphorylation step. beta-sheet or turn structures (band at 1685 cm(-1)) and alpha-helical structures (band at 1653 cm(-1)) seem to be involved. Spectra of the model compound EDTA for Ca(2+) chelation indicate the assignment of bands at 1570, 1554, 1411 and 1399 cm(-1) to Ca(2+) chelating Asp and Glu carboxylate groups partially shielded from the aqueous environment. In addition, an E(2)-P band at 1638 cm(-1) has been tentatively assigned to a carboxylate group in a special environment. A Tyr residue seems to be involved in the reaction (band at 1517 cm(-1) in H(2)O and 1515 cm(-1) in (2)H(2)O). A band at 1192 cm(-1) was shown by isotopic replacement in the gamma-phosphate of ATP to originate from the E(2)-P phosphate group. This is a clear indication that the immediate environment of the phosphoenzyme phosphate group changes in the conversion reaction, altering phosphate geometry and/or electron distribution. (+info)
Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.
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Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein. (+info)