Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N3Z5
Submitted 11 July 2003 ; accepted in final form 10 August 2003
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ABSTRACT |
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free radicals; Ca2+-Mg2+-ATPase; ischemia; coronary artery; vascular smooth muscle; sarco(endo)plasmic reticulum Ca2+ pumps
Reactive oxygen species (ROS) are needed for cellular processes such as mitochondrial function and cell signaling (6, 17, 26, 41). However, excess ROS may accumulate in ischemia reperfusion and in vascular pathologies such as atherogenesis and inflammation (1, 6, 26, 41). They can produce vascular dysfunctions by causing oxidation of lipids and various proteins by peroxides, superoxide, and hydroxyl radicals. In pig coronary artery smooth muscle cells, peroxide and superoxide damage the SERCA2b pump, but the plasma membrane Ca2+ pump (PMCA) is relatively resistant (2, 19, 21, 22, 36). Isoforms of SERCA pumps may also differ in their susceptibility to damage by hydrogen peroxide and superoxide (5, 23). The SERCA pump in endothelium is relatively resistant to peroxide in part because this tissue expresses SERCA3a and has a high level of catalase (18, 20). Incubation in the presence of sodium azide (catalase inhibitor), of isolated microsomes prepared from SERCA2b, and of SERCA3a overexpressing cells has shown that the SERCA3a pump is more resistant to hydrogen peroxide than that of SERCA2b.
Peroxynitrite is produced in blood vessels from nitric oxide, and superoxide is generated by macrophages during injury, atherogenic inflammatory responses, or during ischemia reperfusion (1, 12, 16, 18, 20). In the isolated perfused rat heart, peroxynitrite induces both vasodilation and impaired vascular contraction (8, 12, 39). Peroxynitrite differs from peroxide and superoxide in that it can act directly on proteins by S-nitrosation or tyrosine nitration rather than by simply oxidizing cysteine residues in proteins. In cardiac muscle, slow-twitch skeletal muscle, and pig coronary artery smooth muscle, the peroxynitrite treatment produces a loss of the SERCA pump ATPase activity (24, 39, 40, 42). All of these tissues express SERCA2 gene products. Peroxynitrite has also been shown to cross link proteins via a 3-3'-dityrosine linkage (35, 40). The cross linking may also lead to formation of oligomers. Here, we overexpress the SERCA2b and SERCA3a proteins in HEK-293T cells and examine the effects of incubating microsomes with peroxynitrite on different aspects of SERCA activities and oligomer formation.
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EXPERIMENTAL PROCEDURES |
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Ca2+ uptake. Ca2+ uptake, using trace amounts of 45Ca, was carried out in an ethylene glycol-bis(-aminoethyl ether)-N,N,N',N' tetraacetatic acid (EGTA)-Ca2+ buffer system and an ATP-regenerating system as described earlier (23). The final reaction mixture consisted of creatine kinase (70 units/ml) and the following concentrations (in mM) of the other components: 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, and 0 or 5 K-oxalate. Typically, the reaction was carried out for 2 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.
Ca2+-Mg2+-ATPase. ATPase assays were carried out by following the hydrolysis of the terminal phosphate from [-33P]ATP by a modification of a published method (45). The microsomes were preincubated with peroxynitrite for 10 min at 37°C, followed by the addition of DTT as described above, and then placed at 0°C until used. The microsomal suspension (50 µl) was added to a tube on ice, and then 50 µl of a chilled reaction solution were added. The reaction mixture contained (in mM) 5 Na-azide, 1 MgCl2, 1 ATP (plus trace amounts of 33P-
-ATP), 0.85 EGTA, 1 CaCl2, 30 imidazole-HCl (pH 6.8 at 37°C), 0.33 DTT, 10-20 µg microsomal protein, and 10 µM Ca2+ ionophore A23187
[GenBank]
. After an incubation for 10 min at 37°C, the reaction was stopped by placing the samples on ice and adding 100 µl of ice-cold TCA-ATP (10% trichloroacetic acid and 1 mM ATP). The samples were mixed using a vortex mixer, and then 200 µl of an ammonium molybdate solution (1% ammonium molybdate and 0.25 M sulfuric acid) were added and the samples were mixed again. Finally, 1 ml of butyl acetate was added and the samples were mixed and then centrifuged at 14,000 rpm for 10 min. Butyl acetate (200 µl) was taken for scintillation counting. Cyclopiazonic acid (CPA) (30 µM) was included in some tubes to inhibit the Ca2+-Mg2+-ATPase activity due to the SERCA pumps. The CPA-sensitive component was used as the Ca2+-Mg2+-ATPase activity of the SERCA pumps.
Acylphosphates. The Ca2+-dependent formation of acylphosphate levels was examined by using microsomes that had been preincubated as described above for the other assays by a method described previously (23). Typically, 160 µl of the microsomal suspension were added to 20 µl of a buffer solution and incubated over ice water for 10 min, and then an equal volume of radioactive ATP was added such that the samples contained 100 mM KCl, 30 mM imidazole-HCl, pH 6.8, 50 µM CaCl2, 5 µM unlabeled ATP, 20 µCi of [-33P]ATP, and 50-100 µg of the microsomal protein. The reactions started by adding ATP were stopped after 60 s with 250 µl of ice-cold TCAP (10% trichloroacetic acid, 50 mM phosphoric acid, and 1 mM ATP). The samples were vortexed, placed on ice for 5 min, and centrifuged at 4°C. The pellets were washed two times each with 1,500 µl TCAP and then suspended in 70 µl of freshly prepared sample buffer containing 10 mM MOPS-Na pH 5.5, 1 mM EDTA, 3% SDS, 10% sucrose, 20 mM dithiothreitol, and 0.1% of the tracking dye methylene green. The samples were vortexed vigorously for 15-20 min and then electrophoresed immediately in 7.5% polyacrylamide gels at pH 4. The gels were dried and autoradiographed at room temperature, and the optical densities of the acylphosphate bands were monitored by using a PhosphorImager.
Western blots. Electrophoresis of microsomal proteins was carried out as described previously. Two primary antibodies were used for the detection of SERCA pump proteins: 4G5, which was a mouse monoclonal anti-SERCA pump antibody (a gift from Dr. L. R. Jones, Krannert Institute of Cardiology, Indianapolis, IN), and a rabbit anti-SERCA3a serum (a gift from Dr. F. Wuytack, KUL, Leuven, Belgium) (28, 44). The cardiac SR Ca2+-Mg2+-ATPase binding to the anti-SERCA2 antibody 4G5 was used as a molecular weight standard. Under the experimental conditions used in these experiments, the SERCA2b and SERCA3a migration in the gels was similar to that of the cardiac SR Ca2+-Mg2+-ATPase. The primary antibody binding was visualized by enhanced chemiluminescence by using an Amersham kit following the instructions of the manufacturer.
Data analysis. Values comparing the peroxide-treated microsomes to the control values given here are means ± SE of specified number of replicates. The curve fitting was carried out by using FigP software (Biosoft, Ancaster, ON, Canada). Null hypotheses were tested using a Student's t-test, and P values <0.05 were considered to be statistically significant. Each experiment was replicated three to five times.
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RESULTS |
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Effect of peroxynitrite on Ca2+ uptake. The ATP-dependent uptake was examined in the presence of Na-azide to inhibit uptake into the mitochondria. Ideally, one would determine an influx Ca2+ into the vesicles as a measure of this uptake. However, due to practical considerations, one simply blocks the efflux of Ca2 by using oxalate, which diminishes backflux of Ca2+ by precipitating it inside the vesicles. Preliminary experiments for which (data not shown) demonstrated that 1) Ca2+ uptake was very low in the absence of ATP or oxalate, 2) Ca2+ uptake and the expression of SERCA proteins was very low in untransfected cells and increased in the transfected cells, 3) in transfected cells the uptake was inhibited nearly completely by the SERCA pump inhibitor cyclopiazonic acid, and 4) incubation with peroxynitrite for periods >10 min did not result in any further inhibition of the uptake.
Figure 1A shows the time course of the 45Ca2+ uptake by microsomes from HEK-293T cells overexpressing SERCA2b and from mock transfected cells. The microsomes from the transfected cells were pretreated with 0 or 50 µM peroxynitrite for 10 min. As expected, the uptake was much greater in the microsomes from the transfected cells than in those from the untransfected cells. Treatment with peroxynitrite decreased the 45Ca2+-uptake. Similarly, Fig. 1B shows the time course of the 45Ca2+ uptake by microsomes from HEK-293T cells overexpressing SERCA3a. The uptake was much greater in the microsomes from the transfected cells than in those from the untransfected cells. Treatment with 50 µM peroxynitrite decreased the 45Ca2+ uptake, but the inhibition for SERCA3a was less than that for the SERCA2b in Fig. 1A. In Fig. 1, the 45Ca2+ uptake was approximately linear with time for 2 min. Therefore, in the uptake over 2 min was examined in the subsequent experiments. Figure 2 shows the inhibition of the 45Ca2+ uptake from microsomes preincubated with different concentrations of peroxynitrite. The IC50 values (concentration for 50% inhibition), computed for each curve in several experiments as the one shown in Fig. 2, were significantly (P < 0.05) smaller for SERCA2b (57 ± 13 µM, means ± SE of 5 experiments) than that for SERCA3a (233 ± 44 µM, means ± SE of 5 experiments). SERCA2b expression was slightly higher than SERCA3a in some transfection experiments and lower in the others. However, the IC50 for SERCA3a for peroxynitrite was higher than that for SERCA2b in every experiment. Thus SERCA2b pump was more sensitive to damage by peroxynitrite than the SERCA3a pump.
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Effect of peroxynitrite on Ca2+-Mg2+ ATPase. Peroxynitrite treatment of the microsomes inhibited the CPA-sensitive Ca2+-Mg2+-ATPase activity in a concentration-dependent manner (Fig. 3). The IC50 value for SERCA2b (53 ± 6 µM) for the ATPase inhibition compared well with the similar value (57 ± 8 µM) obtained for inhibition of the Ca2+ uptake. Similarly, the IC50 value for SERCA3a (152 ± 29 µM) for the ATPase inhibition compared well with the similar value (233 ± 44 µM) obtained for inhibition of the Ca2+ uptake. These data suggest that the loss of Ca2+ uptake was mainly due to the inhibition of the pump rather than an increase in the membrane leakiness.
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Effect of peroxynitrite on acylphosphate formation. In the presence of Ca2+, SERCA proteins form an acid-stable acylphosphate with an aspartyl residue in their active site from the terminal phosphate group of ATP. The formation of this high-energy acylphosphate is the first reaction of the pump. Subsequent steps include hydrolysis of this intermediate in the presence of Mg2+ and translocation of Ca2+ across the membrane. We have shown previously that the formation of this acylphosphate band is Ca2+ dependent, i. e, it does not occur in the presence of excess EGTA (21). Figure 4 shows the effects of peroxynitrite on the formation of the acylphosphate from [-33P]ATP. Microsomes from cells overexpressing SERCA2b and SERCA3a showed the primary acylphosphate bands at 110 kDa. SERCA2b showed the monomeric band (band 1, 110 kDa) and another fainter slower moving band (band 2 in Fig. 4A), possibly a dimer. Treatment with peroxynitrite decreased the intensity of band 1 in a concentration-dependent manner, whereas it somewhat increased the intensity of band 2 and resulted in the formation of band 3, which migrated even slower than band 2 (Fig. 4B). By using microsomes treated with increasing concentrations of peroxynitrite, the intensity of band 1 correlated extremely well with the corresponding changes in Ca2+ uptake (Fig. 4C). The slope between the total acylphosphate formed (bands 1-3) vs. Ca2+ uptake values was less steep (0.53 ± 0.17 µM, r2 = 0.84) than with the acylphosphate formed in band 1 vs. the Ca2+ uptake (0.97 ± 0.07 µM, r2 = 0.98). Thus the Ca2+ uptake values were related mainly to the acylphosphate formation of the monomeric protein, indicating that the acylphosphate formed from the aggregates may not contribute to the Ca2+ transport. In contrast, the treatment of SERCA3a-overexpressing microsomes with different concentrations of peroxynitrite did not show band 2 formation, but it may have produced very faint bands corresponding to band 3 (Fig. 4, D and E). The acylphosphate formation of the monomeric band correlated very well (slope = 0.94 ± 0.03 µM, r2 = 0.99, Fig. 4F) with the Ca2+ uptake. Thus, for both SERCA2b and SERCA3a, the monomeric acylphosphate band formation correlated with Ca2+ uptake and SERCA2b gave additional higher molecular weight acylphosphates, whose formation may not contribute to the subsequent steps in the transport.
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Western blots of peroxynitrite-treated samples. Figure 5 shows Western blots of peroxynitrite-treated samples using anti-SERCA antibodies. The untreated samples of microsomes overexpressing SERCA2b or SERCA3a proteins showed mainly one band (band 1 in Fig. 5) corresponding to the monomeric form, as confirmed by the molecular weight markers used. SERCA2b samples treated with peroxynitrite showed two additional higher molecular weight bands (bands 2 and 3 in Fig. 5A). The intensity of band 1 decreased with the increasing peroxynitrite concentrations, band 2 intensity remained at a steady level, and band 3 intensity increased. The total intensity of bands 1-3 did not change with the peroxynitrite treatment, establishing that there is no proteolysis. This suggests that the reactivity of the SERCA2b protein bands was not oligomerization dependent. The lack of proteolysis was also confirmed by the absence of any bands smaller in size than band 1. For band 1, the intensity of acylphosphate formation from Fig. 4 correlated very well with that in the Western blots (slope = 0.90 ± 0.03 µM, r2 = 0.99) (Fig. 5C). This relationship was poorer for bands 2 (slope = 0.65 ± 0.13 µM, r2 = 0.59) and 3 (slope = 0.25 ± 0.04 µM, r2 = 0.78), suggesting that bands 2 and 3 were less efficient in the acylphosphate formation than band 1. For SERCA3a, the peroxynitrite treatment of microsomes resulted in an oligomer band corresponding to band 3 and possibly a much fainter higher molecular weight band without the formation of band 2 (Fig. 5, D and F). The total intensity of bands 1 and 3 did not change, suggesting that oligomerization did not alter the antibody reactivity. The intensity of band 1 decreased only slightly with the increasing peroxynitrite concentration. The slope of the acylphosphate vs. Western intensities for band 1 (0.81 ± 0.08 µM, r2 = 0.81) was less than that for band 1 for SERCA2b. The slope for band 3 for SERCA3a (0.17 ± 0.04 µM, r2 = 0.85) was even smaller. Thus, for both SERCA2b and SERCA3a, the peroxynitrite treatment leads to oligomerization but with different patterns for SERCA2b (formation of bands 1-3) and SERCA3a (formation of bands 1 and 3 but not band 2). The oligomers for both the isoforms are less efficient at forming acylphosphates. In SERCA2b, the acylphosphate formation per monomer does not decrease with the peroxynitrite treatment, but it does decrease for SERCA3a.
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DISCUSSION |
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Correlation of parameters monitored. We monitored different activities of the SERCA pump because each contributes to understanding the mechanism of inhibition. For instance, a loss of the 45Ca2+ uptake may represent an increased leakiness of the microsomal vesicles or inhibition of the pump. It is not sufficient to monitor the ATPase reaction alone. Peroxide treatment is known to uncouple the ATP hydrolysis by Na+-K+-ATPase from the subsequent transport reaction (15). Peroxynitrite may also cause similar uncoupling of the hydrolytic reaction of the SERCA proteins from their transport activity. The inhibition of the acylphosphate formation is indicative of which part of the reaction cycle of the pump is affected. In all of these assays, SERCA3a was more resistant to peroxynitrite than the SERCA2b pump. We have shown earlier that SERCA3a is also more resistant to peroxide and superoxide (5, 23). Peroxynitrite can also cause nitration of tyrosines and S-nitrosation of cysteines (35, 40). There are some differences in tyrosine residues between the SERCA2b and SERCA3a proteins. It is not clear whether these differences play a role in the difference in peroxynitrite susceptibility of the two pumps. The difference in susceptibility to peroxide and superoxide may be due to a key cysteine residue, as proposed earlier (23). Whether this cysteine also contributes to the peroxynitrite sensitivity is not clear. In microsomes treated with different concentrations of peroxynitrite, there was a good correlation between acylphosphate formation of the monomer, ATPase, and the 45Ca2+ uptake for the SERCA2b pump. Thus peroxynitrite treatment led to inhibition of the acylphosphate formation, which was the main mechanism of the effect of peroxynitrite.
Western blot and acylphoshate gel data together show that peroxynitrite also caused oligomerization of the SERCA2b pump, with the oligomers being poorer at forming acylphosphates and even less efficient in carrying out 45Ca2+ transport. In contrast, the level of oligomerization observed with SERCA3a was lower, but after treatment with peroxynitrite even the monomer was less efficient in the acylphosphate formation. Peroxynitrite treatment gave only the very high molecular weight oligomers that were extremely inefficient in forming acylphosphates. Thus SERCA2b and SERCA3a differed in their oligomerization patterns and the inhibition of the acylphosphate formation by the monomers. The pattern of oligomerization observed with SERCA2b differs from that observed previously in the pig coronary artery smooth muscle and is closer to that observed upon peroxynitrite treatment of SERCA2a in slow-twitch muscle (24, 40). Thus it is likely that the oligomerization pattern may also be tissue dependent.
Pathophysiological implications. SERCA2b is expressed ubiquitously and SERCA3a is expressed widely in several tissues, but the exact functional differences between the two proteins are not known. The two proteins differ in their regulation by phospholamban because SERCA3a lacks the phospholamban-binding sequence present in SERCA2 (37). However, phospholamban expression in different tissues also varies (14). SERCA2a and SERCA2b are also known to be regulated by a calmodulin-dependent protein kinase (25, 45), but it is not known whether SERCA3a can be similarly regulated. In isolated microsomes, SERCA3a has a slightly lower affinity for Ca2+ than does SERCA2b (30). Vascular endothelium, mast cells, lymphoid cells, platelets, and Purkinje cells in cerebellum express SERCA3a isoforms in greater abundance than do most other tissues (3, 13, 27, 44). Peroxynitrite is produced in blood vessels from nitric oxide and superoxide, both of which may be generated in large amounts by lymphocytes during sepsis or atherosclerotic inflammation (1, 2, 16, 41). Because endothelial cells express SERCA3a, the greater resistance of SERCA3a to peroxynitrite is consistent with being part of a coping mechanism against oxidative stress in the tissues expressing it.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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