cAMP-dependent protein kinase phosphorylates and activates nuclear Ca2+-ATPase

A Ca2+-pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca2+-ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca2+-ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated/mol of partially purified enzyme. Measurement of ATP-dependent 45Ca2+ uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca2+-pumping activity of NCA. We show that PKA phosphorylation of Ca2+-ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP/PKA- and Ca2+-dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope.     

Ontogeny of sarcoplasmic reticulum protein phosphorylation by Ca2+--calmodulin-dependent protein kinase

In the adult myocardium the Ca2+ uptake and release functions of the sarcoplasmic reticulum (SR) are known to be regulated by a membrane-associated Ca2+-calmodulin-dependent protein kinase (CaM kinase) which phosphorylates the Ca2+-pumping ATPase (Ca2+ pump), Ca2+ release channel (ryanodine receptor) and the Ca2+ pump-regulatory protein, phospholamban. The role of CaM kinase during development, however, has not been examined previously. The present study investigated the ontogenetic expression of SR-associated CaM kinase in the rabbit myocardium as well as development-related changes in CaM kinase-mediated phosphorylation of the SR proteins (Ca2+ pump, Ca2+ release channel and phospholamban) involved in transmembrane Ca2+ cycling. For these experiments, cardiac muscle homogenate and SR-enriched membrane fraction derived from fetal (21- and 28-days gestation), newborn (2 days postnatal) and adult New Zealand White rabbits were used. Western immunoblotting analysis detected the presence of phospholamban, Ca2+ pump and Ca2+ release channel in homogenate and SR at all ages tested. The amount of these proteins in the SR increased substantially during fetal and postnatal development. Phosphorylation studies revealed the presence of CaM kinase-dependent phosphorylation of the Ca2+ pump, Ca2+ release channel and phospholamban as early as 21-days gestation. This phosphorylation could be elicited with the addition of only Ca2+ and calmodulin indicating the presence of a SR-associated CaM kinase as early as 21-days gestation. This was confirmed using a delta-CaM kinase II-specific antibody. Phosphorylation per unit amount of each substrate was greater in the fetus and newborn compared to adult. Phosphorylation of phospholamban could be elicited by exogenous cAMP-dependent protein kinase (PKA) at all developmental stages studied. Activation of SR CaM kinase with Ca2+ and calmodulin, or induction of phospholamban phosphorylation by exogenous PKA, resulted in stimulation of the Ca2+ uptake activity of SR in fetal, newborn and adult heart. These results demonstrate early ontogenetic expression of the Ca2+ cycling proteins and CaM kinase in the SR and the concurrent development of phosphorylation-dependent regulation of SR Ca2+ cycling.     

Targeted overexpression of phospholamban to mouse atrium depresses Ca2+ transport and contractility

Phospholamban is a small phosphoprotein regulator of the Ca2+-pump of cardiac sarcoplasmic reticulum. Dephosphorylated phospholamban inhibits the Ca2+-pump and depresses contractility, whereas phosphorylation of phospholamban by cAMP-activated mechanisms relieves this inhibition and increases contractility. In order to better understand the function of phospholamban in living systems, a transgenic mouse model was established employing targeted overexpression of phospholamban to the atrium, which normally expresses low levels of the protein. Overexpression was achieved by fusing the alpha-MHC-promoter or the ANF-promoter to the phospholamban gene. Double transgenic mice were created by mating mice positive for each transgene. In single transgenic lineages, phospholamban was overexpressed four to six-fold in left atrium. In the double transgenic mice, phospholamban was overexpressed eight- to nine-fold. In the three transgenic strains. Ca2+ uptake by the sarcoplasmic reticulum was depressed to 22-30% of control values at low ionized calcium. This depression of Ca2+ uptake was largely reversed by addition of a phospholamban monoclonal antibody. In the atrial muscle strips, the time course of contraction was increased in a concentration-dependent manner by overexpression of phospholamban, whereas the basal developed tension was decreased up to 85% by phospholamban-overexpression. In all transgenic lineages, isoproterenol, a beta-adrenoceptor agonist, reversed the depression of contractility caused by overexpression of phospholamban and significantly shortened time parameters to levels approaching control values. These data demonstrate that overexpression of phospholamban in a mammalian myocardial tissue normally deficient in the protein substantially inhibits basal contractility, and furthermore suggest that in myocardial tissues containing high levels of the protein, phosphorylation of phospholamban can account for many of the positive inotropic and lusitropic effects of beta-adrenergic stimulation.     

Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.     

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.     

The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.     

Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy produced by doxorubicin in rabbits

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.     

Mechanism of action of sarcoplasmic reticulum calcium-uptake activators--discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction

The Ca2+ uptake by the sarcoplasmic reticulum (SR) can be affected by direct modulation of the Ca2+ pump or by removing the inhibitory effect of dephosphorylated phospholamban. The effect of these mechanisms was assessed using ellagic acid and 1-(3,4-dimethoxyphenyl)-3-dodecanone. Both compounds (30 micromol/l) enhanced SR-Ca2+ uptake in rabbit cardiomyocytes by 65.3 +/- 13% and 44.3 +/- 6.7% for 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid, respectively (at pCa 6.2). A similar effect was observed in cardiac SR microsomes (59.5 +/- 7.4% and 45.1 +/- 6.7) with 30 micromol/l 1-(3,4-dimethodoxyphenyl)-3-dodecanone and ellagic acid, respectively. 1-(3,4-Dimethoxyphenyl)-3-dodecanone increased Ca2+ storage by cardiac SR microsomes mainly at high [Ca2+] with a 57% increase of Vmax, whereas ellagic acid increased Vmax to a smaller extent (22%) and stimulated Ca2+ uptake at lower [Ca2+] with a leftward-shift of the pCa/ATPase relationship by pCa 0.24. Ellagic acid also differed from 1-(3,4-dimethoxylphenyl)-3-dodecanone in that it produced a Ca2+ sensitizing effect only in cardiac SR microsomes (by pCa 0.3) whereas 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulated the ATPase, at saturating Ca2+, in both cardiac and skeletal muscle SR vesicles. It is suggested that 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulates directly the Ca2+-ATPase activity, in contrast to ellagic acid which enhances the cardiac SR-Ca2+ uptake by interacting with phospholamban, as confirmed by the lack of additive effect between ellagic acid and monoclonal antibodies raised against phospholamban. 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid constitute attractive pharmacological tools to investigate the functional consequences of enhancing SR Ca2+, uptake by affecting different mechanisms.     

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.     

Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition

The highly conserved lysine residue Lys758 in the fifth stalk segment of the sarcoplasmic reticulum Ca2+-ATPase was substituted with either isoleucine or arginine by site-directed mutagenesis. The substitution with arginine was without significant effects on Ca2+-ATPase function, whereas multiple changes of functional characteristics were observed with the Lys758 --> Ile mutant. These included insensitivity of ATPase activity to the calcium ionophore A23187, an alkaline shift of the pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinities for Ca2+ and inorganic phosphate, as well as increased sensitivity to inhibition by vanadate. Analysis of the partial reaction steps of the enzyme cycle traced these changes to two steps. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) was increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the dephosphoenzyme with low Ca2+ affinity (E2) to the Ca2+-bound form activated for phosphorylation (E1Ca2) was reduced in the mutant, and the ATP-induced rate enhancement of this step required higher ATP concentrations in the mutant compared with the wild type.     

Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Transgenic approaches to define the functional role of dual site phospholamban phosphorylation

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 --> Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.     

The impact of rufloxacin given as prophylaxis to patients with cancer on their oral and faecal microflora

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.     

Bepridil reducing levothyroxine-induced enhancement of mitochondria Ca2+ Mg(2+)-ATPase activity in rat cerebrum

AIM: To study if bepridil (Bep) could affect the enhancement of activity of cerebral mitochondria Ca2+ Mg(2+)-ATPase caused by levothyroxine (Lev) in relation to ischemic overload calcium cerebrum injury. METHODS: The experimental hyperthyroidism model with ischemic cerebrum was developed in rats by ig Lev 1 mg.kg-1.d-1 for 7 d. Ca2+ Mg(2+)-ATPase activity and its kinetic parameters were assayed. RESULTS: The activity, Vmax and Km of cerebral mitochondria Ca2+ Mg(2+)-ATPase in control rats were 3.1 +/- 0.8, 5.1 +/- 2.3 mmol.P(i).h-1/g protein and 0.81 +/- 0.08 mmol.L-1 (ATP) respectively, whereas those of hyperthyroid rats were significantly altered to 4.6 +/- 0.5, 8.5 +/- 1.9 mmol.P(i).h-1/g protein and 0.49 +/- 0.11 mmol.L-1 (ATP) respectively. After treated with Bep 10 or 20 mg.kg-1.d-1 ig for 3 d, allabove 3 parameters of the enzyme were very significantly reduced vs those of either control or hyperthyroid. CONCLUSION: Bep, via decreasing Ca2+ Mg(2+)-ATPase activity and increasing the affinity of Ca2+ Mg(2+)-ATPase to ATP, could prevent rat cerebrum from ATP depletion and ischemic overload calcium injury.     

Sarcoplasmic reticular Ca2+ pump ATPase activity in congestive heart failure due to myocardial infarction

OBJECTIVE: Earlier studies have shown a depression in the sarcoplasmic reticular (SR) Ca2+ uptake and gene expression in Ca2+ pump ATPase protein in congestive heart failure subsequent to myocardial infarction. It is the objective of this study to understand further the mechanisms of depressed SR Ca2+ pump activity in the failing heart. METHODS: Heart failure in rats was induced by occluding the left coronary artery for 16 weeks and the viable left ventricle was processed for the isolation of SR membranes. Sham-operated animals were used as control. The characteristics of SR Ca2+ pump ATPase in the presence of different concentrations of K+, Ca2+ and ATP were examined and the purity of these membranes was monitored by determining the marker enzyme activities. In addition to measuring changes in cyclic adenosine monophosphate (cAMP) protein kinase and Ca(2+)-calmodulin induced phosphorylation, alterations in SR phospholipid composition as well as sulfhydryl (SH) group content were investigated. RESULTS: Ca(2+)-stimulated ATPase activity, unlike Mg(2+)-ATPase activity, was depressed in the left ventricular SR from failing hearts as compared to control. The decrease in Ca(2+)-stimulated ATPase activity was seen at different concentrations of Ca2+, K+ and ATP but no changes in the affinities of the enzyme for Ca2+ and ATP were evident. The SR Ca(2+)-stimulated ATPase activities in the presence of both cAMP-dependent protein kinase and Ca(2+)-calmodulin were markedly decreased in the failing hearts when compared to control preparations. Furthermore, the 32P incorporation in the presence of cAMP-dependent protein kinase or Ca(2+)-calmodulin was also reduced in the experimental heart SR membranes. The phospholipid composition of the SR membranes from the failing heart was markedly altered. No changes in SH-group or the degree of cross contamination with other membranes were apparent in the failing heart SR. CONCLUSIONS: These results suggest that abnormalities in membrane phospholipid composition and phosphorylation of the enzyme may partly explain the observed depression in SR Ca2+ pump ATPase activity in heart failure following myocardial infarction.     

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium

The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

A high-affinity Ca2+ pump, ECA1, from the endoplasmic reticulum is inhibited by cyclopiazonic acid but not by thapsigargin

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (Km = 30 nM) and displayed two affinities for ATP (Km of 20 and 235 microM). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 microM) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.     

Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation

Cardiac troponin (Tn) I (CTnI), compared with skeletal TnI, contains extra amino acids (32 to 33) at its amino terminus, including two adjacent serine residues. These two serine residues are believed to be phosphorylated by protein kinase A (PKA) upon stimulation of the heart by beta-agonists. In this study, we found that phosphorylation of a cardiac skinned muscle preparation by PKA, mainly at CTnI, results in a decrease in the Ca2+ sensitivity of muscle contraction. The pCa50 decreased by approximately 0.27 +/- 0.06 pCa units upon phosphorylation. To study cardiac muscle relaxation, we used diazo-2, a photolabile Ca2+ chelator with a low Ca2+ affinity in its intact form that is converted to a high-affinity form after photolysis. We found that the rate of cardiac muscle relaxation increased from a time of half-relaxation (t1/2) = 110 +/- 10 milliseconds to t1/2 = 70 +/- 8 milliseconds after CTnI phosphorylation. This result demonstrates that CTnI phosphorylation can be linked with the increased rate of muscle relaxation in a relatively intact muscle preparation. Since CTnI phosphorylation has been shown previously to affect the Ca2+ affinity and Ca2+ off-rate of CTnC in vitro, it is likely that the faster relaxation seen here reflects faster dissociation of Ca2+ from cardiac TnC (CTnC). Model calculations show that increased dissociation of Ca2+ from CTnC, coupled with the faster uptake of Ca2+ by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, can account for the faster relaxation seen in the inotropic response of the heart to catecholamines.     

Propranolol and bepridil attenuating levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation

AIM: To study the effects of propranolol and bepridil on levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation. METHODS: Rat heart hypertrophy was induced by i.p., levothyroxine 1 mg.kg-1.d-1 x 10 d. Then rats were treated by ig propranolol (Pro) or bepridil (Bep) 10 mg.kg-1 daily. Ca2+ Mg(2+)-ATPase activity and enzyme kinetic parameters were assayed. RESULTS: The activity and Vmax of mitochondrial Ca2+ Mg(2+)-ATPase isolated from hypertrophic left ventricle were 25 +/- 4 and 35.1 +/- 0.8 mumol Pi.h-1/mg protein, respectively, those of normal were 6.7 +/- 1.8 and 10 +/- 4 mumol Pi.h-1/mg protein, respectively. Apparent K(m) of the hypertrophic group Ca2+ Mg(2+)-ATPase was 0.4 +/- 0.12 mmol.L-1 ATP, and that of normal was 0.59 +/- 0.22 mmol.L-1 ATP. The total protein quantity of hypertrophic left ventricle was 80 +/- 30 mg, and that of normal was 47 +/- 9 mg. After treated with Pro or Bep (both 10 mg.kg-1 ig), the cardiac hypertrophy was attenuated, the enzyme activity and Vmax as well as total protein quantity of hypertrophic left ventricle were reduced to normal level, but apparent K(m) was not affected. CONCLUSION: Both Pro and Bep prevented the myocardium and its mitochondria from ischemia and overload calcium injury.