Rat Cardiac Sarcoplasmic Reticulum Research Articles

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45μmol/g of body weight) twice a day for a period of 2weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-uptake, endowed with antioxidant and L-type Ca2+ channel blocking activities.In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico.In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner.DTBHA, at concentrations higher than 10 µM, increased Ca2+ uptake in cardiac SR without affecting Ca2+-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca2+ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein.In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

Rats fed linseed oil in microemulsion forms enriches the cardiac sarcoplasmic reticulum lipids with docosahexaenoic acid and lower calcium transport

The effect of feeding rats with whey protein or lipoid microemulsion of n-3 rich linseed oil (LSO), and n-6 rich sunflower oil (SNO) compared to native or microemulsion forms of each oil for 60days on fatty acid composition, Ca2+Mg2+ ATPase, Ca2+ uptake and membrane fluidity of rat cardiac sarcoplasmic reticulum (SR) were assessed. Rats consuming LSO in lipoid microemulsion showed higher levels of ALA (18:3, n-3) conversion to DHA (22:6, n-3) when compared to rats given LSO in native form. Compared to all other dietary treatments, the alterations in the n-6/n-3 PUFA composition of SR of rats given LSO in lipoid microemulsion effected the greatest changes in Ca2+Mg2+ ATPase activity and Ca2+ uptake which effectively reduced Ca2+ transport. Therefore, the LSO in lipoid microemulsion confers similar heart healthy fatty acid compositional and calcium transport changes and potentially anti-arrhythmic effects similar to fish oils.

Hydrogen sulfide regulates cardiac sarcoplasmic reticulum Ca2 + uptake via KATP channel and PI3K/Akt pathway

AimsTo investigate the effects of hydrogen sulfide (H2S) on calcium uptake activity of the rat cardiac sarcoplasmic reticulum (SR) and possible signaling. Main methodsCrude SR was isolated after treatment with H2S, then SR Ca2+ uptake and SR Ca2+-ATPase (SERCA) activity was measured by the isotopic tracer method. The possible roles of the KATP channel and PI3K/Akt and SR-membrane protein phospholamban (PLB) pathway were analyzed by specific blockers, and target protein activation was assayed by measuring protein phosphorylation. Key findingsExogenous H2S lowered Ca2+ uptake into the SR time or concentration dependently, which was associated with decreased SERCA activity. Inhibiting endogenous H2S production by DL-propargylglycine increased SR Ca2+ uptake and SERCA activity. H2S inhibition of PLB phosphorylation was through SERCA activity and was reversed by two PI3K inhibitors, wortmannin and LY294002. Glibenclamide (a KATP channel blocker) blocked the inhibitory effects of H2S on PLB and Akt phosphorylation. Pinacidil (a KATP channel opener) reduced the phosphorylation of PLB and reversed the effects of DL-propargylglycine. H2S preconditioning increased PLB phosphorylation but did not affect SERCA activity. SignificanceEndogenous H2S transiently and reversibly inhibits SR Ca2+ uptake in rat heart SR because of downregulated SERCA activity associated with PLB phosphorylation by the PI3K/Akt or KATP channel. The transient negative regulation of SR Ca2+ uptake and the L-type Ca2+ channel contributes to Ca2+ cycle homeostasis, which might be an important molecular mechanism in ischemic diseases.

Calumenin Inhibits Ca 2+ Release from Sarcoplasmic Reticulum in Murine Cardiomyocytes through a Direct Interaction with RyR2

Sarcoplasmic reticulum (SR) luminal proteins play an important role in calcium buffering and in regulation of Ca2+ release. Recently, we showed that knockdown of calumenin enhanced Ca2+ cycling in the SR (Sahoo et al., (2009) J. Biol.Chem. 284, 31109-31121). To identify the molecular mechanism behind the enhanced Ca2+ release in calumenin knockdown cells, we investigated the role of calumenin on RyR2-mediated Ca2+ release. Using immunprecipitation assay we found that calumenin interacts with RyR2 in mouse heart and calumenin and RyR2 are co-localized in cardiomyocytes. GST-pull down assay of calumenin deletion fragments and GST-RyR2 intra-luminal loops suggested that the 132-222 amino acid region of calumenin and RyR2-Loop I (4520-4577 amino acids) are the major binding partners. Knockdown of calumenin enhanced the binding of ryanodine in calumenin knockdown HL-1 cells, as depicted by [3H]ryanodine binding assay. Single channel recordings of RyR2 in rat cardiac SR showed that addition of purified GST-calumenin or GST-calumenin-B (132-222 amino acid) to the trans side of the lipid bilayer decreased open probability of RyR2 channels. Taken together, the present results show that calumenin inhibits RyR2 activity in cardiomyocytes by a direct interaction with RyR2. Supported by Korea NRF Grant (2010-0002159), GIST Systems Biology Infrastructure Establishment Grant (2010) and KISTI-KREONET (2010).

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca(2+)-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265-269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca(2+)-cycling proteins. But it enhanced Ca(2+) transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca(2+) uptake showed increased Ca(2+) sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 mum Ca(2+), suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132-222 amino acids of calumenin and 853-892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca(2+)-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca(2+)-cycling of cardiomyocytes.

Calumenin Knock-down (kd) Enhances Ca2+ Cycling Ability In Hl-1 Cells

Calumenin is a multiple EF-hand Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) lumen. Evidence of the interaction between calumenin and SERCA2 in rat cardiac SR was shown recently (Mol. Cells, 26:265-269, 2008). To elucidate the possible role of calumenin in cardiac excitation-contraction (E-C) coupling, calumenin was knocked down by transfection of mouse cardiac cell line (HL-1 cells) with calumenin specific siRNA oligonucleotides. After 72 hrs of transfection, calumenin protein level was reduced by 75% without any obvious changes in the expression levels of other E-C coupling proteins such as RyR2, SERCA2, NCX, CSQ and PLB. A field stimulation (1Hz) of KD cells (n = 58) led to significantly increased Ca2+ transient peak height (1.02 ± 0.02 vs. 0.82 ± 0.03 fura-2 ratio at 340 and 380 nm, p < 0.05), decreased time to peak (0.093 ± 0.001 vs. 0.107 ± 0.003 s, p < 0.05) and time to 50% baseline (0.172 ± 0.005 vs. 0.235 ± 0.006 s, p < 0.05) as compared to control cells (n = 44). On the other hand, the SR Ca2+ load remained unchanged in KD cells. Pull-down experiments with GST fusion proteins showed that calumenin interacts with both RyR2 and SERCA2 in a Ca2+ dependent manner. Taken together, the present results suggest that calumenin is related to SR Ca2+ homeostasis. Currently, the molecular interactions between calumenin and SERCA2 or RyR2 are being examined by using various deletion mutants.

Calumenin Interacts with SERCA2 in Rat Cardiac Sarcoplasmic Reticulum

Calumenin Interacts with SERCA2 in Rat Cardiac Sarcoplasmic Reticulum

Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

Aromatic indolinic aminoxyls as antioxidants in cardiac sarcoplasmic reticulum lipid and protein oxidation.

The results presented demonstrate the influence of aromatic indolinic aminoxyls: 1,2-dihydro-2-ethyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C2) and 1,2-dihydro-2-octadecyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C18) on oxidation of lipids and proteins of cardiac sarcoplasmic reticulum membranes. We have used doxorubicin and t-butyl hydroperoxide as agents inducing oxidative stress in isolated rat cardiac sarcoplasmic reticulum membrane system. Carbonyl groups were measured as the end product of membrane protein oxidation, and thiobarbituric acid reactive substances were assessed as a marker of lipid peroxidation. Inhibition of peroxidation of certain membrane components depends on the length of acyl chain. Aminoxyl IA-C2 inhibits the lipid peroxidation process while IA-C18 is an efficient protector against protein oxidation.

Direct and indirect modulation of rat cardiac sarcoplasmic reticulum function by n-3 polyunsaturated fatty acids.

Measurements were made of trans-sarcolemmal Ca(2+) fluxes and intracellular [Ca(2+)](i) in rat ventricular myocytes loaded with Indo-1 to determine how the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca(2+) release. We report that in 10 microM EPA, the Ca(2+) efflux generated by individual waves increased by 11.3 +/- 4.9 % over control levels. However, wave-generated efflux per unit time fell overall by 19 +/- 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca(2+) efflux was greater than normal and the cell lost 28.0 +/- 10.6 micromol l(-1) Ca(2+). This probably represents the loss of extra Ca(2+) accumulated by the sarcoplasmic reticulum (SR), while Ca(2+) release was inhibited. These results are evidence of inhibition of the SR Ca(2+)-release mechanism and reduced availability of Ca(2+) to the SR. From the relationship between average intracellular Ca(2+) and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 +/- 8.1 %, the majority of this (29.2 +/- 8.8 %) is due to inhibition of the Ca(2+)-release mechanism. In EPA, the rate of fall of Ca(2+) in the caffeine response (an indicator of surface membrane Ca(2+) efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca(2+) observed in EPA is due to a lower influx of Ca(2+) across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti-arrhythmic actions of EPA is discussed.

Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100µM Ca2+; however, the interactions were interrupted in the presence of 20mM EGTA, 1µM rapamycin or 1µM FK506, suggesting that the interaction is Ca2+-dependent, and is mediated by FKBP12.6. The Ca2+-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20µM deltamethrin, 10µM cyclosporin A (CsA), or 10µM FK506 led to Ca2+ oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20µM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca2+ oscillations, probably due to the leakiness of the RyR. However, Ca2+ oscillations by rapamycin were not further affected by 10µM CsA or 10µM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca2+-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10µM ryanodine-treated cardiomyocytes, CsA failed to induce Ca2+ oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca2+ release in rat heart.

Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100 microM Ca(2+); however, the interactions were interrupted in the presence of 20 mM EGTA, 1 microM rapamycin or 1 microM FK506, suggesting that the interaction is Ca(2+)-dependent, and is mediated by FKBP12.6. The Ca(2+)-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20 microM deltamethrin, 10 microM cyclosporin A (CsA), or 10 mciroM FK506 led to Ca(2+) oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20 microM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca(2+) oscillations, probably due to the leakiness of the RyR. However, Ca(2+) oscillations by rapamycin were not further affected by 10 microM CsA or 10 mciroM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca(2+)-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10 microM ryanodine-treated cardiomyocytes, CsA failed to induce Ca(2+) oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca(2+) release in rat heart.

Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae.

This study was designed to measure the Ca2+ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca2+ (100 nM), in the absence and presence of caffeine. Measurements of [Ca2+] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5-40 mM) into this volume caused an initial release of Ca2+ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca2+. Ca2+ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca2+ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5-40 mM) and 100 nM Ca2+ indicated that the SR's ability to retain Ca2+ depends inversely on the dose of caffeine. The relative Ca2+ content of the SR after preincubation with caffeine was 86.7+/-3.5% at a caffeine concentration of 5 mM, 62.5+/-5.1% at 10 mM caffeine, 37.8+/-8.1% at 20 mM caffeine and 7. 1+/-1.9% at 40 mM caffeine. Measurement of the SR Ca2+ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca2+-binding capacity of the preparation (equivalent to 245+/-10 microM BAPTA) and (2) the Ca2+ content of the SR accessed by caffeine after equilibration with 100 nM Ca2+ (186+/-11 micromol/l cell volume or 5.6 mmol/l SR volume).

Sarcoplasmic Reticulum-Associated and Protein Kinase C-Regulated ADP-Ribosyl Cyclase in Cardiac Muscle

Two types of ADP-ribosyl cyclase activity were distinguished in dog and rat cardiac muscles by measuring the enzymatic conversion of NGD (as an NAD analog) into the fluorescent product cyclic GDP-ribose in cardiac muscle subcellular fractions. Both types of activity were confined to membrane fractions isolated from microsomes by sucrose gradient centrifugation. One of the activities co-purified with fractions that were enriched in sarcolemma (SLM), as evidenced by immunodetection of the dihydropyridine receptor, while the other activity was found to co-precipitate with the sarcoplasmic reticulum (SR), that was identified on the basis of its immuno-staining with a ryanodine receptor monoclonal antibody. In certain aspects, the plasma membrane-bound ADP-ribosyl cyclase activity resembled the characteristics of CD38 or CD38-like proteins: it was sensitive to thiols and lectins and was recognized by a monoclonal anti CD38 antibody. The SR enzyme had apparently distinct properties, as it was insensitive to both thiols and lectins and was not recognized by the CD38 antibody. In addition, the SR-associated ADP-ribosyl cyclase was inhibited by endogenous protein kinase C (PKC)-dependent phosphorylation in both dog and rat cardiac SR. The PKC-modulated SR ADP-ribosyl cyclase we describe here might be a principal component of the signal transduction machinery that is responsible for regulation of the intracellular levels of cADPR.

The MgATPase activity of rat cardiac sarcoplasmic reticulum is a function of the calcium ATPase protein

Magnesium-dependent ATPase (MgATPase) activity is associated with many E 1-E 2 or P-type transport ATPases including the sarcoplasmic reticulum (SR) calcium ATPase. The SR isolated from rat heart has a MgATPase activity which is 6–12 times faster than the MgATPase activity of the SR isolated from dog heart. To determine the origin of the high MgATPase activity of rat heart SR, we compared and contrasted cardiac SR isolated from both species. The preparations were similar in the following ways: (i) contamination by other organelles; (ii) the comigration of MgATPase activity with calcium-dependent ATPase (CaATPase) activity through a sucrose gradient; (iii) a similar ATPase activity sensitivity to pH and ATP concentration; (iv) the high and similar of sensitivity of ATPase activity to detergent; and (v) a similar protein profile. In both preparations, a single protein in the 105,000-Da region of polyacrylamide gels was phosphorylated by ATP, and the phosphorylated species was an acylphosphate formed in the presence and absence of calcium. Dimethyl sulfoxide, which slows acylphosphoenzyme breakdown, markedly inhibited both CaATPase and MgATPase activities of both preparations but not other enzyme activities. Importantly, the specific inhibitor of the SR calcium pump, thapsigargin, completely inhibited the CaATPase activity with an I 50 of 6–7 n m; however, a higher concentration (I 50 of 2 μ m) was required to inhibit the MgATPase activity of the rat cardiac SR. These results provide evidence that the Mg-ATPase activity of rat cardiac SR is part of the enzyme cycle of the calcium ATPase protein.

Inhibition of rat cardiac sarcoplasmic reticulum Ca 2+ ATPase (SERCa2) gene expression by phorbol esters and TGF-β

Inhibition of rat cardiac sarcoplasmic reticulum Ca 2+ ATPase (SERCa2) gene expression by phorbol esters and TGF-β

Intramolecular oscillation of the phosphorylation domain of rat cardiac sarcoplasmic reticulum titrated with arachidonoyl phosphatidylcholine.

Sarcoplasmic reticulum vesicles were prepared from rat myocardium. The intramolecular oscillation of the phosphorylation domain of Ca(2+)-ATPase in control vesicles and in vesicles titrated with diarachidonoyl phosphatidylcholine was studied with a nanosecond time-resolved fluorometer. The membrane viscosity of the lipid domain was decreased by the lipid titration. The phosphorylation domain was labeled with a fluorophore, anilinonaphthylmaleimide (ANM). The time course of anisotropy decay of ANM fluorescence reflects the localized oscillation in the protein structure. The half-decay time of the anisotropy was decreased by diarachidonoyl titration from 77 nsec in control vesicles to 66 nsec, suggesting an increase in the intramolecular oscillation. Concomitantly observed decreases in membrane viscosity and Ca(2+)-ATPase activity suggest that the decreased membrane viscosity destabilized the Ca(2+)-ATPase protein structure causing a reduction in Ca(2+)-ATPase activity.