Skip Navigation

Cardiovascular Research 1997 36(1):67-77; doi:10.1016/S0008-6363(97)00183-1
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Antipenko, A. Y
Right arrow Articles by Kirchberger, M. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Antipenko, A. Y
Right arrow Articles by Kirchberger, M. A
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Membrane phosphorylation protects the cardiac sarcoplasmic reticulum Ca2+-ATPase against chlorinated oxidants in vitro

Alexander Y Antipenko and Madeleine A Kirchberger*

Department of Physiology and Biophysics, Box 1218, The Mount Sinai School of Medicine of the City University of New York, 1 Gustave L. Levy Place, New York, NY 10029, USA

* Corresponding author. Tel. (+1-212) 241-6543; fax (+1-212) 860-3369.

Received 10 April 1997; accepted 26 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of β-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. Methods: Ca-uptake and Ca2+-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. Results and Conclusions: Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 µM NaOCl or NH2Cl, which inhibit Ca-uptake by 21–41% when assayed at 25 or 37°C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.

KEYWORDS Sarcoplasmic reticulum; Ca2+-ATPase; Phospholamban; Beta-adrenergic agonists; Myocardial ischemia; Myocardial infarction; Dog, skeletal muscle microsomes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The calcium (Ca) pump protein (Ca2+-ATPase) of the cardiac sarcoplasmic reticulum (SR) plays a critical role in supplying activator Ca2+ to the contractile proteins and accounts for the major portion of Ca2+ removed from the cytoplasm during cardiac relaxation [1]. Chlorinated oxidants produced as a result of neutrophil activation in inflammatory states [2]inhibit the pump when tested in microsome, single myocyte, or isolated heart preparations [3–5], hence may be capable of impairing myocardial function in vivo. Activation of neutrophils has been demonstrated in patients with angina [6–8], myocardial infarction [8, 9], after coronary angioplasty [10], and in cardiac surgery [11, 12], although not, as was found more recently, in myocardial stunning after brief ischemia [13]or during early reperfusion of ischemic tissue [14].

Normally, oxidants participate in the destruction of foreign organisms in the body and hence are beneficial. However, in inflammatory states, oxidants are likely to be produced within the myocardium, attack intracellular targets, and disrupt normal cellular metabolism. Some oxidants diffuse to their targets from activated neutrophils attached on the endothelium or on myocytes after having emigrated from the blood vessels [15]. Activated neutrophils produce large amounts of hydrogen peroxide (H2O2) that may be converted to other potentially injurious oxidants in tissues [16]. In the presence of chloride ions, the enzyme myeloperoxidase, also released by the neutrophils, catalyzes the formation of hypochlorous acid (HOCl) (reaction 1):


Formula 1

(1)
Hypochlorous acid/hypochlorite is rapidly converted to monochloramine (NH2Cl) in the presence of urea, whose concentration in the myocardium is increased in ischemia [17](reaction 2).


Formula 2

(2)
Hypochlorite and monochloramine are considered among the most potent oxidants believed capable of producing myocardial injury.

Inhibition of SR membrane Ca uptake by oxidants is attributable to oxidation of one or more critical sulfhydryl groups in the Ca2+-ATPase (Ca pump) protein [18, 19]. In an early study, protein kinase A (PKA)-catalyzed phosphorylation of cardiac microsomes was shown to desensitize the Ca pump to sulfhydryl alkylation by N-ethylmaleimide [20]. Greater than 95% of microsomal phosphorylation is attributable to phosphorylation of phospholamban (PLN), a low molecular weight, amphiphilic protein located on the SR membrane in close proximity to the pump [21, 22]. In vivo, PLN is phosphorylated as a result of β-adrenergically induced activation of PKA, thereby reversing an inhibitory effect of unphosphorylated PLN on the pump. Proteolytic cleavage of the cytoplasmic domain of PLN as a result of trypsin treatment of microsomes also reverses PLN's inhibitory effect in vitro [23]as does incubation of microsomes in the presence of monoclonal antibody against PLN [24]or certain polyanionic compounds [25]. PLN phosphorylation increases Vmax(Ca) of Ca uptake and decreases the Km(Ca) of the pump [26]. The regulation of Ca pump activity over the entire range of intracellular Ca2+ concentrations is consistent with the well-known abbreviation of systole, increased –dP/dtmax, and decreased contractile duration produced by β-adrenergic agonists in the heart. A central role of PLN in the β-agonist induced increase in cardiac contractility was demonstrated in a study of PLN-gene deficient mice, whose hearts exhibit mechanical properties similar to fully isoproterenol-stimulated hearts from wild-type mice [27]. Thus PLN was shown to play a major role in both the inotropic and lusitropic effects of β-adrenergic agonists on the myocardium.

In the present study, we examined whether covalent modification of PLN by phosphorylation is able to protect the SR Ca pump protein in canine cardiac microsomes against oxidative damage. We demonstrate significant protection by phosphorylation against chlorinated products of activated neutrophils.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by National Academy Press (Washington, D.C., 1996).

2.1 Materials
[{gamma}-32P]ATP was from Amersham. NaOCl and PKA catalytic subunit were from Sigma. Unless otherwise stated, the PKA used was a preparation partially purified from bovine heart and dialyzed against 5 mmol/l histidine-HCl, pH 6.8 [28]; the final solution against which the PKA was dialyzed was used as control solution. The molecular weight protein standards for the electrophoretic gels were obtained from Gibco BRL. All other reagents were obtained as reported previously [26].

2.2 Preparation of microsomes
Crude canine cardiac and fast skeletal muscle microsomes derived from vastus lateralis muscle of New Zealand white rabbits were subjected to further purification on a sucrose step gradient as described previously [26]. The light microsomes in the F1 fraction, which was insensitive to 5 µmol/l ruthenium red [29]and hence contained no detectable ryanodine-sensitive Ca release channels are henceforth referred to as purified microsomes. Purified microsomes were stored in liquid nitrogen. Protein concentrations were determined using the biuret procedure with bovine serum albumen as the standard.

2.3 Ca uptake and Ca2+-ATPase assays
Ca uptake assays were run at pH 6.8 in a standard assay mixture at 25 or 37°C in the presence of oxalate and Formula Cl2, as described previously [26]. Microsomes were added to the temperature-equilibrated reaction mixtures containing different concentrations of oxidant, followed by the addition Formula to start the Ca uptake reaction. Reactions were terminated by filtration. All reactions were linear with respect to time and microsomal protein concentration.

In some experiments, the microsomes were treated with trypsin, which cleaves the cytoplasmic domain of PLN and, at the low concentrations used, has little or no direct effect on the transport properties of the Ca pump other than to increase its activity as a result of removal of PLN's inhibitory influence [23]. Briefly, the microsomes (1.5 mg/ml) were treated for 2 min with 0.01 mg/ml trypsin in 40 mmol/l histidine-HCl at pH 6.8 and 25°C, followed by the addition of 0.12 mg/ml of soybean trypsin inhibitor. Control microsomes were incubated under identical conditions except that the trypsin inhibitor was present in the incubation medium already prior to the addition of the microsomes. The microsomes were maintained on ice as aliquots were removed to assay Ca uptake. In other experiments, Ca uptake was measured in phosphorylated and unphosphorylated microsomes as described previously [26]. After 2 min of incubation of the microsomes in the standard Ca uptake reaction mixture that included the phosphorylation or control reagents, NaOCl, NH2Cl, or distilled water was added to a concentration, at this point, of 110% of the final concentration specified in the text, and the incubation was continued for an additional 2 min prior to the final addition of the Formula buffer to start the Ca uptake reaction. The addition of the Ca2+ buffer reduced the concentration of cAMP to 2 µmol/l and that of PKA to 0.05, 0.30, or 0.75 mg/ml, and the remaining reagents to the concentrations in the standard Ca uptake assay mixture. In assays of the effect of 10 µM NaOCl on PKA activity using histone as the substrate [28], no effect was found.

Ca2+-ATPase activity was assayed at 25°C by measuring the rate of decrease in NADH absorbency at 340 nm using an enzyme-linked assay described previously [26]with the following modification. Instead of jasmone, 10 µM NaOCl or distilled water was added 1 min after a 3-min initial temperature equilibration of the cuvette containing the reaction mixture within the spectrophotometer.

2.4 Reversibility of effects of oxidants
To test for the reversibility of the inhibitory effect of oxidants on Ca uptake, microsomes were first treated for 2 or 10 min in 40 mmol/l histidine-HCl, pH 6.8 at 25°C, 120 mmol/l KCl, 5 mmol/l NaN3, and either 10 µmol/l NaOCl or NH2Cl or distilled water (control). An aliquot of the reaction mixture was then transferred to a standard Ca uptake reaction mixture that included 5 mmol/l DTT or distilled water but lacked the Formula buffer, thereby reducing the concentration of oxidant to 1/50 of its concentration in the initial incubation medium. After 5 min of incubation of the microsomes in the presence of the DTT or an equivalent volume of distilled water, the Ca uptake reaction was started by addition of the Formula buffer; samples were taken and processed as described before [23].

2.5 PLN phosphorylation: quantitation and immunoblot analysis
Purified cardiac microsomes (0.57 mg/ml) that had been treated with trypsin or trypsin inhibitor-inactivated trypsin, as described above, were incubated for 1 min in 44 mmol/l histidine-HCl, pH 6.8 at 25°C, 1.2 mmol/l MgCl2, 2.2 µmol/l cAMP, 0.82 mg/ml PKA, 2.2 mmol/l EGTA-Tris, 11 mmol/l NaF, and 0.11 mmol/l [{gamma}-32P]ATP, followed by addition of NaOCl to a final concentration of 10 µM, which decreased the concentration of the remaining reagents by 9.1%. The specific radioactivity of the ATP was 1.0 µCi/nmol. After 1 min of incubation, reactions were stopped by the addition of trichloroacetic acid. The samples were solubilized in a solubilizing mixture containing sodium dodecylsulphate (SDS) and electrophoresed on two 10 or 15% polyacrylamide mini-gels, as described previously [30]. One of the gels was transferred to a nitrocellulose membrane and processed for Western blotting, using a polyclonal antibody raised in rabbits against a synthetic polypeptide containing PLN residues 2 through 30 [30].

The second gel was prepared for quantization of Formula incorporation into SR membrane components with the aid of a Molecular Dynamics PhosphorImager system and ImageQuant software. The dried gel was exposed to a PhosphorImager screen for 3 h and the data stored in the PhosphorImager computer. To relate the density of the bands including PLN to a known amount of radioactivity and to insure linearity of the band density with the amount of radioactivity present, 6 aliquots containing incremental amounts of [{gamma}-32P]ATP ranging from 1.2 to 40 nCi were applied to filter paper and processed together with the dried gel on the PhosphorImager system in order to obtain the density of each of these spots, corrected for background. An appropriate background value was subtracted from all densities.

2.6 Preparation of oxidants
The hypochlorite concentration was estimated from the decrease in absorbency of 5-thionitrobenzoic acid, using a molar extinction coefficient of 13 600 M–1cm–1 at 412 nm [31]. The 5-thionitrobenzoic acid was prepared by reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) with sodium borohydride at 37°C [32]. NH2Cl was prepared at 4°C by adding a 5–6% solution of NaOCl to a solution of ammonium chloride in 0.05 mol/l potassium phosphate buffer (pH 8.0) at a ratio of NaOCl to ammonia of 1 to 1.5 [33]. NH2Cl was separated from unreacted HOCl/OCl– on a Sephadex G-50 column [34]and its concentration determined by measuring its absorbence at 242 nm using a molar extinction coefficient of 429 M–1 cm–1. The NH2Cl was considered free of unreacted NaOCl based on a lack of decrease in absorbency with lowered pH. It was maintained at 4°C and used within two days of preparation.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of NaOCl on Ca uptake
In an initial set of experiments, we determined the effect of NaOCl on Ca uptake by cardiac microsomes that had been trypsin-treated and/or phosphorylated by PKA (Fig. 1A). NaOCl was found to produce a virtually identical concentration-dependent decrease in Ca uptake in the trypsin-treated microsomes and their controls. No significant difference in Ca uptake was produced by up to 1 µmol/l NaOCl; higher concentrations were markedly inhibitory with half-maximal inhibition occurring at about 30 µmol/l. However in the trypsin control microsomes treated with PKA, no inhibition of Ca uptake was observed at NaOCl concentrations as high as 10 µmol/l. At 10 µmol/l NaOCl, Ca uptake by the non-phosphorylated microsomes or trypsin-treated or control microsomes was already markedly reduced. Above 10 µmol/l NaOCl, Ca uptake by phosphorylated microsomes also decreased sharply but remained less sensitive to increasing oxidant concentration than the unphosphorylated microsomes. Also tested were microsomes that had been treated in the presence of PKA catalytic subunit or control solution (Fig. 1A). The catalytic subunit preparation must be reconstituted in a high concentration of DTT, which interferes with the oxidants being tested, hence it was necessary to centrifuge, resuspend the microsomes, and determine their protein concentration after phosphorylation [26]prior to assaying Ca uptake. Since no significant difference was found in the results obtained with the catalytic subunit and the holoenzyme, further studies were carried out with the holoenzyme in order to avoid centrifugation, resuspension of microsomes, and redetermination of the protein concentration. Incubation of fast skeletal muscle microsomes, which contain no PLN [35], under conditions favorable for PKA-catalyzed phosphorylation had no effect on Ca uptake (Fig. 1B). The skeletal muscle microsomes showed essentially the same sensitivity to NaOCl as the unphosphorylated or trypsin-treated cardiac microsomes (cf. Fig. 1A and B).


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effect of NaOCl on Ca uptake by purified cardiac (A) and light fast skeletal muscle (B) microsomes. Cardiac microsomes were treated with trypsin ({blacktriangleup}) or trypsin inhibitor-inactivated trypsin (control solution) ({triangleup}) and assayed for Ca uptake in a standard assay medium containing the indicated NaOCl concentrations. Also, the cardiac microsomes (circles) that had been treated with control solution as well as fast skeletal muscle microsomes that had received no prior treatment (squares) were incubated in the presence (bullet, {blacksquare}) and absence ({circ}, {square}) of 2 µmol/l cAMP and 0.75 mg/ml PKA under conditions favorable for phosphorylation. After a 1-min incubation at 25°C, NaOCl was added to the reaction mixture and after 2 more min, 11 µmol/l Ca2+ was added to start the Ca uptake reaction. Additionally, the microsomes that had been treated with trypsin control solution were further treated with 0.15 U/µl PKA catalytic subunit ({blacktriangledown}) or control solution ({triangledown}), centrifuged, and resuspended in buffer A [26]prior to assay of Ca uptake at the indicated NaOCl concentrations. In this case, microsomes were added to the temperature-equilibrated reaction mixture and 2 min later, Ca2+ was added to start the reaction. See Section 2for additional experimental detail. 100% Ca uptake corresponds to: ({triangleup}) 0.40, ({blacktriangleup}) 0.62, ({circ}) 0.44, (bullet) 0.64, ({triangledown}) 0.38, ({blacktriangledown}) 0.50, ({square}) 1.58±0.25, and ({blacksquare}) 1.58±29 µmol Ca/mg·min. The data identified by different open and closed symbols in panel A represent 3 independent single experiments; the data shown in panel B are the means±SD of 3 experiments with different microsome preparations.

 
The relationship between the stimulatory effect of PKA on the Ca pump and the inhibitory effect of 10 µmol/l NaOCl was evaluated further in the following series of experiments. Microsomes were treated with trypsin or trypsin inhibitor-inactivated trypsin and then incubated in the presence and absence of cAMP and saturating and subsaturating concentrations of PKA (Table 1). Control (non-trypsin treated) microsomes incubated with both cAMP and 0.3 mg/ml PKA exhibited the expected increase in Ca uptake (i.e. 30%) when assayed at 11 µmol/l Ca2+ and a smaller increase in the presence of a subsaturating concentration of PKA. Trypsin treatment of microsomes, also as expected [36], increased Ca uptake by 33%, whereas incubation of trypsin-treated microsomes with cAMP and 0.3 mg/ml PKA increased Ca uptake to a considerably lesser extent (i.e. by 6%) compared to the non-trypsin-treated microsomes. Inhibition of Ca uptake by 10 µmol/l NaOCl was highest (i.e. 33 or 32%) in the microsomes incubated in the absence of cAMP and PKA (no additions), irrespective of whether the microsomes had been pretreated with trypsin, and decreased with increasing PKA concentration in control microsomes (11% and 3% inhibition at 0.05 and 0.3 mg/ml PKA, respectively). At 0.3 mg/ml PKA, the apparent 3% decrease in Ca uptake by the non-trypsin treated microsomes with NaOCl lacked statistical significance (p = 0.3) in contrast to trypsin-treated microsomes incubated similarly, in which a 24% inhibition was observed.


View this table:
[in this window]
[in a new window]

  		Table 1 Effect of cAMP/protein kinase A (PKA) on Ca uptake by trypsin-treated and control microsomes assayed in the presence and absence of 10 µM NaOCl

 
The percent inhibition of Ca uptake produced by 10 µmol/l NaOCl under the different conditions listed in Table 1 varied inversely with the percent increase in Ca uptake attributable to treatment of the microsomes with cAMP and a saturating or subsaturating concentration of PKA (Fig. 2). In summary, only in the case of the microsomes that were treated with cAMP and a saturating concentration of PKA was protection against 10 µmol/l NaOCl essentially complete. Trypsin-treated microsomes were significantly inhibited by the oxidant even though their rates of Ca uptake approached those seen in non-trypsin-treated, phosphorylated microsomes. Hence the full protective effect observed upon incubating cardiac microsomes under conditions favorable for phosphorylation appears to require uncleaved PLN. No significant stimulation of Ca uptake was observed in the presence of cAMP alone and a mild stimulation of about 9% was seen with PKA alone (data not shown). However when the PKA was denatured by boiling, no increase in Ca uptake was observed, and 10 µM NaOCl produced the expected inhibition (i.e. 31%). Hence a small increase in Ca uptake observed in the presence of PKA alone may be attributable to the generation of endogenous cAMP by the presence of sarcolemmal membrane contaminants. Such a stimulation was seen only at the relatively high PKA concentrations used in the Ca uptake assay but not at the low concentration used in the Ca2+-ATPase assay described below.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Relationship between the magnitude of the increase in Ca uptake produced by cAMP and PKA and the decrease produced by 10 µmol/l NaOCl. The data were derived from the 5 data sets presented in Table 1. Shown are the p value [37]and correlation coefficient (r) obtained in a t-test of the regression with 3 degrees of freedom.

 
3.2 Effect of NaOCl on Ca2+-ATPase activity
The identical conditions used in the Ca uptake assay were technically unsuitable for use in our assay of microsomal Ca2+-ATPase activity. Accordingly, the concentrations of microsomes and PKA were reduced to 2.4 µg/ml and 0.11 mg/ml, respectively, oxalate was omitted, and a Ca2+ ionophore was added to the reaction mixture, as were the enzymes and substrate for monitoring NADH oxidation. Prior to the addition of test reagents to the reaction mixture, it was established that 10 µmol/l NaOCl, 3% dimethylsufoxide, and 0.3 µg/ml A23187 [GenBank] do not interfere in the assay system.

In this series of experiments, treatment of the microsomes with cAMP and a predetermined saturating concentration of PKA under conditions favorable for phosphorylation produced a maximum stimulation of Ca2+-ATPase activity of 42% (Table 2). Subsequent addition of 10 µmol/l NaOCl to the reaction mixture had no effect on the ATPase activity. The same concentration of oxidant, however, reduced the Ca2+-ATPase activity of unphosphorylated control microsomes by 25%. No statistically significant increase in Ca2+-ATPase activity was observed in the presence of cAMP alone or PKA alone. In both cases, 10 µM NaOCl decreased enzyme activity to a value close to that observed in control microsomes.


View this table:
[in this window]
[in a new window]

  		Table 2 Effect of 10 µM NaOCl on Ca2+-ATPase activity in phosphorylated and control microsomes

 
3.3 Effect of NaOCl on the Ca2+ concentration dependence of Ca uptake
In this series of experiments, treatment of microsomes with cAMP and PKA produced a 36% increase in Vmax(Ca) of Ca uptake and a 7% decrease in Km(Ca) (Fig. 3 and Table 3). Effects of phosphorylation or trypsin treatment on both Vmax(Ca) and Km(Ca), the former one predominating, are found consistently when purified microsomes are assayed at 25°C under standard incubation conditions [26], in contrast to crude microsomes in which the effect seen with phosphorylation or trypsin treatment is primarily to decrease Km(Ca) and to increase Vmax(Ca) slightly (e.g. Ref. [23]) or to a variable extent (cf. Refs [23]and [36]. NaOCl (10 µmol/l) produced no significant change in the Ca uptake parameters for phosphorylated microsomes (Table 3), but in control microsomes, it decreased Vmax(Ca) by 23% without significantly affecting Km(Ca) or the Hill coefficient.


Figure 3
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Ca2+ concentration dependence of Ca uptake by phosphorylated and control cardiac microsomes incubated in the presence and absence of 10 µmol/l NaOCl. Microsomes were incubated in the presence ({circ}, {triangleup}) and absence (bullet, {blacktriangleup}) of 2 µmol/l cAMP and 0.30 mg/ml PKA under conditions favorable for phosphorylation followed by the addition to the reaction mixture of either 10 µmol/l NaOCl ({circ}, {triangleup}) or distilled water (bullet, {blacktriangleup}). Ca uptake was initiated after 2 more min by the addition of a CaCl2–EGTA buffer solution to yield the indicated Ca2+ concentrations. Each data point represents an average of 4 different experiments±SD (error bars are shown in one direction only). The means±SD of the optimized kinetic parameters, obtained in 4 separate fits of the data sets to the equation V = Vmax/[1+(Km/[Ca2+])N] by a nonlinear least-squares procedure, where N is the Hill coefficient, are shown in Table 3.

 

View this table:
[in this window]
[in a new window]

  		Table 3 Kinetic parameters for Ca uptake by control and phosphorylated cardiac microsomes: effect of 10 µM NaOCl

 
3.4 Effect of monochloramine
NH2Cl, a second chlorinated oxidant associated with neutrophil activation, produced a concentration-dependent inhibition of microsomal Ca uptake resembling that of NaOCl (Fig. 4). The inhibition was negligible at concentrations between 0.1 and 1 µmol/l but at 10 µmol/l, the mean Ca uptake rate was only 59% of the activity observed in the absence of NH2Cl. However in contrast to control microsomes, Ca uptake was identical in the presence and absence of 10 µmol/l NH2Cl in microsomes that had been incubated in the presence of cAMP and PKA. Above 10 µmol/l NH2Cl, Ca uptake decreased sharply yet remained less sensitive to NH2Cl than unphosphorylated microsomes.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effect of NH2Cl on Ca uptake by phosphorylated and control cardiac microsomes. Microsomes were incubated in the presence (bullet) and absence ({circ}) of 2 µmol/l cAMP and 0.75 mg/ml PKA under conditions favorable for phosphorylation and, after the addition of the indicated concentrations of NH2Cl to the reaction mixture, were assayed for calcium uptake at 11 µmol/l Ca2+. The data represent the means±SD of calcium uptake rates obtained in 3 experiments carried out with different microsome preparations. See Section 2for experimental detail. 100% calcium uptake corresponds to: ({triangleup}) 0.52, ({blacktriangleup}) 0.75 µmol Ca/mg·min.

 
3.5 Temperature and pH dependence
At 37°C, the sensitivity of Ca uptake to NaOCl and NH2Cl was indistinguishable (Fig. 5). Inhibition by either oxidant became apparent between 0.1 and 1 µmol/l and was half maximal at about 25 µmol/l. At 10 µmol/l oxidant, Ca uptake by the unphosphorylated (control) microsomes was only about 73% of the rate found in phosphorylated microsomes, which were insensitive to either oxidant at the same concentration. As was observed at 25°C, Ca uptake by the phosphorylated microsomes remained less sensitive to all concentrations of oxidant tested until the highest concentration (1 mmol/l) where the inhibition was virtually complete. The protective effect of phosphorylation was also tested at an approximately physiological intracellar pH of 7.3 at 37°C. NaOCl at 10 µM reduced the Ca uptake rate from 1.58±0.49 to 1.11±0.43 µmol/mg·min, a 30% decrease. However in phosphorylated microsomes, the Ca uptake rates in absence and presence of 10 µM NaOCl were identical (1.93±0.56 and 1.92±0.55 µmol Ca/mg·min, respectively).


Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effect of oxidants on Ca uptake by phosphorylated and control cardiac microsomes assayed at 37°C. Microsomes were incubated in the presence (bullet, {blacktriangleup}) and absence ({circ}, {triangleup}) of 2 µmol/l cAMP and 0.75 mg/ml PKA followed by the addition to the reaction mixture of either 10 µM NaOCl (bullet, {circ}) or NH2Cl ({blacktriangleup}, {triangleup}). After a 2-min incubation with the oxidant, 11 µmol/l Ca2+ was added to initiate Ca uptake. A single experiment with each oxidant is shown. See Section 2for experimental details. 100% Ca uptake corresponds to: ({circ}) 1.05, ({triangleup}) 1.08, (bullet) 1.27, and ({blacktriangleup}) 1.24 µmol Ca/mg·min.

 
3.6 Reversibility of oxidant effects
In microsomes that exhibited a reduction in Ca uptake to 79% and 76% of the control level as a result of treatment with 10 µmol/l NaOCl for 2 and 10 min, respectively, a subsequent 5-min incubation of the microsomes in 5 mmol/l DTT restored their rate of Ca uptake to 100% and 101%, respectively, of the value in control microsomes that had been incubated in the absence of oxidant and DTT. As an additional control, microsomes were incubated for 2 and 10 min in the absence of oxidant and then for 5 min in the presence of DTT. In this case, the Ca uptake rates were 97% and 95% of the rates obtained in microsomes incubated for similar periods in the absence of oxidant and DTT. The same experiment was carried out using 10 µmol/l NH2Cl, which reduced Ca uptake to 73% and 75% of the control value (no oxidant and no DTT) after 2- and 10-min incubations, respectively. Treatment of these microsomes with DTT restored the Ca uptake rates to 100% in both cases. Treatment of the microsomes under control conditions in the absence of oxidant, followed by the addition of DTT as before, resulted in Ca uptake rates that were 98% and 97%, respectively, of the control values. These data indicate that the above-described effects of 10 µmol/l NaOCl and NH2Cl are fully reversible under the conditions tested.

3.7 Effect of 10 µmol/l NaOCl on SR membrane phosphorylation
In order to investigate the role of PLN or other proteins in the protective effect of cAMP and PKA against oxidant-induced damage of the Ca, the extent of Formula incorporation into the microsomes was determined under conditions similar, although, for technical reasons, not identical to those used to treat microsomes prior to measurement of Ca uptake. On the electrophoretic gel shown in Fig. 6A, the band that shows by far the highest amount of Formula incorporation corresponds to pentameric PLN. Monomeric PLN also was radiolabelled. Of the two minor phosphorylated proteins seen on the gel, the one migrating immediately above the level of the ovalbumin molecular weight standard is likely to be the regulatory subunit of PKA whereas the one seen in some of the tracks at the top of the gel could represent phosphorylase a. Irrespective of the identity of the two phosphoproteins, they are present in the samples consisting of trypsin-treated microsomes, which failed to exhibit a protective effect, and hence cannot be associated with the protective effect of phosphorylation against oxidant damage of the pump. A trace amount of Formula incorporation may be seen in the control microsomes in a protein band near the top of the gel and in the region corresponding to a protein of about 15 kDa. This Formula incorporation is either reduced slightly or invisible in the trypsin-treated microsomes. In view of the minor amount of phosphorylation and the fact that no evidence can be adduced to link these bands to the SR Ca pump, the proteins migrating in these particular positions are unlikely to play a role in the protective effect of SR membrane phosphorylation on the Ca pump, although the possibility cannot be completely eliminated.


Figure 6
View larger version (79K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 PhosphorImager (A) and Western blot (B) analysis of electrophoretically separated cardiac microsomes. Control (trypsin inhibitor-inactivated trypsin-treated) microsomes (tracks 1 through 5) or TT (trypsin-treated) microsomes (tracks 6 through 9) were incubated in the presence of [{gamma}-32P]-ATP and the following additions to the reaction mixture: tracks 1 and 9, no additions; 2 and 6, 2 µmol/l cAMP and 0.75 mg/ml PKA (PK-A); 3 and 7, same as 2 and 6 except that 10 µmol/l NaOCl was present; 4 and 8, 2 µmol/l cAMP alone; and 5, 0.75 mg/ml PKA alone. The microsomes were then applied to two 15% SDS–polyacrylamide gels and electrophoresed. One gel was used to obtain the PhosphorImager image and the other the Western blot. Each track contained 10 µg of microsomal protein. TG, top of separating gel; BG, bottom of gel; P-PLN, pentameric phospholamban; M-PLN, monomeric PLN; P, phosphorylated; UN, unphosphorylated. The apparent molecular sizes of the protein markers used are (kDa): (a) 44.6, ovalbumin; (b) 30.5, carbonic anhydrase; (c) 17.8, β-lactoglobulin; (d) 12.8, lysozyme; (e) 7.2, bovine trypsin inhibitor. The amount of PLN phosphorylation obtained by quantitation of the PhosphorImager image is (nmol P/mg·min): tracks 1, 0.07; 2, 2.68; 3, 2.55; 4, 0.21; 5, 0.23; 6, 0.14; 7, 0.13; 8, 0.01; 9, 0.00.

 
The largest Formula incorporation into PLN was obtained in the presence of both cAMP and PKA, namely 2.68 nmol Formula of microsomal protein, taken as 100%, when quantitated by PhosphorImager analysis (Fig. 6A). cAMP alone, PKA alone, or no further additions to the basic phosphorylation reaction mixture produced a much reduced Formula incorporation (8%, 9%, or 3%, respectively), which is consistent with the observed minimal increase in Ca2+-ATPase activity (Table 2) and Ca uptake (see text above) with cAMP alone and PKA alone. However the percent change in Formula incorporation into PLN cannot be directly correlated with the increases in the rate of Ca uptake or Ca2+-ATPase activity in the presence of cAMP and/or PKA since different experimental conditions were required in the determinations. The addition of 10 µmol/l NaOCl to the microsomes phosphorylated in the presence of both cAMP and PKA produced no significant decrease in the level of phosphorylation (6%). In trypsin-treated microsomes, Formula incorporation into PLN was undetectable when microsomes were incubated in the absence cAMP and PKA and was low (4%) when incubated in the combined presence of both reagents. As before, further treatment of these microsomes with 10 µmol/l NaOCl caused no significant change in Formula content of PLN.

The above findings were obtained using a 15% polyacrylamide gel, which allowed entry into the gel of proteins as large as approximately 90 000 daltons. An identical analysis was carried out using a 10% gel in order to detect potential phosphorylation of the Ca2+-ATPase protein and other higher molecular weight proteins. No PKA-catalyzed phosphorylation of the Ca2+-ATPase protein was detected, whether in trypsin-treated or control microsomes (gel not shown).

In Western immunoblot analysis of native canine cardiac microsomes, polyclonal antiserum against a synthetic peptide analog of PLN recognized a single major band corresponding to pentameric PLN, a 27 kDa protein, and a more diffuse band of higher mobility that corresponds to monomeric PLN of approximately 6 kDa [21](Fig. 6B, track 1). Upon phosphorylation, a slight decrease is observed in the mobility of pentameric PLN (track 2). The presence of a single band corresponding to unphosphorylated pentameric PLN in track 1 indicates that no endogenously phosphorylated PLN was detectable in the microsomes by Western blot analysis. Pentameric PLN in microsomes incubated in the presence of cAMP alone (track 4) and PKA alone (track 5) migrates largely as an unphosphorylated protein. Pretreatment of the microsomes with trypsin under conditions previously shown to cleave cytoplasmic fragment of PLN from the membrane [23]eliminated all visible bands corresponding to PLN on the Western blot shown in Fig. 6B (tracks 6 through 9), although a small amount of PLN is detectable by Formula incorporation (Fig. 6A), a more sensitive indication of the presence of any remaining PLN. This residual amount of uncleaved PLN may remain as a result of the mild conditions chosen for trypsin treatment in order to avoid hydrolysis of the Ca2+-ATPase protein at the T2 site.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present data demonstrate a positive correlation between a PKA-mediated increase in Ca uptake by cardiac SR membranes that is associated with PLN phosphorylation and a decreased inhibition of Ca uptake by physiologically attainable concentrations (see below) of NaOCl or NH2Cl. The Ca pump of microsomes phosphorylated in vitro was fully protected against a 10 µmol/l concentration of these oxidants and to a lesser extent at increasing oxidant concentrations (Figs. 1–5GoGoGoGo, Tables 1–3GoGo). This protective effect of phosphorylation cannot be attributed to the exogenous PKA acting as a hypochlorite scavenger for the following reasons. (i) In assays of Ca2+-ATPase activity (Table 2), microsomes incubated in the presence of PKA alone showed no protection against 10 µmol/l NaOCl. (ii) No protective effect was observed in fast skeletal muscle microsomes, which lack PLN, upon incubation in the presence of cAMP and PKA under conditions favorable for phosphorylation. (iii) The full protective effect was absent in trypsin-treated cardiac microsomes incubated similarly. In the latter case, a minor effect of microsomal treatment with cAMP and PKA under phosphorylation conditions can be accounted for by a much-reduced increase in phosphorylation compared to the non-trypsin-treated microsomes (Table 1). As seen on the gel shown in Fig. 6A, small amounts of uncleaved PLN may remain after mild trypsin treatment.

The observed decrease in SR membrane Ca uptake produced by NaOCl or NH2Cl may result from an inhibition of one or more steps in the Ca2+-ATPase reaction cycle. In early studies, the rate of the E1P to E2P conversion during the catalytic cycle of the Ca2+-ATPase was shown to be decreased by N-ethylmaleimide [38, 39], a sulfhydryl reactive reagent. It was subsequently suggested that differences in the degree of oxidation of essential thiol groups in the ATPase protein, which appear to reduce the rate of the E1'PCa2 -> E2'PCa2 or E1'PMgATPCa2 -> E2'PMgATPCa2 transition, can account for a two-fold variability in ATPase activity in purified fast skeletal muscle Ca2+-ATPase preparations that cannot be attributed to differences in enzyme homogeneity [40]. Oxidation of essential thiol groups may also account for differences in the degree of stimulation of the E1'P Ca2 -> E2' PCa2 transition by regulatory ATP [41]and hence to variability in enzyme activity.

In the fast skeletal muscle Ca2+-ATPase protein, covalent modification of either Cys 344 or Cys 364 by sulfhydryl-reactive reagents is tolerated whereas modification of both cysteines produces Ca pump inhibition [19]. The corresponding cysteine residues in the cardiac Ca2+-ATPase protein occur within a stretch of amino acid residues (residues 336 to 412) necessary for a functional interaction between PLN and the Ca2+-ATPase [42]. A putative conformational change in the Ca2+-ATPase protein associated with increased activity as a result of PLN phosphorylation or trypsin treatment could produce a shielded environment for the critical cysteine(s). In this case, in order to explain the lack of protection with trypsin treatment, one may invoke the fact that in addition to cleaving PLN, trypsin also cleaves the Ca2+-ATPase protein at the T1 site [23], which could interfere with any protective effect associated with a PLN-related conformational change. However it is important to note that hydrolysis of the Ca2+-ATPase protein at the T1 site has no effect on ATP-supported Ca uptake whether in fast skeletal [43]or cardiac [23]SR. The simpler explanation for the observed phosphorylation-induced protective effect is shielding of the reactive cysteine residue(s) on the Ca2+-ATPase by uncleaved, phosphorylated PLN. Thus it is conceivable that a phosphorylation-induced conformational change in uncleaved PLN [44], the Ca2+-ATPase, or both proteins produces shielding of reactive cysteine(s) on the Ca2+-ATPase from mild exposure to certain sulfhydryl-reactive molecules.

The inhibition of the cardiac SR Ca pump produced by 10 µmol/l NaOCl or NH2Cl under the experimental conditions studied was fully reversible upon brief treatment of the microsomes with DTT (see Results). Our findings are consistent with previous reports on the reversibility of the inhibitory effects on the SR Ca pump produced by relatively mild treatment with hypochlorous acid [3–5, 45]. In one study [5]the reversal of inhibition by DTT was correlated with the restoration of contractility in perfused hearts and in another study [4]with restoration of normal cytoplasmic Ca2+ concentrations after a 3-fold increase caused by exposure of ventricular myocytes to hypochlorous acid.

Hypochlorous acid, NH2Cl, and other oxidants are produced as a result of activation of neutrophils, which may adhere to endothelial cells or cardiomyocytes. Some oxidants, like hydroxyl radicals or hypochlorite ions, are so highly reactive that they bind already extracellularly to the nearest target. However hypochlorous acid, which has a pK of approximately 7.5 under physiological conditions [46], exists extracellularly to a large extent in the undissociated form, allowing it to cross the cell membrane, partially dissociate, and become reactive intracellularly. Eley et al. [5]have presented evidence based on perfusion of isolated rat hearts with a physiologically relevant concentration of hypochlorous acid to indicate that the oxidant can cross both the endothelium and sarcolemma of cardiomyocytes and inhibit the Ca2+-ATPase in a reversible reaction with Ca2+-ATPase sulfhydryls. NH2Cl, which is lipophilic, is readily able to oxidize sulfhydryl groups in intracellular proteins and is therefore considered even more toxic [47].

Ten to 20 µmol/l hypochlorite oxidizes sulfhydryl groups of target proteins while concentrations above 50 µM oxidize methionine and tryptophan residues as well [48]. In vivo, a local concentration of 60 to 90 µM hypochlorous acid is estimated to be produced by activated neutrophils and a concentration of 10 µM, at which full protection by phosphorylation was observed in the present study, is considered attainable at some distance from the release site [49].

The pathophysiological and clinical implications of the in vitro findings described in this report are potentially considerable. Our findings of a protective effect of PLN phosphorylation against Ca pump oxidation by chlorinated oxidants provide an understanding of some of the factors that may contribute to myocardial damage or protection at the cellular and molecular levels under conditions associated with neutrophil activation [50]when locally produced levels of norepinephrine may be elevated [51]. Although β-adrenergic agonists increase cardiac workload, which can lead to demand ischemia (e.g. Ref. [52]), they may, at least in the limited capacity described in the present communication, serve as an "endogenous myocardial protective substance" [53]against oxidative stress. It will be important to determine whether the reported protective effect of SR membrane phosphorylation toward reactive cysteine residues in the Ca pump protein against chlorinated oxidants extends also to other types of oxidants. The study by Limas [20], using N-ethylmaleimide (see Section 1), would suggest this to be the case. Since oxidants are likely to be produced upon reperfusion of ischemic myocardium [54], oxidation of reactive sulfhydryls in the Ca pump protein might account for the depression of Ca2+-ATPase activity observed in SR preparations obtained from nonischemic and stunned canine myocardium, particularly at high Ca2+ concentrations [55]. This pattern of inhibition is similar to that observed in the present study with chlorinated oxidants (cf. Fig. 3, Table 3). Moreover, the presently reported protection against oxidative damage to the SR Ca pump by SR membrane phosphorylation may contribute to the salutary effects of β-adrenergic agonists reported occasionally in the literature. Examples are the ability of transient β-adrenergic stimulation to precondition the rat heart against postischaemic contractile dysfunction [52]or the ability of epinephrine and amrinone, a phosphodiesterase inhibitor, to improve contractile function when administered together to patients without a history of heart failure who have underdone cardiopulmonary bypass [56]. The protective effect conferred by phosphorylation of SR membrane proteins, of which the major one appears to be PLN, may suggest new strategies for protecting the heart against oxidative damage.

Time for primary review 26 days.


   Acknowledgements
 
This study was supported by grant HL15764 from the National Institutes of Health. We thank Dr. I. Karpichev and M. Casey for assistance with the PhosphorImager analysis.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Negretti N, O'Neill S.C, Eisner D.A. The relative contributions of different intracellular and sarcolemmal systems to relaxation in rat ventricular myocytes. Cardiovasc Res (1993) 27:1826–1830.[Abstract/Free Full Text]
  2. Weiss S.J. Tissue destruction by neutrophils. N Engl J Med (1989) 320:365–376.[Web of Science][Medline]
  3. Kukreja R.C, Weaver A.B, Hess M.L. Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants. Biochim Biophys Acta (1989) 990:198–205.[Medline]
  4. Fukui K, Kaneda M, Takahashi E, Washio M, Doi K. Protective effects of sulfhydryl compounds on HOCl-induced intracellular Ca2+ increase in single rat ventricular myocytes. J Mol Cell Cardiol (1994) 26:455–461.[CrossRef][Web of Science][Medline]
  5. Eley D.W, Eley J.M, Korecky B, Fliss H. Impairment of cardiac contractility and sarcoplasmic reticulum Ca2+ ATPase activity by hypochlorous acid: reversal by dithiothreitol. Can J Physiol Pharmacol (1991) 69:1677–1685.[Web of Science][Medline]
  6. Mehta J, Dinerman J, Mehta P, Saldeen T.G, Lawson D, Donnelly W.H, Wallin R. Neutrophil function in ischaemic heart disease. Circulation (1989) 79:549–556.[Abstract/Free Full Text]
  7. De Servi S, Ricevuti G, Mazzone A, Pasotti D, Bramucci E, Angoli L, Specchia G. Transcardiac release of leukotriene C4 by neutrophils in patients with coronary artery disease. J Am Coll Cardiol (1991) 17:1125–1128.[Abstract]
  8. Dinerman J.L, Mehta J.L, Saldeen T.G, Emerson S, Wallin R, Davda R, Davidson A. Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction. J Am Coll Cardiol (1990) 15:1559–1563.[Abstract]
  9. Bell D, Jackson M, Nicoll J.J, Millar A, Dawes J, Muir A.L. Inflammatory response, neutrophil activation and free radical production after acute myocardial infarction: effect of thrombolytic treatment. Br Heart J (1990) 63:82–87.[Abstract/Free Full Text]
  10. De Servi A, Mazzone A, Ricevuti G, Fioravanti A, Bramucci E, Angoli L, Stefano G, Specchia G. Granulocyte activation after coronary angioplasty in humans. Circulation (1990) 82:140–146.[Abstract/Free Full Text]
  11. Semb A.G, Gabrielson T.O, Halstensen T.S, Fagerhol M.K, Brandtzaeg P, Vaage J. Cardiac surgery and distribution of the leukocyte L1 protein-calprotectin. Eur J Cardiothorac Surg (1991) 5:363–367.[Abstract]
  12. Semb A.G, Forsdahl K, Vaage J. Granulocyte and eicosanoid gradients across the coronary circulation during myocardial reperfusion in cardiac surgery. Eur J Cardiothorac Surg (1990) 81:201–211.
  13. Bolli R. Role of neutrophils in myocardial stunning after brief ischaemia: the end of a six year old controversy (1987–1993). Cardiovasc Res (1993) 27:728–729.[Web of Science][Medline]
  14. Curello S, Ceconi C, de Giuli F, Panzali A.F, Milanesi B, Calarco M, Pardini A, Marzollo P, Alfieri O, Messineo F, Ferrari R. Oxidative stress during reperfusion of human hearts: potential sources of oxgen free radicals. Cardiovasc Res (1995) 29:118–125.[Abstract/Free Full Text]
  15. Entman M.L, Youker K, Shappell S.B, Siegel C, Rothlein R, Dreyer W.J, Schmalstieg F.C, Smith C.W. Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism. J Clin Invest (1991) 85:1497–1506.[CrossRef][Web of Science]
  16. Kukreja RC, Hess ML, Free Radicals, Cardiovascular Dysfunction and Protection Strategies. Austin: RG Landes Company, 1993.
  17. Hacker T.A, Renstrom B, Paulson D, Liedtke A.J, Stanley W.C. Ischemia produces an increase in ammonia output in swine myocardium. Cardioscience (1994) 5:255–260.[Web of Science][Medline]
  18. Scherer N.M, Deamer D.W. Oxidative stress impairs the function of sarcoplasmic reticulum by oxidation of sulfhydryl groups in the Ca2+-ATPase. Arch Biochem Biophys (1986) 246:589–601.[CrossRef][Web of Science][Medline]
  19. Kawakita M, Yamashita T. Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. III. Identification of cysteine residues whose modification with N-ethylmaleimide leads to loss of the Ca2+-transporting activity. J Biochem (1987) 102:103–109.[Abstract/Free Full Text]
  20. Limas C.J. Effect of cyclic AMP-dependent phosphorylation on the reactivity of sulfhydryl groups in the cardiac sarcoplasmic reticulum. Biochem J (1980) 192:867–872.[Web of Science][Medline]
  21. Tada M. Molecular structure and function of phospholamban in regulating the calcium pump from sarcoplasmic reticulum. Ann NY Acad Sci (1992) 671:92–102.[Web of Science][Medline]
  22. MacLennan DG, Toyofuku T, Regulatory interactions between calcium ATPases and phospholamban. In: Clapham DE, Ehrlich BE (Eds.). Organellar Ion Channels and Transporters. New York: The Rockefeller University Press, 1996:89-103.
  23. Kirchberger M.A, Borchman D, Kasinathan C. Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump. Biochemistry (1986) 25:5484–5492.[CrossRef][Web of Science][Medline]
  24. Suzuki T, Wang J.H. Stimulation of bovine cardiac sarcoplasmic reticulum Ca2+ pump and blocking of phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody. J Biol Chem (1986) 261:7018–7023.[Abstract/Free Full Text]
  25. Xu Z.-C, Kirchberger M.A. Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban. J Biol Chem (1989) 264:16644–16651.[Abstract/Free Full Text]
  26. Antipenko A.Y, Spielman A.I, Kirchberger M.A. Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum: Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax(Ca) of calcium transport. J Biol Chem (1997) 272:2852–2860.[Abstract/Free Full Text]
  27. Luo W, Grupp I.L, Harrer J, Ponniah S, Grupp G, Duffy J.J, Doetschman T, Kranias E.G. Molecular structure and function of phospholamban in regulating the calcium pump from sarcoplasmic reticulum. Circ Res (1994) 75:401–409.[Abstract/Free Full Text]
  28. Kirchberger M.A, Tada M. Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac slow and fast contracting skeletal muscles. J Biol Chem (1976) 251:725–729.[Abstract/Free Full Text]
  29. Meissner G, Henderson J.S. Rapid calcium release from cardiac sarcoplasmic reticlum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem (1987) 262:3065–3073.[Abstract/Free Full Text]
  30. Kasinathan C, Xu Z.-C, Kirchberger M.A. Differences in calcium uptake in native canine cardiac microsomes are correlated with the ratio of unphosphorylated to phosphorylated phospholamban as determined by Western blot analysis. Biochem Biophys Res Commun (1988) 157:1296–1301.[CrossRef][Web of Science][Medline]
  31. Ellman G.L. Tissue sulfhydryl groups. Arch Biochem Biophys (1959) 82:70–77.[CrossRef][Web of Science][Medline]
  32. Aune T.M, Thomas E.L. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanite ion. Eur J Biochem (1977) 80:209–214.[CrossRef][Web of Science][Medline]
  33. Gaginella T.S, Grisham M.B, Thomas D.B, Walsh R, Moummi C. Oxidant-evoked release of acetylcholine from enteric neurons of the rat colon. J Pharmacol Exper Ther (1992) 263:1068–1073.[Abstract/Free Full Text]
  34. Thomas E.L, Grisham M.B, Jefferson M.M. Preparation and characterization of chloramines. Methods Enzymol (1986) 132:569–584.[Medline]
  35. Jorgensen A.O, Jones L.R. Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study. J Biol Chem (1986) 261:3775–3781.[Abstract/Free Full Text]
  36. Lu Y.-Z, Kirchberger M.A. Effect of a nonionic detergent on calcium uptake by cardiac microsomes. Biochemistry (1994) 33:5056–5062.[CrossRef][Web of Science][Medline]
  37. Fisher RA, Yates F, Table VII. Statistical Tables for Biological, Agricultural and Medical Research, New York: Hafner Publishing Co. 1963: 63.
  38. Kawakita M, Yasuoka K, Kaziro Y. Selective modification of functionally distinct sulfhydryl groups of sarcoplasmic reticulum Ca2+, Mg2+-adenosine triphosphatase with N-ethylmaleimide. J. Biochem. (Tokyo) (1980) 87:609–617.[Abstract/Free Full Text]
  39. Nakamura Y, Tonomura Y. Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+, Mg2+-dependent ATPase of sarcoplasmic reticulum. J. Biochem. (Tokyo) (1982) 91:449–461.[Abstract/Free Full Text]
  40. Stefanova H.I, Napier R.M, East J.M, Lee A.G. Effects of Mg2+, anions and cations on the Ca2+, Mg2+-activated ATPase of sarcoplasmic reticulum. Biochem J (1987) 245:723–730.[Web of Science][Medline]
  41. Gould G.W, East J.M, Froud R.J, McWhirter J.M, Stefanova H.I, Lee A.G. A kinetic model for the Ca2++Mg2+-activated ATPase of sarcoplasmic reticulum. Biochem J (1987) 245:723–730.[Web of Science][Medline]
  42. Toyofuku T, Kurzydlowski K, Tada M, MacLennan D.H. Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca2+-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban. J Biol Chem (1994) 269:22929–22932.[Abstract/Free Full Text]
  43. Scott T.L, Shamoo A.E. Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2+-Mg2+)-ATPase. J Membrane Biol (1982) 64:137–144.[CrossRef][Web of Science][Medline]
  44. Mortishire-Smith R.J, Pitzenberger S.M, Burke C.J, Middaugh C.R, Garsky V.M, Johnson R.G. Solution structure of the cytoplasmic domain of phospholamban: phosphorylation leads to a local perturbation in secondary structure. Biochemistry (1995) 34:7603–7613.[CrossRef][Web of Science][Medline]
  45. Yanagishita T, Matsuoka T, Kako K.J. Pharmocological intervention in oxidant-induced calcium pump dysfunction of dog heart. Biochem Internat (1989) 18:1111–1119.[Web of Science][Medline]
  46. Morris J.C. The acid ionization constant of HOCl from 5 to 35°. J Phys Chem (1966) 70:3798–3805.[CrossRef][Web of Science]
  47. Grisham M.B, Jefferson M.M, Thomas E.L. Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils. J Biol Chem (1984) 259:6766–6772.[Abstract/Free Full Text]
  48. Schraufstatter I.U, Browne K, Harris A, Hyslop P.A, Jackson J.H, Quehenberger O, Cochrane C.G. Mechanisms of hypochlorite injury of target cells. J Clin Invest (1990) 85:554–562.[Web of Science][Medline]
  49. MacFarlane N.G, Miller D.J. Effect of the reactive oxygen species hypochlorous acid and hydrogen peroxide on force production and calcium sensitivity of rat cardiac myofilaments. Eur J Physiol (1994) 428:561–568.[CrossRef][Web of Science][Medline]
  50. Entman M.L, Smith C.W. Postreperfusion inflammation: a model for reaction to injury in cardiovascular disease. Cardiovasc Res (1994) 28:1301–1311.[Free Full Text]
  51. Schömig A, Dart A.M, Dietz R, Mayer E, Kübler W. Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A. Locally mediated release. Circ Res (1984) 55:689–701.[Abstract/Free Full Text]
  52. Asimakis G.K, Inners-McBride K, Conti V.R, Yang C.-J. Transient β adrenergic stimulation can precondition the rat heart against postischaemic contractile dysfunction. Cardiovasc Res (1994) 28:1726–1734.[Abstract/Free Full Text]
  53. Parratt J. Endogenous myocardial protective (antiarrhythmic) substances. Cardiovasc Res (1993) 27:693–792.[Free Full Text]
  54. Sun J.-Z, Tan X.-L, Park S.-W, Qui Y, Turrens J.F, Bolli R. Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs. J Clin Invest (1996) 97:562–576.[Web of Science][Medline]
  55. Krause S.M, Jacobus W.E, Becker L.C. Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic "stunned" myocardium. Circ Res (1989) 65:526–530.[Abstract/Free Full Text]
  56. Royster R.L, Butterworth J.F IV, Prielipp R.C, Zaloga G.P, Lawless S.G, Spray B.J, Kon N.D, Wallenhaupt S.L, Cordell A.R. Combined inotropic effects of amrinone and epinephrine after cardiopulmonary bypass in humans. Anesth Analg (1993) 77:662–672.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Antipenko, A. Y
Right arrow Articles by Kirchberger, M. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Antipenko, A. Y
Right arrow Articles by Kirchberger, M. A
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?