ATPase Activity Of Sarcoplasmic Reticulum Research Articles

The effect of chlorpyrifos upon ATPase activity of sarcoplasmic reticulum and biomechanics of skeleta l muscle contraction.

We investigated the effect of chlorpyrifos, an organophosphate insecticide, on Ca2+,Mg2+-ATPase activity of sarcoplasmic reticulum and on contraction dynamics (force and length changes) of Rana temporaria m. tibialis anterior muscle fiber bundles. All of the used concentrations of chlorpyrifos (10-6 to 10-5 M) caused decrease of Ca2+,Mg2+-ATPase activity. The inhibition of Ca2+,Mg2+-ATPase activity by chlorpyriphos in concentrations of 10-6 M to 7.5·10-6 M is due to permeation of sarcoplasmic reticulum rather than due to direct enzyme inhibition by organophosphate insecticides. The inhibitory properties of the compound were higher at increased concentration and exposure timeframes. Chlorpyrifos in concentration range of 10-6 to 7.5·10-6 M causes changes in muscle fiber response force that were more pronounced than changes in contractile length. We demonstrated inhibition of Ca2+,Mg2+-ATPase activity caused by noncholinergic effects of chlorpyriphos. It is possible to conclude that influence of organophosphate insecticides happens not only in the neuromuscular transmission but also on the level of subcellular structures.

THE EFFECT OF THE ALUMINUM CHLORIDE--QUERCETIN COMPLEX ON Ca2+, Mg(2+)-ATPase ACTIVITY AND CONTRACTION DYNAMIC PROPERTIES OF MUSCLE TIBIALIS ANTERIOR FROM RANA TEMPORARIA.

Combined effect of aluminum chloride and quercetin solutions on the enzymatic activity and contraction dynamics of muscle fiber bundles of the Rana temporaria m. tibialis anterior was investigated. It was shown that these complexes inhibit muscle contraction. Linear reduction of Ca2+, Mg(2+)-ATPase activity induced by all of the used concentrations of AlCl3 - quercetin was demonstrated. It was found that complex of quercetin with AlCl3 has a greater inhibitory effect on muscle contraction dynamic and causes greater reduction during all periods of stimulation in comparison to the separate effect of the investigated compounds. All the studied concentrations of AlCl3 and quercetin solutions (AlCl3: 10(-4)-10(-2) M; quercetin: 10(-6)-10(-5) M) caused concentration depended contraction strengths and lengths reduction. The decrease in strength and length of muscle contractions was of constant and mostly linear nature within observed timeframe as well as within each periods of contraction. The changes were least pronounced within pretetanic period, but were profound within terminal period of muscle activity. The changes in dynamic contraction properties and Ca2+, Mg(2+)-ATPase activity of sarcoplasmic reticulum under effect of the investigated compounds was minimal in the beginning of the muscle's response to stimulus, prior to muscle strength reaching stable contraction level.

ALUMINUM CHLORIDE EFFECT ON Ca2+,Mg(2+)-ATPase ACTIVITY AND DYNAMIC PARAMETERS OF SKELETAL MUSCLE CONTRACTION.

We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10(-4) M Increasing the concentration of AlCl3 to 10(-2) M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg(2+)-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg(2+)-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

Suppressive effect of ATP on the denaturation of sarcoplasmic reticulum (Ca2+)-ATPase from southern bluefin tuna Thunnus maccoyii and its biochemical properties

Sarcoplasmic reticulum (SR) was prepared from ordinary white muscle of southern bluefin tuna. The chymotryptic digestibility, pH and temperature dependent Ca2+-ATPase activity of SR, as well as the thermal and freezing stability with different additives including ATP were examined. A major component of SR Ca2+-ATPase, which was about 100 kDa by SDS-PAGE analysis, was digested to 60 kDa and 38 kDa fragments by α-chymotrypsin. The SR Ca2+-ATPase activity was 0.3 μmol Pi/min/mg at 25 °C. There was no break point in the Arrhenius plot of temperature-dependent Ca2+-ATPase activity. The activation energy of Ca2+-ATPase was 35.44 kJ/mol. The pH dependence of SR Ca2+-ATPase activity of southern bluefin tuna showed a clear peak with a maximum activity at pH 6.5, and SR Ca2+-ATPase was half inactivated when heated at 45 °C for 4 min. SR Ca2+-ATPase was almost inactivated by pH 5.5 acid treatment, for 2 h at 0 °C. Denaturation by acid treatment was strongly suppressed by the addition of 2 mM ATP. ATP also showed a strong suppressive effect against denaturation of SR Ca2+-ATPase by freezing and heating. These results indicate that ATP reduces denaturation of SR Ca2+-ATPase and that enhancement of ATP levels during freezing has the potential to improve meat quality.

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

15N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at Ser-16 in aligned phospholipid bilayers

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically 15N-Ala-11- and 15N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl- sn-glycero-3-phosphocholine/2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned 15N-labeled Ala-11 and Leu-7 WT-PLB samples show 15N resonance peaks at approximately 71 ppm and 75 ppm, respectively, while the corresponding phosphorylated forms P-PLB show 15N peaks at 92 ppm and 99 ppm, respectively. These 15N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

Modeling calcium waves in cardiac myocytes: importance of calcium diffusion.

Under certain conditions, cardiac myocytes engage in a mode of calcium signaling in which calcium release from the sarcoplasmic reticulum (SR) to myoplasm occurs in self-propagating succession along the length of the cell. This event is called a calcium wave and is fundamentally a diffusion-reaction phenomenon. We present a simple, continuum mathematical model that simulates calcium waves. The framework features calcium diffusion within the SR and myoplasm, and dual modulation of ryanodine receptor (RyR) release channels by myoplasmic and SR calcium. The model is used to illustrate the effect of varying RyR permeability, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity and calcium ion mobility in myoplasm and SR on wave velocity. The model successfully reproduces calcium waves using experimentally-derived variables. It also supports the proposal for wave propagation driven by the diffusive spread of myoplasmic calcium, and highlights the importance of SR calcium load on wave propagation.

Sarcoplasmic reticulum ATPase activity in type I and II skeletal muscle fibres of chronic heart failure patients

Background Reduced exercise tolerance and muscle weakness are present in patients with CHF. Altered metabolism, histology and function in skeletal muscle of patients with CHF have been reported. The sarcoplasmic reticulum (SR) has control of Ca ++ release and uptake required for contraction and relaxation, respectively, and uses a considerable amount of energy. Little is known about SR alterations in CHF. We determined sarcoplasmic reticulum adenosine triphosphatase (SR ATPase) activity in different types of skeletal muscle fibres of CHF patients. Methods SR ATPase activity, succinate dehydrogenase (SDH) activity and myofibrillar adenosine triphosphatase (M ATPase) activity in single fibres of the vastus lateralis muscle in 16 CHF patients and 5 controls was determined using quantitative enzyme histochemistry. Results SR ATPase activity of type II skeletal muscle fibers was significantly higher compared to type I fibres. SR ATPase activity in type II skeletal muscle fibres of CHF patients was higher than in control subjects. Conclusion Increased skeletal muscle SR ATPase activity contributes to reduced exercise tolerance in CHF patients.

Mechanisms of cardiodepression by an Na+–H+exchange inhibitor methyl-N-isobutyl amiloride (MIA) on the heart: lack of beneficial effects in ischemia–reperfusion injuryThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

Although Na+-H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3- -free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia-reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia-reperfusion-induced myocardial injury.

Gene Therapy in Congestive Heart Failure

Although research and development of gene therapy have encountered several difficulties in recent years, they have also begun to show potential for the treatment of acquired and inherited human diseases. Gene therapy may have wide applicability for the treatment of numerous cardiovascular diseases, including congestive heart failure (CHF), ischemic heart disease, and cardiomyopathy. CHF remains a leading cause of death and morbidity in industrialized nations, and the number of patients with CHF continues to increase as the population ages. Pharmacological therapies control symptoms, reduce left ventricular (LV) dilation, improve function, and reduce mortality. These therapies control the disease but do not cure it. Currently, however, the growing knowledge of molecular mechanisms involved in myocardial dysfunction has allowed the identification of potential therapeutic targets designed with the aim of curing CHF. Several studies in animal models have provided hope that gene therapy may be one of the future strategies for the treatment of clinical CHF. In these studies, myocardial gene transfer has been used to target at least 3 different biological pathways that play a crucial role in the pathophysiology of CHF: Intracellular calcium signaling, β1-adrenergic receptor (β1-AR) signaling, and antiapoptotic signaling. The activity of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) is decreased in failing myocardium, and this results in abnormal Ca2+ handling.1 In an animal model of heart failure, Miyamoto et al2 showed that in vivo overexpression of SERCA2a, achieved by catheter injection of an adenovirus carrying the SERCA2a gene into the aortic root, restored systolic and diastolic function to normal levels. In another study, Muller et al3 analyzed the chronic overexpression of SERCA2a in both normal and pressure-overloaded hearts (induced by abdominal aortic banding of transgenic rats). Overexpression of SERCA2a resulted in a …

The effects of phenothiazines and other calmodulin antagonists on the sarcoplasmic and endoplasmic reticulum Ca 2+ pumps

The effects of a number of phenothiazines and other calmodulin antagonists on the Ca 2+-ATPase activity of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) were investigated. The drugs used in this study were trifluoperazine, calmidazolium, fluphenazine, chlorpromazine, W-7, and calmodulin-binding peptide. Our results showed that calmidazolium and calmodulin-binding peptide were the most potent inhibitors of skeletal muscle SR Ca 2+-ATPase activity (isoform SERCA 1) ( ic 50 values of 0.5 and 7 μM, respectively), while W-7 was the least potent inhibitor ( ic 50, 125 μM). All of the antagonists had little effect on the cerebellar ER Ca 2+-ATPase activity (isoform SERCA 2b), except for trifluoperazine, which had a biphasic effect, causing stimulation at low concentrations and inhibition at higher concentrations. Our results suggest that the effects of these calmodulin antagonists are independent of calmodulin and that they inhibit the Ca 2+-ATPase in an isoform-specific manner. It was found that these antagonists inhibit the skeletal muscle isoform of the Ca 2+ pump by altering the Ca 2+ affinity and the associated Ca 2+-binding steps, as well as possibly stabilising the E1 conformational state of the enzyme.

Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca(2+) uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca(2+)-ATPase activity in SR and changing of character of Ca(2+) release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca(2+) transport of SR.

Reversible Inhibition of the Calcium-pumping ATPase in Native Cardiac Sarcoplasmic Reticulum by a Calmodulin-binding Peptide: EVIDENCE FOR CALMODULIN-DEPENDENT REGULATION OF THEVmax OF CALCIUM TRANSPORT

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.

Restoration of contractile function in isolated failing human ventricular cardiac myocytes by gene transfer of SERCA2a

Myocardial cells from failing (F) human hearts are characterized by abnormal Ca 2+ handling, a negative force frequency relationship (FFR), and decreased sarcoplasmic reticulum (SR) ATPase activity. We have previously shown that overexpmssion of SERCA2a by adenoviral gene transfer increases SR ATPase activity and enhances Ca 2+ handling both in vitro and in vivo. To test whether contractile function can be restored by overexpression of SERCA2a in human hearts, myocardial cells isolated from 10 patients with end-stage heart failure were infected with adenoviruses encoding either the SERCA2a gene or reporter genes green fluorescent protein (GFP) and /3-galactosidase for 24 hrs. Cells isolated from 2 non-failing hearts were used as controls (C). As shown below, compared to C myocytes, F myocardial cells had prolonged contraction and calcium transient detected by Fura-2. Overexpression of SERCA2a in F myocytes decreased the time course of both contractions and Ca z+ by 19+2% and 27-+4%. F myocytes had decreased Ca 2+ release and an increase in diastolic Ca 2+ with increasing frequency (0.1-1.5Hz). Overexpression of SERCA2a restored the FFR in the F cells to normal: increasing frequency resulted in enhanced SR Ca 2+ release and contraction. These results show that gene transfer of SERCA2a improves the contractile function in F human myocytes. Targeting SERCA2a may provide therapeutic benefits in heart failure.

Effect of non-selective endothelin blockade, TAK-044, on the ischemic cellular injury of rat heart.

The aim of this study is to evaluate the role of non-selective endothelin blockade (TAK-044) in ischemic myocardial injury. Forty anesthetized rats were separated into four groups: 1) TAK-I group, after preinjection of TAK-044 (3 mg/kg), LAD was ligated for 60 min and reperfused for 60 min; 2) Saline (SAL)-I group, LAD ligation and reperfusion without TAK-044; 3) TAK-C group, sham operated TAK group; 4) SAL-C group, sham-operated SAL group. Myocardium from each group was separated and analyzed by biochemical and ultrastructural procedures. Reperfusion arrhythmia (VT) was observed in 88% of the SAL-I group, in contrast to only 36% of the TAK-I group. At the end of reperfusion, hemodynamics indicated no significant differences between these two groups. The Ca(++)-ATPase activity of sarcoplasmic reticulum (SR) was 3.9 mumoles Pi/mg protein/h (39% of SAL-C group) in the SAL-I group, while that in the TAK-I group was significantly higher at 6.1 (54%). The ratio of infarct/risk area was 58% in the SAL-I group and 36% in the TAK-I group. In the ultrastructural observations, irreversibly injured cells of the ischemic portion were reduced significantly from 35% (SAL-I group) to 14% (TAK-I group). Thus, our results indicated that endothelin blockade reduced ischemic cellular injury. The mechanism of this reduction was speculated to be a resistance to ischemic injury in the subcellular levels of the myocardium conferred by a reduction of vascular constriction and improvement of imbalance in the energy supply and demand.

Inhibition of skeletal sarcoplasmic reticulum Ca2+-ATPase activity by deferoxamine nitroxide free radical.

Deferoxamine is an inhibitor of iron-dependent free radical reactions. Despite the antioxidant roles, prolonged clinical use of the chelator is far from benign, and paradoxically, deferoxamine has been shown to promote lipid peroxidation. The possible toxicity of the drug's metabolites, such as deferoxamine nitroxide free radical, deserves attention. We, therefore, tested the hypothesis that deferoxamine nitroxide radicals produced as a result of enzymatic one-electron oxidation of deferoxamine by horseradish peroxidase in the presence of H2O2 are capable of inactivating Ca2+-ATPase of skeletal sarcoplasmic reticulum microsomes as a model system with which to explore the effect of the radical on a biological membrane. Ca2+-ATPase activity of sarcoplasmic reticulum was depressed by exposure to Fenton's reagent (H2O2/FeSO4); the observed effect was significantly enhanced by deferoxamine. We found that the Fenton reaction produced hydroxyl radical, as determined by electron spin resonance spectroscopy. The formation of hydroxyl radical was completely inhibited by deferoxamine; instead, under the same experimental conditions (in the presence of sarcoplasmic reticulum vesicles with or without FeSO4 but without spin trap 5, 5-dimethyl-1-pyrroline N-oxide), the spectral shape and hyperfine coupling constants of electron spin resonance signals confirmed to be long-lived deferoxamine radical were obtained. Furthermore, exposure of sarcoplasmic reticulum vesicles to deferoxamine radical formed by horseradish peroxidase via reaction with H2O2 caused an inhibition of the Ca2+-ATPase activity. The findings show that the sarcoplasmic reticulum vesicles can act as peroxidases and suggest that deferoxamine enhances the decreased Ca2+-ATPase activity afforded by H2O2/FeSO4 due to formation of its metabolites, possibly deferoxamine nitroxide free radical.

Contribution of Abnormal Sarcoplasmic Reticulum ATPase Activity to Systolic and Diastolic Dysfunction in Human Heart Failure

Two of the most significant characteristics of failing human myocardium are an increased diastolic [Ca2+]i and a prolonged diastolic relaxation. These abnormalities are more pronounced at higher frequencies of stimulation and may be caused by an altered Ca2+ resequestration into the sarcoplasmic reticulum (SR). The force-frequency relationship was determined in multicellular preparations obtained from non-failing (n=6) and failing human myocardium (n=11). The active force in non-failing tissue increased as a function of the frequency of stimulation. In failing myocardium, an increase in frequency of stimulation (>1 Hz) was accompanied by a decrease in active force. Changes in the frequency of stimulation and active force were also associated with changes in intracellular calcium concentrations. The diastolic force in failing myocardium was augmented following an increase in frequency of stimulation, whereas in non-failing tissue, no increase in diastolic force was observed. Associated with the increase in diastolic force was an increase in intracellular diastolic calcium concentrations. The SR Ca2+ ATPase activity was reduced in failing compared to non-failing myocardium. SR Ca2+ ATPase was positively correlated with diastolic force in non-failing myocardium. The relationship between Ca2+ ATPase activity at 1 micromol/l [Ca2+] and active force between 0.5 and 2.0 Hz was different between failing and non-failing myocardium. The diastolic force demonstrate an inverse relationship with the SR Ca2+ ATPase activity in failing myocardium. These data suggest that a reduction in SR Ca2+ ATPase activity contributes to the impairment in both systolic and diastolic function of failing human hearts.

An antibody to annexin V blocks Ca(2+)-dependent ATPase activity of sarcoplasmic reticulum in human failing hearts.

An antibody to annexin V blocks Ca(2+)-dependent ATPase activity of sarcoplasmic reticulum in human failing hearts.

Phencyclidine block of Ca 2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca 2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 μM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca 2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 μM PCP in vitro. A significant reduction of SR Ca 2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca 2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca 2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca 2+ activation. These results indicate that PCP alters the myocardial Ca 2+ homeostasis by inhibiting the Ca 2+ ATPase in cardiac SR in rats. Inhibition of SR Ca 2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.

Sarcoplasmic reticulum responses to repeated sprints are affected by conditioning of horses.

Sarcoplasmic reticulum (SR) responses to repeated sprints and to physical conditioning were studied in 10 Quarter Horses. Exercise tests (four repeated sprints on a treadmill) were conducted before and after 12 wk of sprint conditioning. Muscle samples from the middle gluteal muscle were taken before and after each exercise test, and SR vesicles were isolated. Calcium uptake was determined spectrophotometrically using antipyrylazo III, and Ca2+-ATPase activity was determined using an enzyme-linked optical assay. Conditioning increased calcium uptake rate and Ca2+-ATPase activity by 14 and 38%, respectively, before exercise and by 25 and 26% after exercise. Exercise decreased calcium uptake rate and Ca2+-ATPase activity by 37 and 27%, respectively, before conditioning and by 28 and 21% after conditioning. Decreases in calcium uptake and Ca2+-ATPase activity of SR have been associated with fatigue during exercise, and this association is strengthened by the moderating effect of conditioning.