Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle

Ashok K. Grover1,2, Sue E. Samson1, Sarah Robinson1, and Chiu Yin Kwan1

Departments of 1 Medicine and 2 Biology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxynitrite generated in arteries from superoxide and NO may damage Ca2+ pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca2+ into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca2+ uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca2+ pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca2+ uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca2+ pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.

sarco(endo)reticulum Ca2+ pump; adenosinetriphosphatase; free radicals; oxidative stress; vascular diseases; ischemia-reperfusion


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REACTIVE OXYGEN SPECIES (ROS) are necessary for cellular processes such as mitochondrial function and play a role in signal transduction. However, excess ROS may accumulate during ischemia-reperfusion and in vascular pathologies such as atherogenesis and inflammation (5, 7, 8, 10-12, 14, 25, 28). Excess ROS can produce vascular dysfunctions as a result of oxidation of lipids and cysteine residues in various proteins by peroxides, superoxide, and hydroxyl radicals. Of particular interest is the damage caused by ROS to transport processes as they disturb cellular homeostasis in blood vessels. For instance, ROS can severely damage the Ca2+-dependent K+ channels, although the voltage-sensitive Ca2+-channels in arteries are relatively resistant to ROS (8, 21). In pig coronary artery smooth muscle, peroxide and superoxide damage the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump, but the plasma membrane Ca2+ (PMCA) pump is relatively resistant (17, 20). Even different isoforms of SERCA pumps such as SERCA2 and SERCA3 differ in their susceptibility to damage by hydrogen peroxide (23). SERCA pump in endothelium is relatively resistant to peroxide because this tissue expresses SERCA3 and has a high level of catalase (16, 18).

Peroxynitrite is produced in the arteries from nitric oxide and superoxide generated by macrophages during injury or atherogenic inflammatory responses (10-12). Peroxynitrite may also be produced in the arteries during ischemia-reperfusion. In the isolated perfused rat heart, it induces both vasodilation and impaired vascular relaxation (8, 10, 11, 34). The effects of peroxynitrite on SERCA and PMCA pumps in vascular smooth muscle have not been reported, perhaps because it is presumed that they would act like the ROS peroxide and superoxide, which are known to damage these transporters. Peroxynitrite, however, can act by S-nitrosylation or tyrosine nitration, or it may decompose rapidly into superoxide and nitric oxide. In slow-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+ pump, peroxynitrite treatment produces a loss of the ATPase activity (8, 10, 11, 34, 35). Peroxynitrite has also been shown to cross-link proteins via a 3-3'-dityrosine linkage (8, 10, 11, 33-35). It is clear that peroxynitrite does not act like just another ROS. Therefore, we examined the effects of peroxynitrite pretreatment on SERCA and PMCA pumps in membranes isolated from the smooth muscle of pig coronary artery.


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Membrane isolation. Pig hearts were obtained from a slaughterhouse, and smooth muscle from the left anterior descending coronary artery was dissected and used for subcellular membrane fractionation as previously reported (22). The tissue used in the membrane preparation was predominantly smooth muscle devoid of endothelium, adventitia, erythrocytes, and cardiac myocytes (22). On the basis of the biochemical markers previously described, the gradient fraction F2 (band just above 28% sucrose) is enriched in PM, and gradient fraction F3 (band just above 40%) is rich in SR (22).

Peroxynitrite pretreatment. Aliquots of peroxynitrite (Calbiochem) under nitrogen were stored at -80°C and used within 3 mo. The peroxynitrite concentration was determined by its absorbance at 302 nm (extinction coefficient = 1,670 M-1 · cm-1) in 0.1 M NaOH immediately before use. Peroxynitrite was used as a solution in 0.1 M NaOH, and all the control samples also contained the same concentration of this base. Creatine kinase, which was used to regenerate ATP in the Ca2+ uptake assays, is inhibited by peroxynitrite and its decomposition product, hydrogen peroxide (30, 33). To avoid such interference from peroxynitrite in the subsequent assays, after incubation for 30 min at 37°C the peroxynitrite and any hydrogen peroxide produced from its decomposition were quenched by adding DTT to a final concentration of 1 mM and placing the samples on ice.

Ca2+ uptake. Ca2+ uptake was carried out using trace amounts of 45Ca in an EGTA-Ca2+ buffer system and an ATP-regenerating system as described earlier (13). The final reaction mixture consisted of creatine kinase (70 U/ml) and the following concentrations of the other components (in mM): 100 KCl, 5 Na-azide, 5 MgCl2, 5 ATP, 10 creatine phosphate, 1 EGTA, 0.85 CaCl2 (plus trace amounts of 45Ca), 30 imidazole-HCl (pH 6.8 at 37°C), 0.33 DTT, 0 or 5 K-oxalate (with F3 fraction), and 0 or 5 K-phosphate (with F2 fraction, pH 6.8 at 37°C). This resulted in 5 µM free [Ca2+] as described earlier (17). Typically, the reaction was carried out for 60 min, and then the reaction mixture was filtered through 0.45-µm nitrocellulose filters under suction and washed three times with ice-cold solution containing (in mM): 250 sucrose, 30 imidazole-HCl (pH 7.1), and 0.5 EGTA.

Catalase assays. Preincubation mixtures were prepared containing catalase (25 µg catalase) plus F3 as described in Peroxynitrite pretreatment except that DTT was not added after the preincubation. Aliquots (10 µl) of the preincubated samples were used in kinetic assays for catalase in a 1-ml reaction volume by following the decomposition of 10 mM hydrogen peroxide as previously described (16).

Western blots. Western blots were carried out typically in 9% acrylamide gels using the Laemmli gel system and were electroblotted to nitrocellulose (20). RPN800 Rainbow marker (Amersham Pharmacia Biotech) and laminin (subunits 200 and 400 kDa; GIBCO) were used as molecular weight markers (4). The primary antibodies used were 5F10 (mouse monoclonal anti-PMCA pump antibody; Affinity BioReagents, Golden, CO) and 4G5 (mouse monoclonal anti-SERCA pump antibody; gift from Dr. L. R. Jones, Univ. of Virginia). The bound primary antibody was detected by enhanced chemiluminescence using an Amersham kit.

Acylphosphate analysis. The acid-stable acylphosphates for SERCA2b (110 kDa) and PMCA (140 kDa) pumps can be formed from ATP in the presence of Ca2+ or from inorganic orthophosphate (Pi) in the presence of EGTA (17, 20, 24). For the acylphosphate formation of SERCA pump from ATP, the F3 membranes were pretreated with peroxynitrite and then the peroxynitrite was quenched on ice with a solution containing DTT. The acylphosphate formation from ATP was carried out at 0°C for 60 s in a reaction mixture (100 µl) containing (in mM) 100 KCl, 30 imidazole-HCl (pH 6.8), 50 CaCl2, 0.8 DTT, and 5 µM ATP (plus trace amounts of [gamma -32P]ATP). The samples were precipitated and used for acid gel electrophoresis as described previously (20). The gels were dried and analyzed using a PhosphorImager. When the F2 membranes were used to examine the PMCA pump, 500 µM LaCl3 was also included in the reaction mixture to stabilize the PMCA acylphosphates. Acylphosphate formation from Pi was carried out as described previously (24). Briefly, membranes were treated with peroxynitrite followed by DTT and then placed on ice with dimethyl sulfoxide (40%, vol/vol). After 10 min on ice, the tubes were placed at 22-24°C, and a Pi solution was added so that final concentrations of the various components were (in mM) 50 morpholinopropane sulfonate-KOH (pH 6.8), 10 EGTA, 5 MgCl2, 0.8 DTT, and 0.05 dipotassium phosphate (plus trace amounts of [32P]orthophosphoric acid). The samples were mixed and incubated at 22-24°C for 10 min and then processed for acid gel electrophoresis.

Data analysis. Curve fitting was carried out by nonlinear regression using the FigP software from BioSoft (Ancaster, Canada). Statistical significance was determined using Student's t-test. All experiments were replicated three to five times.


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Comparison of effects of peroxynitrite on PM- and SR-enriched membrane fractions. Two membrane fractions were used in the initial work, F2 and F3. Previous characterization of these fractions using biochemical markers shows that F2 is enriched in PM and F3 in SR (22). We used PMCA- and SERCA-selective antibodies in Western blots to confirm that F2 is more enriched in PMCA and F3 in SERCA (not shown). Figure 1 shows the ATP-dependent azide-insensitive (nonmitochondrial) Ca2+ uptake by F2 and F3 fractions. Because both phosphate and oxalate prevent back flux of Ca2+ by promoting its crystallization inside the vesicles but oxalate works preferentially in the SR membranes, we examined the uptake in the presence of 5 mM phosphate with F2 and 5 mM oxalate with F3. Figure 1 shows that the uptake was linear with time for up to 120 min for F2 and F3 in control and peroxynitrite (200 µM)-pretreated membranes. The pretreatment with 200 µM peroxynitrite decreased the uptake by 75 ± 2% (from 13.4 ± 0.5 to 3.3 ± 0.3 nmol · mg protein-1 · min-1) in the F3 fraction but by only 30 ± 5% (from 2.0 ± 0.1 to 1.4 ± 0.1 nmol · mg protein-1 · min-1) in F2. An uptake time of 60 min was used in all subsequent experiments even though the uptake was linear up to 120 min.


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Fig. 1.   Time course of Ca2+ uptake by F2 [plasma membrane (PM) enriched] and F3 [sarcoplasmic reticulum (SR) enriched] fractions pretreated with 200 µM peroxynitrite. Each value is the means ± SE of 6 replicates. A: with the F2 fraction, the slope of the graph was 2.0 ± 0.1 nmol Ca2+ uptake · min-1 · mg protein-1 for the control and 1.4 ± 0.1 nmol Ca2+ uptake · min-1 · mg protein-1 for the peroxynitrite-treated membranes. The two slopes differ significantly from each other (P < 0.05). With or without the treatment, the Ca2+ uptake was linear over the 120-min period (r2 = 0.9785 and 0.9867, respectively). B: with the F3 fraction, the slope of the graph was 13.4 ± 0.3 nmol Ca2+ uptake · min-1 · mg protein-1 for the control and 3.3 ± 0.3 nmol Ca2+ uptake · min-1 · mg protein-1 for the peroxynitrite-treated membranes. The two slopes differ significantly from each other (P < 0.05). With or without the treatment, the Ca2+ uptake was linear over the 120-min period (r2 = 0.9951 and 0.8824, respectively).

Figure 2A shows a summary of five experiments on the effects of different concentrations of peroxynitrite on the Ca2+ uptake by F2 and F3 fractions. The inhibition was more severe for F3 than for F2. The best fit for the inhibition data for F3 gave an inhibition constant (Ki) of 56 ± 4 µM and a Hill coefficient of 1.49 ± 0.15. For F2, the data fit best with a model in which F2 contains some SR as an impurity, and the inhibition observed in this fraction stems solely from this impurity, i.e., has the same Ki and Hill coefficient values but only a small fraction of the uptake can be inhibited by peroxynitrite (Fig. 2A). To confirm this finding, we inhibited the Ca2+ uptake due to the SERCA pump with 1 µM thapsigargin. This concentration of thapsigargin inhibited the Ca2+ uptake in the F3 fraction by 93%. Using 10 µM thapsigargin does not produce significantly greater inhibition (P > 0.05). In the F2 fraction, however, the thapsigargin inhibition is not as pronounced (Fig. 2B). Thapsigargin inhibits by ~60% with the thapsigargin-insensitive component being ~40%. When the samples are pretreated with 250 µM peroxynitrite as in Fig. 2A, the peroxynitrite inhibits most of the thapsigargin-sensitive uptake but only 20-25% of the thapsigargin-insensitive uptake (Fig. 2C). These results confirm that the SERCA pump is significantly more susceptible to damage by peroxynitrite than the PMCA pump.


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Fig. 2.   Ca2+ uptake by membranes pretreated with different concentrations of peroxynitrite. A: F2 and F3 fractions were pretreated with different concentrations of peroxynitrite. Data are means ± SE of 5 experiments on different days, each with a separate membrane preparation. In each experiment, the mean value of the Ca2+ uptake in samples treated without peroxynitrite was taken as 100%, and all other values for F2 were compared with this value. Similarly, the mean value of the Ca2+ uptake by the F3 fraction treated without peroxynitrite was taken as 100%, and all other values for F3 were compared with this value. Data for the F3 fraction fit best with a Ki of 56 ± 4 µM and a Hill coefficient of 1.49 ± 0.15. Fitting the data for F2 using the same Hill coefficient and Ki as for F3 gave values of 0.9731 for r2 and 33 ± 2% for maximum inhibition by peroxynitrite. These data did not fit well when maximum inhibition was fixed to 100% and Ki and the Hill coefficient were varied. B: effect of 1 µM thapsigargin on Ca2+ uptake in the F2 fraction. C: effect of pretreatment with 250 µM peroxynitrite on thapsigargin-sensitive and -insensitive Ca2+ uptake in the F2 fraction.

The Ca2+-dependent formation of acylphosphates from ATP is the first step in the reaction cycle of both the PMCA and SERCA pumps, but the acylphosphates formed from the PMCA are 140 kDa compared with 110 kDa formed from the SERCA pump. Figure 3 shows that the F2 fraction gave acylphosphates for both sizes, confirming that it also contains the SERCA pump. Pretreatment of F2 with increasing concentrations of peroxynitrite inhibited mainly the 110-kDa acylphosphate band with very little effect on the 140-kDa band. Thus, at these concentrations, peroxynitrite inhibited mainly the SERCA pump. Hence, all subsequent experiments were conducted on the F3 fraction.


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Fig. 3.   Effect of peroxynitrite on acylphosphate formation from ATP in the F2 fraction. A: phosphorimage showing bands for plasma membrane Ca2+ pump (PMCA; 140 kDa) and sarco(endo)plasmic reticulum Ca2+ pump (SERCA; 110 kDa) acylphosphate. B: relative intensities of the bands.

Other ROS as possible intermediates. Peroxynitrite at neutral pH can spontaneously decompose to produce superoxide, catalase, and low concentrations of hydroxyl radicals (15, 31). Therefore, we determined whether the inhibition of Ca2+ uptake in F3 by peroxynitrite was due to the generation of hydrogen peroxide, superoxide, or hydroxyl radicals formed during the reaction. We have shown previously that catalase plus SOD can overcome the effect of superoxide and that catalase alone can abolish the effect of hydrogen peroxide in this system (17, 20). Including excess catalase plus SOD with or without mannitol (to quench hydroxyl radicals) does not prevent the inhibition by peroxynitrite (Fig. 4, P > 0.05). There remained the possibility that the pretreatment with peroxynitrite could inactivate catalase, thus giving artifactual results. Therefore, we also tested catalase activities in identical experiments. To minimize the carryover of any decomposition products into the assay solutions, 10 µl of the samples were diluted 100-fold during the assay. Theoretically, the maximum amount of peroxide produced from 500 µM peroxynitrite during the preincubation would thus be diluted to 5 µM in the final assay. Thus the carryover amount would be negligible compared with the 10 mM hydrogen peroxide during the assays. Table 1 shows that preincubating catalase with peroxynitrite does not alter its activity.


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Fig. 4.   Catalase, SOD, and mannitol controls. The F3 fraction was incubated in the presence of 100 µM peroxynitrite plus excess SOD (38 units), catalase (22 units), and mannitol (50 mM) as shown. Inclusion of catalase plus SOD with or without mannitol did not prevent the inhibition due to peroxynitrite (P > 0.05).


                              
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Table 1.   Catalase activity after preincubation with peroxynitrite

Ca2+ uptake, acylphosphates, and protein oligomerization. The Ca2+ pump cycles between two conformations: E1 and E2 (26). E1 binds Ca2+ with high affinity, and in the presence of Ca2+ it can form an acid-stable acylphosphate intermediate from ATP (forward reaction). In contrast, E2 has a low affinity for Ca2+ and forms acylphosphate from Pi in the absence of Ca2+ (reverse reaction). We treated the F3 fraction with different concentrations of peroxynitrite and compared the Ca2+ uptake and the formation of acylphosphates in both the reactions. Acylphosphates formed from ATP gave a major band at 105-110 kDa. Peroxynitrite pretreatment inhibited the formation of acylphosphates from ATP (Fig. 5A). The inhibition of the uptake and acylphosphate formation correlated well with a slope that did not differ significantly from 1 (P > 0.05, Fig. 6D). The presence of very high activities of other interfering ATPases prevents direct assays of Ca2+-Mg2+-ATPase in this tissue. Hence, this relationship is important in that it demonstrates a correspondence between the inhibition of the Ca2+ uptake and that of the SERCA2 Ca2+-Mg2+-ATPase rather than an inability of the membrane vesicles to retain Ca2+ due to an increased leakiness. Acylphosphates formed from Pi gave a major band at 105-110 kDa (reverse reaction, Fig. 5B). Peroxynitrite treatment also inhibited the formation of acylphosphates from Pi. However, the inhibition was poorer than that of the Ca2+ uptake (Fig. 5E). Further analysis also showed that the inhibition of the acylphosphates from Pi was poorer than that from ATP (P < 0.05).


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Fig. 5.   Correlation between peroxynitrite treatment inhibition of Ca2+ uptake, acylphosphate formation, and protein aggregation in the F3 fraction. A: phosphorimage showing acylphosphate formation from ATP. B: phosphorimage showing acylphosphate formation from Pi. C: autoradiogram of Western blot using anti-SERCA pump antibody 4G5. Numbers below lanes are peroxynitrite concentration (µM). Lines at left of gel are positions of molecular mass markers (kDa). D: correlation between acylphosphate formation from ATP with the Ca2+ uptake from experiments on 2 membrane preparations in gels such as that shown in A. Data were fit by linear regression (r2 = 0.9291) with a fixed intercept of 0. A slope of 0.94 ± 0.05 was obtained. The expected line has a slope of 1. The slope obtained did not differ significantly from 1 (P > 0.05). E: correlation between acylphosphate formation from Pi with the Ca2+ uptake from 2 experiments from gel shown in B. Data were fit by linear regression (r2 = 0.8156) with a fixed intercept of 0. The slope (0.62 ± 0.05) of the line differed significantly from 1 (P < 0.05). F: correlation between protein oligomerization from the gel shown in C with the Ca2+ uptake. Data were fit by linear regression (r2 = 0.8057) with a fixed intercept of 0. The slope of the line (0.67 ± 0.09) differed significantly from 1 (P < 0.05).



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Fig. 6.   Hydrogen peroxide (500 µM) treatment induced inhibition of Ca2+ uptake (A), acylphosphate formation from ATP (B) or Pi (C), and protein aggregation (D) in the F3 fraction. 1, Incubation without hydrogen peroxide but with 1 mM DTT added after the incubation; 2, incubation with hydrogen peroxide and DTT added after the incubation; 3, incubation with hydrogen peroxide and no DTT added after the incubation. Values are means ± SE of 3 replicates for the Ca2+ uptake. Two replicates were used for each gel. Percent inhibition of the acylphosphate formation or the percent oligomers formed did not differ significantly from that of the Ca2+ uptake (P > 0.05). Adding DTT after the incubation with peroxide did not reverse the inhibition of the uptake, acylphosphate formation, or the oligomerization (P > 0.05).

We also examined the effects of peroxynitrite treatment on the Ca2+ pump protein in Western blots by using a SERCA2-specific monoclonal antibody, 4G5. Samples treated without peroxynitrite gave a major band at 105-110 kDa as expected (Fig. 5C). However, the treatment with peroxynitrite gave an additional very high molecular mass band (Fig. 5C). The expected size of the dimer would be 210-220 kDa. However, the oligomers moved considerably slower than the 250-kDa molecular mass marker, indicating their size to be much larger than dimers. We analyzed the cross-linked samples in 6% acrylamide gels, running the gels for 4 h even though the dye band had left the gels at 2.5 h and using laminin (200 and 400 kDa) as an additional marker. The oligomer had a mobility similar to that of the 400-kDa laminin subunit. The intensity of the oligomers increased with the increasing peroxynitrite concentration (Fig. 5C). Figure 5C also shows that no dimers (220 kDa) were observed even at low concentrations of peroxynitrite when only a small fraction of the SERCA2 protein had formed the oligomers. For each lane we computed the reactivity of the oligomers with the antibody as a percentage of the total reactivity with oligomers plus the monomer (Fig. 5F). This value increased with the inhibition of the Ca2+ uptake observed in these membranes, but its slope was significantly less than 1 (P < 0.05). The slope of the percent oligomers formed was also significantly different from the acylphosphates formed from ATP but not those formed from Pi.

Effect of hydrogen peroxide. We have reported previously that hydrogen peroxide treatment inactivates the SERCA pump in the coronary artery smooth muscle (18, 20). However, it is not known if DTT can reverse the effect of this treatment, how this treatment affects the formation of acylphosphates from Pi, and whether it causes protein aggregation. Therefore, we repeated the experiment as in Fig. 5 but used only one concentration (500 µM) of hydrogen peroxide. Sodium azide was included in the reaction mixture to prevent the breakdown of peroxide by intrinsic catalase or myeloperoxidase. Peroxide treatment produced an inhibition of the uptake and 1 mM DTT added after incubation with peroxide did not reverse this effect (Fig. 6A). There was also a parallel inhibition of the acylphosphate formation from ATP (Fig. 6B) and inhibition of the acylphosphate did not appear to lag behind significantly (Fig. 6C). The treatment also produced an aggregation of the pump protein. The aggregates had the same pattern and size as those produced on the peroxynitrite treatment. Percent protein in the aggregate did not appear to lag behind the inhibition of the uptake (Fig. 6D).


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The results show that 1) peroxynitrite treatment inhibits the SERCA pump preferentially over the PMCA pump; 2) this effect is not prevented by including catalase, SOD, and mannitol with the peroxynitrite; 3) inhibition of the Ca2+ uptake parallels that of the acylphosphate formation from ATP; 4) inhibition of the acylphosphate formation from Pi is slightly poorer; 5) the treatment leads to formation of oligomers with a molecular weight significantly higher than that of a dimer; 6) formation of the oligomers is slightly poorer than the inhibition of the Ca2+ uptake; 7) treatment with hydrogen peroxide also leads to inhibitions of Ca2+ uptake and the acylphosphate formation, and it gives oligomers of the same molecular weight as the peroxynitrite pretreatment; and 8) these effects of peroxynitrite or hydrogen peroxide cannot be reversed with excess DTT. Below, we focus on the methods used in this study, their possible implications on the pump structure and function, and the biological implications of these findings.

Because pure PM or SR membranes cannot be obtained from coronary artery smooth muscle, we used the effect of oxalate on the inhibition of back flux of Ca2+ to identify the activity in the SR, the acylphosphates from ATP to distinguish between the effects on the PM (140 kDa) and SR (110 kDa), and antibodies to verify these results. We also confirmed, using thapsigargin in the F2 fraction, that SERCA pump is more sensitive to peroxynitrite than is the PMCA pump. The membrane fractions used contain high levels of interfering ATPases and much smaller amounts of the SERCA pump protein compared with tissues such as cardiac and skeletal muscle. Therefore, direct assays of Ca2+-Mg2+-ATPases are not possible. Hence, we have verified our results at the levels of acylphosphate formation in forward and reverse directions to establish that the observed effects of peroxynitrite are on the pump rather than on membrane leakiness. Furthermore, in experiments using catalase, SOD, and mannitol we show that the effect of peroxynitrite on the pump does not involve peroxide, superoxide, or hydroxyl radicals. Also, these effects are not reversed by excess DTT, suggesting that the reaction goes beyond simple disulfide bond formation or S-nitrosylation. It is attractive to think of a tyrosine nitration pathway, because Y294, Y295, and Y793 may be nitrated in SERCA2a by peroxynitrite (36). More recently, it has been shown that tyrosine nitration has also been demonstrated in the SERCA pump in aortic smooth muscle (1). However, hydrogen peroxide also produced inhibition of the pump that was not reversed by DTT, and it produced oligomers similar to those produced by peroxynitrite. Peroxynitrite may produce protein cross-linking via dityrosine (32), but dityrosine is also formed upon free radical treatment of SERCA1 (32, 37). Thus reactions with hydrogen peroxide and peroxynitrite may occur via different pathways but produce similar end results.

Correlation of parameters monitored. The peroxynitrite treatment of the coronary artery F3 fraction produced inhibition of the SERCA pump, acylphosphate formation from ATP and Pi, and also caused oligomerization of the pump protein. However, the inhibition of the acylphosphate formation from Pi was slightly poorer. There are several possible explanations. The acylphosphate formation from ATP would be inhibited when any one of the following are inhibited: high-affinity Ca2+ binding site, ATP binding site, or the acylphosphate formation site. In contrast, the inhibition of the acylphosphate from Pi would require only one site to be damaged. The reaction with Pi was carried out in the presence of 40% DMSO, which may have solubilized the lipid annulus of this protein. The treatment may also confer a preference of E2 conformation over the other. Thapsigargin and melittinin produce such conformational preferences (3, 39). Aggregation of the pump protein also accompanied the melittinin inhibition, but the size of individual oligomer species was not determined (38). Peroxynitrite treatment of SERCA2a in slow-twitch muscle followed by Western blots also shows the loss of monomeric species (36).

Quaternary structure of the SERCA pump. Current models on interactions between Ca2+ pump subunits are based on skeletal muscle (2, 26). SERCA1, when in the membrane, has been proposed to be a dimer, although in the presence of detergents monomers it may be fully functional (2, 26). Radiation inactivation of SERCA1 in fast-twitch skeletal muscle and of SERCA2a in heart give target sizes consistent with a dimeric structure (2, 9). Cross-linking of SERCA1 also gives mainly dimers but also trimers and tetramers (6). Figure 5 clearly shows that no SERCA2 dimers are formed even upon treatment of the coronary artery membranes at low peroxynitrite concentrations that cause oligomerization of only a fraction of this protein. Only higher molecular weight oligomers are formed, and they appear to be tetramers. This result suggests that the organization of the Ca2+ pump protein in the coronary artery SR differs from that in the skeletal and cardiac SR. In radiation inactivation experiments, the target size of the SERCA pump protein in the coronary artery is much larger than in the cardiac muscle (19). Therefore, we speculate that the SERCA pump in the coronary artery may be present as a much larger oligomeric complex than in the skeletal or cardiac muscle, but it is not known whether it contains many subunits of this protein or also regulatory proteins.

Physiological implications. The effects of peroxynitrite may be important in aging, inflammation, and atherosclerosis. The SERCA pump activity in rat heart decreases with aging. SERCA2a in rat twitch skeletal muscle shows that nitrated tyrosine residues increase with aging (35, 36). Such data are not currently available for the coronary artery. Protein aggregates formed at high concentrations of peroxynitrite may represent irreversible tissue damage, and hence it should be investigated whether a "denitrase" activity may prevent the loss at an earlier stage (27). The sensitivity of the SERCA pump to peroxynitrite and other ROS in a cell may depend on their levels of antioxidants (such as glutathione and ascorbate), the type of ROS degrading enzymes, and the SERCA isoforms they express. The SERCA pump in endothelium is relatively insensitive to peroxide because endothelium is enriched in catalase and also expresses SERCA3 gene in addition to the SERCA2 gene expressed in the coronary artery smooth muscle (16, 18, 29). The tyrosine residues (Y294 and Y295) shown to be nitrated in SERCA2a are flanked by similar sequences in SERCA2 (GAIYYFKIAV) and SERCA3 (GAVYYFKIAV) (GenBank accession nos. X58291 and AF068220). Similarly, the tyrosine residues (Y753) shown to be nitrosylated in SERCA2a are flanked by similar sequences in SERCA2 (AIYNNMKQFI) and SERCA3 (AIYSNMKQFI). In HEK-293 T cells overexpressing the pump proteins, SERCA2 protein is more sensitive to peroxide than is SERCA3 (23). It would be of interest to determine whether the difference in susceptibility between SERCA2 and SERCA3 also holds for peroxynitrite, particularly when exposure to this agent is extremely likely for endothelium. Thus the present study has resolved some issues on the effects of peroxynitrite on the coronary artery SERCA pump and raised several other questions of biological and mechanistic importance.


    ACKNOWLEDGEMENTS

This work was funded by Heart & Stroke Foundation of Ontario Grant-in-Aid T-4815. A. K. Grover received a Career Investigator Award from the Heart & Stroke Foundation of Ontario.


    FOOTNOTES

Address for reprint requests and other correspondence: A. K. Grover, Dept. of Medicine, HSC 4N41, McMaster Univ., 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: groverak{at}mcmaster.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 6, 2002;10.1152/ajpcell.00297.2002

Received 26 June 2002; accepted in final form 6 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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