Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

Ashok K. Grover and Sue E. Samson

Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated 45Ca2+ uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 ± 13 µM, but up to 500 µM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 ± 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.

free radicals; adenosinetriphosphatase; bradykinin; angiotensin; cyclopiazonic acid; thapsigargin; fluorescence; ischemia ; endoplasmic reticulum

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE SARCO(ENDO)PLASMIC reticulum Ca2+ (SERCA) pool plays a key role in signal transduction in vascular smooth muscle and endothelial cells (21, 25). Several agents such as angiotensin II, endothelins, and prostanoids act on vascular smooth muscle, at least in part, by producing second messengers [such as inositol 1,4,5-trisphosphate (IP3)] that release Ca2+ stored in the endoplasmic reticulum, whereas other agents may act mainly via entry of extracellular Ca2+ (24, 29). The net result is an increase in intracellular Ca2+ concentration ([Ca2+]i) and contraction of the smooth muscle. In contrast, agents such as acetylcholine and bradykinin act on endothelium by releasing Ca2+ from the endoplasmic reticulum and via extracellular entry (21). The consequence in this instance is an increase in [Ca2+]i in the endothelial cells. This results in the activation of pathways that may lead to the release of endothelial relaxation factors, especially nitric oxide (NO) because endothelial cells contain a Ca2+/calmodulin-stimulated NO synthase (6). The deactivation of smooth muscle and endothelial cells involves a decrease in [Ca2+]i, and the SERCA pumps play an important role in it. The SERCA pumps also play a role in refilling the endoplasmic reticulum along with the extracellular Ca2+ entry (24, 29).

The SERCA pump in the arterial smooth muscle can be readily damaged by reactive oxygen species (ROS, free radicals) that are generated during ischemia and reperfusion (17-19, 33). Similar damage also occurs in the sarcoplasmic reticulum Ca2+ pump in the heart (6). The SERCA pump in smooth muscle is more sensitive to ROS than are the IP3-sensitive Ca2+ channels, the L-type voltage-sensitive Ca2+ channels, or the Na+ pump (9, 10). Damage to the SERCA pump in smooth muscle leads to an impairment of the ability of the arteries to contract with agents such as angiotensin II (16). The effects of ROS on some of the functions of endothelium have also been reported (7, 8, 23, 30). Many agents working on endothelium also act by mobilizing an intracellular Ca2+ pool (7, 8, 13). Although the smooth muscle contains SERCA2b, endothelium has been suggested to express the SERCA pump isoform SERCA3 (1-3, 19, 31, 32), which is approximately three times more resistant to ROS than SERCA2b (18). However, to our knowledge this is the first study comparing the effects of ROS on the SERCA pumps in endothelium and smooth muscle.

    EXPERIMENTAL METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Coronary artery smooth muscle and endothelial cell cultures. Pig coronary artery smooth muscle cells were isolated and plated in Dulbecco's modified Eagle's medium (DMEM) (GIBCO 12800-017) supplemented with 0.5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 2 mM glutamine, 50 mg/l gentamicin, 0.125 mg/l amphotericin B, and 10% fetal bovine serum and then allowed to grow to confluence (16). The cells were removed from the plates by trypsinization (0.25% trypsin, 1 mM EDTA in Ca2+- and Mg2+-free Hanks' balanced salt solution, GIBCO) for 4 min at 37°C and then replated. At the third passage, a large stock of cells was frozen into aliquots each containing 2 × 106 cells. An aliquot of cells was thawed and grown. After the cells had grown to confluence, they were trypsinized and replated at a density of 2 × 104 cells/cm2. The medium was changed after 2 days. The cells were used 5-8 days after plating. Thus all of the experiments on smooth muscle cells were performed on cells in passage 4. Endothelial cells were dislodged from the inner surface of an artery by using a sterile cotton swab. All the cells were cultured in DMEM. Aliquots of the frozen cells were thawed and used in passage 5.

Western blots. Western blot studies were carried out either using whole cell material or microsomes. For whole cell studies, typically 107 cells were suspended in 100 µl of the Laemmli sample buffer and boiled for 2 min. From this suspension, 1 µl (105 cells) was applied on a gel. Electrophoresis was carried out in 7.5% Laemmli gels, which were then electroblotted onto nitrocellulose, and the blots were treated with a 500-2,000× dilution of the primary antibodies. Three primary antibodies were used: a mouse monoclonal anti-smooth muscle alpha -actin, the mouse anti-endothelial NO synthase, and a rabbit anti-von Willebrand factor. The antibody binding was visualized by enhanced chemiluminescence using an Amersham kit, following the instructions of the manufacturer.

Peroxide treatment and permeabilization of cells. The cultured smooth muscle cells were harvested by trypsinizing for 4 min as described above and suspended in physiological saline solution (PSS) containing (in mM) 140 NaCl, 5 KCl, 10 NaHEPES at pH 7.4, 1 MgCl2, and 1.5 CaCl2. The cells were diluted at a concentration of ~106 cells/ml in PSS containing specified concentrations of hydrogen peroxide. The cell suspensions were incubated for 30 min at 37°C with different concentrations of peroxide and then centrifuged to remove the peroxide. The cells were resuspended in the skinning solution without saponin and then washed in it. The skinning solution contained (in mM) 165 KCl, 0.4 MgCl2, 5 sodium azide, 1 dithiothreitol (DTT), and 20 sodium 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 6.8 (11). The cells were permeabilized at a concentration of 107 cells/ml in the skinning solution containing 250 µg saponin/ml for 15 min at 25°C. The same method was also used for permeabilizing the endothelial cells, with the only difference being that a higher concentration of endothelial cells (2 × 107 cells/ml) was found to be optimum, since these cells are much smaller than smooth muscle cells. The permeabilized cells (>95%) were permeable to trypan blue. The permeabilized cells were placed on ice and used within several minutes.

Ca2+ transport. Typically, 50 µl of the permeabilized cells (25-75 µg protein) were added to 150 µl of a 45Ca2+ uptake medium so that the final composition of the solutions was 30 mM imidazole-HCl (pH 6.8 at 37°C), 100 mM KCl, 1 mM MgCl2, 5 mM sodium azide, 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.85 mM CaCl2 (plus trace amounts of 45CaCl2), 5 mM ATP, 10 mM creatine phosphate, 50-80 U/ml creatine kinase, 5 mM oxalate, and 15-30 µg of cell protein (11). This resulted in a Ca2+ concentration of 5 µM. In an initial experiment, it was determined that the uptake was linear for up to 30 min. Routinely, the samples were incubated for 30 min at 37°C and then filtered through 0.45-µm nitrocellulose filters under suction. The filters were washed three times with a chilled solution containing 8% sucrose, 0.5 mM EGTA, and 40 mM imidazole-HCl (pH 7.0). The filters were then placed in vials containing Beckman Ready Safe cocktail, and the amount of radioactivity in the filters was determined with a scintillation counter.

Acylphosphates. The acylphosphate experiments were carried out on microsomes prepared from the cultured endothelial or smooth muscle cells (16). The cells were suspended in chilled homogenization solution (8% sucrose, 1 mM phenylmethysulfonyl fluoride, and 2 mM DTT). They were homogenized for 4 × 5 s with a Polytron PT20. The homogenates were centrifuged at 10,000 g for 2 min to remove mitochondria and nuclei. KCl was added to the supernatant to get a final concentration of 0.7 M, and the suspension was placed on ice for 10 min and then centrifuged at 550,000 g for 15 min. The microsomal pellets were rinsed with the suspension solution (8% sucrose and 5 mM sodium azide) to remove traces of DTT and then suspended in it. The microsomes were incubated in different concentrations of hydrogen peroxide for 30 min at 37°C, placed back on ice, and then used immediately. The Ca2+-dependent acylphosphate formation by the microsomes was examined as follows (16). Routinely, 24 µl of the microsomal suspension were added to 12 µl of a buffer solution and incubated in ice water for 10 min, and then an equal volume of radioactive ATP was added. Typically, the final reaction mixtures contained 100 mM KCl, 30 mM imidazole-HCl (pH 6.8), 50 µM CaCl2, 5 µM unlabeled ATP, 20 µCi of [gamma -32P]ATP, and 10-15 µg of the microsomal protein. The reactions were started by adding ATP and stopped after 60 s by adding 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 then centrifuged at 4°C. The pellets were washed once with 1,500 µl TCAP and then suspended in 70 µl of freshly prepared sample buffer containing 10 mM NaMOPS (pH 5.5), 1 mM EDTA, 3% sodium dodecyl sulfate (SDS), 10% sucrose, 20 mM DTT, 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% SDS-polyacrylamide gels at pH 4 as described previously (16). The gels were autoradiographed at room temperature, and the optical densities of the acylphosphate bands were monitored by image analysis.

[Ca2+]i measurements. Cells were cultured on coverslips for the [Ca2+]i measurement experiments (16). The coverslips coated with the cultured cells were removed from the culture medium and rinsed with a solution containing (in mM) 115 NaCl, 5.8 KCl, 2 CaCl2, 0.6 MgCl2, 12 glucose, and 25 NaHEPES (pH 7.4 at 37°C). These coverslips were then preincubated in this buffer at 37°C for 30 min in the presence of specified concentrations of peroxide, washed three times to remove peroxide, incubated for 40 min at 22-23°C in 3 µM fluo 3-acetoxymethyl ester (AM) and 2 mM probenecid dissolved in the above buffer, washed three times to remove the excess dye, and then incubated in a solution without the dye and with probenecid for 30 min at 22-23°C to allow for hydrolysis of the dye. The coverslips were then rinsed with the same buffer containing probenecid but with 1 mM Ca2+ and placed in 2 ml of the same in a stirred thermostated cuvette at 37°C for fluorescence measurement using a SPEX Fluorolog 112. Thus the added peroxide was removed by washing before the cells were loaded with fluo 3-AM and the fluorescence was monitored. This protocol was important because peroxide itself may interact with the added agents. For measurements in Ca2+-free solutions, 1 mM Ca2+ was replaced by 0.5 mM EGTA in the last step. The fluorescence was monitored at excitation and emission wavelengths of 490 and 530 nm, respectively. After the basal fluorescence and the effect of cyclopiazonic acid (CPA) on the fluorescence were monitored, calibration was carried out and the fluorescence values converted to [Ca2+]i as described previously (16).

Contractility experiments. The pig left coronary artery along with the surrounding cardiac cells was excised from the heart and placed in a Krebs solution bubbled with 95% CO2-5% O2 (16). The Krebs solution contained the following (in mM): 115 NaCl, 5 KCl, 22 NaHCO3, 1.7 CaCl2, 1.1 MgCl2, 1.1 KH2PO4, 0.03 EDTA, and 7.7 glucose. The cardiac tissue and fat were removed, and the arteries were dissected as described previously (16). To obtain the deendothelialized arteries, the endothelium was removed by drawing a piece of cotton through each artery. The endothelium removal was monitored as a loss of bradykinin relaxation. Each artery was cut into 3-mm-long rings and suspended between two hooks in an organ bath. The tissues were placed under an initial tension of 3 g, which was corrected 30 min later. A typical contraction experiment using deendothelialized arteries consisted of the following steps: 1) monitor for 30 min the force generated by adding 60 mM KCl to the bathing Krebs solution, 2) over 20-30 min wash the arteries two times in normal Krebs solution, 3) treat the arteries for 30 min with specified concentrations of peroxide, 4) wash three times in normal Krebs solution for over a total of 60 min, and 5) test the effects of CPA or other agents. This experimental protocol was chosen to ensure that the results depended on the damage of peroxide on the tissues and not on the effects of these agents on the assay solutions. The contraction to 60 mM KCl in each tissue was taken as 100%, and the subsequent CPA or KCl was compared with it. For monitoring the endothelium-dependent relaxation, the arteries were not deendothelialized. A typical relaxation experiment consisted of the following steps: 1) monitor for 30 min the force generated by adding 60 mM KCl to the bathing Krebs solution, 2) over 20-30 min wash the arteries two times in normal Krebs solution, 3) treat the arteries for 30 min with specified concentrations of peroxide, 4) wash three times in normal Krebs solution for over a total of 60 min, 5) produce a contraction with 30 mM KCl, and 6) at the peak of this contraction add CPA. The relaxation was expressed as a percentage of the contraction. At the end of the experiment, the tissues were blotted and weighed. In the control experiments with peroxide, the tissues were treated identically except that peroxide was not added during the preincubation period.

Data analysis. The curve fitting was carried out with FigP (Biosoft). Null hypotheses were tested with a Student's t-test, and P values of <0.05 were considered to be statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The effect of peroxide was examined at three different organizational levels: Ca2+ pump activities in permeabilized cells or microsomes, Ca2+ mobilization in intact cells, and contraction and relaxation of artery rings. Except where specified, the cells or tissues were preincubated with peroxide, washed to remove the peroxide, and then assayed. This protocol eliminated any direct interactions between peroxide and the components of the assay systems, thus assuring that only the damage that remained when peroxide was washed off was monitored.

Characterization of cultured cells. The antibody reactivity of the whole cell lysates was examined in Western blots, and the results are shown in Fig. 1. The endothelial cells reacted positively to anti-von Willebrand factor and anti-endothelial NO synthase but negatively to anti-smooth muscle alpha -actin. Conversely, the smooth muscle cells reacted positively to anti-smooth muscle alpha -actin but not to anti-von Willebrand factor or anti-endothelial NO synthase.


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Fig. 1.   Western blots for characterizing cultured cells. A, C, and E: endothelial cells. B, D, and F: smooth muscle cells. Antibodies used were anti-von Willebrand factor (A and B), anti-endothelial nitric oxide synthase (C and D), and anti-smooth muscle alpha -actin (E and F). See EXPERIMENTAL METHODS for further details. The following were also used as positive controls but are not shown here: human platelet lysate for von Willebrand factor, human umbilical vein endothelial cultured cells for endothelial nitric oxide synthase, and freshly isolated smooth muscle from pig coronary artery for smooth muscle alpha -actin.

Characterization of SERCA pump. Figure 2 shows the time course of the ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake by cells that had been cultured from endothelium and smooth muscle of pig coronary artery and then permeabilized. The oxalate-stimulated uptake was linear for up to 30 min. In 30 min, the uptake in the absence of ATP or oxalate was <10% of the total uptake. The value of the uptake observed was consistently about threefold higher in the smooth muscle cells than in the endothelial cells. In all subsequent experiments, the uptake was examined over 30 min. Figure 3 shows the effects of the sarcoplasmic reticulum Ca2+ pump inhibitors thapsigargin and CPA on the ATP-dependent, azide-insensitive, oxalate-stimulated Ca2+ uptake by the permeabilized cells (5, 11, 12, 22). Both agents inhibited the uptake nearly completely, consistent with the uptake being due to the SERCA pumps. Furthermore, consistent with the literature (11, 12), thapsigargin was a more potent inhibitor than CPA (Fig. 3).


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Fig. 2.   Time course of Ca2+ uptake by permeabilized cells. Cultured cells from smooth muscle and endothelium were permeabilized with saponin and used for monitoring uptake of 45Ca2+ in the presence of ATP-regenerating system and sodium azide (to inhibit uptake by mitochondria) with (+) and without (-) 5 mM oxalate. Samples were filtered after the specified intervals of time. Values are means ± SE of 4 replicates. See EXPERIMENTAL METHODS for further details.


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Fig. 3.   Effects of cyclopiazonic acid (CPA) and thapsigargin. A: smooth muscle cells. B: endothelial cells. Permeabilized cells were incubated with the ATP-regenerating system, 5 mM oxalate, and 5 mM sodium azide for 30 min and then filtered. Data are from 2 experiments and represent means ± SE of 8-16 replicates. In each experiment, mean value of the uptake in the absence of CPA or thapsigargin was taken as 100% and all values were expressed relative to it. See EXPERIMENTAL METHODS for further details.

Effect of peroxide on SERCA pump. Two assays of the Ca2+ pump were used: transport of 45Ca2+ and Ca2+-dependent formation of acylphosphates. For the first assay, the cells were permeabilized in the absence of DTT because peroxide reacts instantaneously with this reagent. The permeabilized cells were used for Ca2+ uptake in the presence of different concentrations of peroxide (Fig. 4). Although azide was included in these assays to inhibit the mitochondrial Ca2+ transport, it would also inhibit any intrinsic catalase activities of these cells and hence prevent a rapid degradation of peroxide. Peroxide decreased the Ca2+ uptake in a concentration-dependent manner. At low concentrations of peroxide, however, the inhibition due to peroxide was significantly (P < 0.05) less in the endothelial cells than in the smooth muscle cells (Fig. 4).


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Fig. 4.   Effect of adding peroxide to Ca2+ uptake reaction mixture on Ca2+ transport. For this experiment, cells were permeabilized in the absence of dithiothreitol. Permeabilized cells were used for Ca2+ uptake in the presence of specified concentrations of peroxide, the ATP-regenerating system, 5 mM oxalate, and 5 mM sodium azide. After 30 min, uptake reaction was ended by filtration. Data are from 2 experiments and represent means ± SE of 12 replicates. In each experiment, mean value of the uptake in the absence of peroxide was taken as 100% and all values were expressed relative to it. See EXPERIMENTAL METHODS for further details.

SERCA pumps bind Ca2+ and [gamma -32P]ATP, forming 32P-labeled acylphosphates of 110-115 kDa that are acid stable. Permeabilized cells gave a very faint band compared with the background, and, hence, purified microsomes were used in this experiment. Microsomes prepared from the cultured cells were treated with different concentrations of peroxide and then used for the acylphosphate experiments. Only one major band was seen in autoradiograms of the acylphosphate gels. In the presence of added EGTA instead of CaCl2, no bands were observed, indicating that the acylphosphate formation was Ca2+ dependent. Figure 5 shows that the intensities of these bands decreased when microsomes from the cells treated with peroxide were used. Again, in this assay, the Ca2+ pump was significantly (P < 0.05) more sensitive to peroxide in smooth muscle cells [50% inhibitory concentration (IC50) = 97 ± 36 µM] than in endothelial cells (IC50 = 704 ± 112 µM).


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Fig. 5.   Effect of treating microsomes with peroxide on acylphosphate formation. Microsomes were incubated in peroxide and then used to examine the acylphosphate formation as described in EXPERIMENTAL METHODS. A: each lane showed only one major band at 110-115 kDa. In some gels, molecular mass of this band was established with the cardiac sarcoplasmic reticulum acylphosphates. Under these experimental conditions, mobilities were similar for the acylphosphates formed from the cardiac sarcoplasmic reticulum and the microsomes of smooth muscle and endothelial cells. Optical densities of the acylphosphate bands from gels as the ones shown were monitored. Within each gel, control (without peroxide) was carried out in duplicate. Mean intensity of control lanes was taken as 100%. For B, data are means ± SE of 4 gels for each cell type. See EXPERIMENTAL METHODS for detailed protocols.

In the subsequent experiments, we examined the effects of preincubating intact cells with peroxide and washing them before permeabilizing them to examine the Ca2+ uptake. In these experiments, azide was excluded from the initial incubation steps but included in the Ca2+ uptake solution to prevent the mitochondrial Ca2+ uptake. In one experiment, we incubated the cells at 37°C with 50 µM peroxide for different lengths of time; then we washed and permeabilized these cells and examined the ATP-dependent, azide-insensitive, oxalate-stimulated Ca2+ uptake. The experimental protocol was such that peroxide was present in the incubation mixtures only for the specified length of time, but all the cells were incubated at 37°C for 60 min, and also that peroxide was not present in the Ca2+ uptake solution. There was a decline in the Ca2+ uptake with the preincubation time with peroxide, but this decline was much slower in the endothelial cells than in the smooth muscle cells (Fig. 6).


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Fig. 6.   Effect of pretreating intact cells with 50 µM peroxide for different times before permeabilization and Ca2+ uptake. Cells were trypsinized and then incubated in the presence of 50 µM peroxide for the specified times at 37°C. Cells were then centrifuged, washed, permeabilized, and used for the uptake reaction. Uptake was monitored in the presence of the ATP-regenerating system, 5 mM oxalate, and 5 mM sodium azide for 30 min. Data are from 1 experiment and represent means ± SE of 6 replicates. Mean value of the uptake in the absence of peroxide was taken as 100% for each cell type, and all values were expressed relative to it.

In yet another set of experiments, we preincubated the cells with different concentrations of peroxide at 37°C for 30 min, washed the cells, permeabilized them, and examined the Ca2+ uptake. As shown in Fig. 7, the oxalate-stimulated Ca2+ uptake by the permeabilized smooth muscle cells decreased with increasing concentrations of peroxide (IC50 = 43 ± 10 µM). However, the endothelial cells were significantly (P < 0.05) more resistant to the peroxide treatment (IC50 = 2,215 ± 537 µM).


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Fig. 7.   Effect of pretreating intact cells with different concentrations of peroxide before permeabilization and Ca2+ uptake. Cells were trypsinized and then incubated in the presence of different concentrations of peroxide for 30 min at 37°C. Cells were then centrifuged, washed, permeabilized, and used for the uptake reaction. Uptake was monitored in the presence of the ATP-regenerating system, 5 mM oxalate, and 5 mM sodium azide for 30 min. Data are from 3 experiments and represent means ± SE of 12-24 replicates. In each experiment, mean value of the uptake in the absence of peroxide was taken as 100% and all values were expressed relative to it.

Effect of peroxide on CPA-induced Ca2+ increase. Figure 8 contains representative tracings showing basal [Ca2+]i levels, measured using the fluorescence dye fluo 3, and the effects of inhibiting the SERCA pumps with CPA (11, 23). In the Ca2+-free solution, smooth muscle cells had a basal [Ca2+]i of 110 ± 16 nM and adding 10 µM CPA increased it by 152 ± 17 nM. As reported previously, pretreating the cells with peroxide produced a decrease in basal [Ca2+]i, and it inhibited the CPA-induced increase in [Ca2+]i; 300 µM peroxide produced 77 ± 2% inhibition (Fig. 8). Endothelial cells showed basal [Ca2+]i of 67 ± 9 nM, and CPA produced an increase of 76 ± 15 nM. In endothelial cells pretreated with peroxide, the basal [Ca2+]i (60 ± 10 nM) and the CPA-induced increase (75 ± 12 nM) in [Ca2+]i did not differ significantly from the control (P > 0.05). Thus the peroxide pretreatment inhibited the effect of CPA on the smooth muscle cells but not on the endothelial cells. This result is consistent with the Ca2+ pump being more resistant to peroxide in the endothelial cells than in the smooth muscle cells.


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Fig. 8.   Effect of peroxide on CPA-induced increase in intracellular Ca2+ concentration ([Ca2+]i). Cultured cells were treated with 300 µM peroxide, washed, loaded with fluo 3-acetoxymethyl ester and, for monitoring [Ca2+]i, placed in Ca2+-free solution. A: representative tracings. Arrow indicates the time of addition of 10 µM CPA. For B, values are means ± SE of 18 replicates from smooth muscle cells and 9 from endothelial cells, each carried out in at least 2 different experiments. See EXPERIMENTAL METHODS for detailed protocols.

Effect of peroxide on CPA-induced contraction and relaxation. Inhibiting the SERCA pump with CPA produced an increase in [Ca2+]i in smooth muscle and endothelial cells. However, the increase in [Ca2+]i in the two cell types had different effects on contractility. In deendothelialized arteries, CPA produced a contraction that was 36 ± 6% of that produced by 60 mM KCl (Fig. 9). CPA relaxed the arteries by 41 ± 4% when the endothelium was intact, and the arteries were precontracted with 30 mM KCl (Fig. 9). The CPA-induced relaxation was blocked by the NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) (Table 1), indicating that it involved the NO synthesis pathway.


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Fig. 9.   Effect of peroxide on CPA-induced contraction and relaxation of the coronary artery rings. A: contraction of deendothelialized arteries. Artery rings were contracted with 60 mM KCl, washed, and then treated with 0 or 500 µM peroxide for 30 min and then washed again. At this time, CPA was added to deendothelialized arteries to monitor the resulting contraction. B: relaxation of arteries with intact endothelium. Arteries with intact endothelium were contracted with 30 mM KCl. CPA was then added at the peak of the contraction, and relaxation was monitored. Representative tracings are shown on left. A summary (values are means ± SE) from several experiments is shown on right.

Peroxide, when added to deendothelialized arteries, produced a small (5-10% of the contraction produced by KCl) transient contraction lasting 5-10 min. Pretreating the deendothelialized arteries with peroxide for 30 min, followed by washing them to remove the peroxide, decreased the subsequent contractions by CPA with an IC50 value of 97 ± 13 µM (Fig. 9). Pretreating the deendothelialized rings with 500 µM peroxide diminished this contraction to 4 ± 1% of the 60 mM contraction (90% inhibition). The contraction produced by 30 mM KCl was 93 ± 4% of the 60 mM KCl contraction. Treatment with 500 µM peroxide decreased the contraction significantly (P < 0.05) but only marginally to 73 ± 4% (22% inhibition). Thus the peroxide treatment preferentially inhibited the CPA contractions over the KCl contractions. Figure 9 shows a comparison of the effects of 500 µM peroxide on the CPA-induced contractions in deendothelialized arteries and the CPA-induced endothelium-dependent relaxation. Whereas the peroxide pretreatment inhibited the CPA-induced contraction by 90%, it did not significantly (P > 0.05) alter the endothelium-dependent relaxation (Table 1).

We also examined the effects of peroxide on the action of peptide hormones on the artery. Angiotensin II contracted the deendothelialized arteries, producing 22 ± 3% of the 60 mM KCl contraction. Pretreatment with 500 µM peroxide diminished this contraction to 1.5 ± 0.7% of the 60 mM KCl contraction (93 ± 4% inhibition). Bradykinin relaxed the precontracted arteries with intact endothelium by 52 ± 5%. In arteries pretreated with 500 µM peroxide, the corresponding relaxation was 28 ± 6% (40 ± 13% inhibition). Thus peroxide inhibited the angiotensin II contraction in the deendothelialized arteries significantly (P < 0.05) more than it inhibited the bradykinin relaxation in the arteries with intact endothelium (Table 1).

                              
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Table 1.   Effect of preincubating tissues with 500 µM peroxide on contractility

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the RESULTS, we demonstrated that the SERCA pump is more resistant to peroxide in the endothelial cells than in smooth muscle. We also showed that the increase in [Ca2+]i produced by inhibiting the SERCA pump with CPA is more resistant to peroxide in endothelium than in smooth muscle and that the CPA-induced endothelium-dependent relaxation is more resistant to peroxide than the CPA-induced contraction in deendothelialized artery rings. The DISCUSSION will focus on an interpretation of these observations, the limitations of the methods in obtaining such interpretations, a comparison of these results with those reported previously, and a working model based on these observations and the literature.

ATP-dependent, oxalate-stimulated, azide-insensitive 45Ca2+ uptake by the saponin-permeabilized cells was used to demonstrate that pretreating the cells with peroxide preferentially inhibited the SERCA pump in smooth muscle cells over that in endothelial cells. The higher potency of thapsigargin than that of CPA in inhibiting the 45Ca2+ uptake indicates that it is the SERCA pump that is inhibited (11), since these agents do not affect other pumps. However, a SERCA pump has also been reported to be present in the nuclear membrane, which is present in the permeabilized cell preparation (20). In theory, the SERCA pump activity can also be monitored as Ca2+-Mg2+-ATPase activity, but these measurements are not possible, since the smooth muscle cells contain a very high level of Mg2+-ATPase activity that interferes (17). Therefore, we used the partial Ca2+-Mg2+-ATPase reaction of forming acylphosphates instead of Ca2+-Mg2+-ATPase. Peroxide preferentially inhibited the Ca2+-dependent acylphosphate formation in microsomes isolated from smooth muscle cells over those from the endothelial cells. Because nuclei are discarded during the preparation of the microsomal fraction, the preferentially inhibited SERCA pump is unlikely to be nuclear. Furthermore, because both the acylphosphate formation and the uptake were inhibited, the possibilities that the observed results reflect only an effect of peroxide on membrane permeability or an uncoupling of the pump are ruled out. Because saponin was not used in the experiments with the microsomes, the possibility is also ruled out that the differences observed between the two cell types in the permeabilization experiments are mainly due to differences in their interactions with saponin. The observed effects of peroxide on the CPA-induced [Ca2+]i transients in Ca2+-free solution are also consistent with the greater resistance of the SERCA pump in endothelial cells. The possibility remained that all the transport and the [Ca2+]i measurement studies were simply artifacts due to phenotypic expression changes on culturing the cells. Therefore, it was empiric to carry out the functional studies in the artery rings.

Peroxide inhibited the contractions by agents, such as angiotensin II or CPA, whose actions involved intracellular Ca2+ mobilization (16). Relaxation experiments required precontracted tissues, and hence they could not be carried out in a Ca2+-free solution. The contraction due to extracellular Ca2+ entry on membrane depolarization with KCl can better withstand peroxide (16). Therefore, although the results of the contractility experiments are consistent with greater peroxide resistance of the SERCA pump in the endothelial cells than in smooth muscle cells, this interpretation is not unique, since a possible contribution of Ca2+ entry from extracellular space cannot be ruled out.

In this study, we examined the damage caused by pretreating the tissues with peroxide and not reversed when they are washed. ROS may also produce direct actions on the contractility assays. For instance, ROS may react directly with NO (26). Our results on the damage remaining after the removal of peroxide are consistent with the previous studies in the literature (7, 8, 15, 16, 27). We and others have shown that the SERCA pump in smooth muscle is inactivated by ROS (7, 8, 15, 16, 27). However, this is the first study comparing smooth muscle and endothelium or examining the effect of ROS directly on the SERCA activity. The effect of t-butyl-hydroxy-peroxide on [Ca2+]i has been reported for bovine aortic endothelial cells (8). Whereas t-butyl-hydroxy-peroxide inhibited bradykinin-stimulated Ca2+ influx from extracellular space, it had little effect on the agonist-induced release of Ca2+ from intracellular stores. Our finding that peroxide only partially inhibited the bradykinin relaxation is also consistent with this observation. The SERCA pump inhibitors thapsigargin and 2,5-di-t-butylhydroquinone were shown to produce an increase in [Ca2+]i in endothelial cells in Ca2+-free solution, but they also caused a Ca2+ entry when Ca2+ was reintroduced to the solution (7). ROS generated by t-butyl-hydroxy-peroxide affected the SERCA inhibitor-induced Ca2+ entry only on prolonged exposure to ROS, but the Ca2+ entry was affected more readily. These results are also consistent with the resistance of the endothelial SERCA pump to ROS. It has been reported that, in isolated ischemic- and hypoxic-perfused hearts, the endothelium-dependent relaxation was impaired before the endothelium-independent relaxation (14, 28). These studies are consistent with ours, since the contractile apparatus (as shown by contractions to KCl) is relatively resistant to peroxide. It is the ability to contract by mobilizing intracellular Ca2+ that is impaired in the smooth muscle, and this was not examined in these studies. Furthermore, it should be taken into consideration that the present study has been carried out with only peroxide; the resistance to other ROS may be different.

What is the cause of the greater resistance of SERCA pumps in endothelium than in smooth muscle? Whereas different smooth muscles express the splice variant SERCA2b coded by the gene SERCA2, rat aortic endothelium has been reported to express the gene SERCA3 (1-3, 19, 31, 32). The SERCA3 gene is also expressed in other surface cells such as tracheal epithelium, mast cells, lymphoid cells, and platelets (32). In microsomes from HEK293 cells overexpressing SERCA3, the IC50 value for peroxide to inhibit the Ca2+ pump is approximately three times higher than that for the cells overexpressing SERCA2b (18). Therefore, one of the possible reasons for the differences in the sensitivity to peroxide between endothelial and smooth muscle cells may be the expression of different SERCA isoforms. However, there are some caveats with this interpretation. First, SERCA3 expression has not yet been demonstrated in the pig coronary artery endothelium and it has been suggested that not all the vascular endothelium may express SERCA3 (2). Second, the only evidence for the existence of SERCA3 in the endothelium is in the in situ hybridization studies (1, 2) and no other evidence has been presented, although it has been stated in another study that vascular endothelium expresses SERCA3 (31). The third and final caveat is that we observed a larger difference in the resistance to peroxide between endothelium and smooth muscle in the studies with intact cells or artery rings than in those with permeabilized cells or microsomes. These caveats detract from the simple interpretation that endothelium expresses SERCA3. Smooth muscle expresses SERCA2b, and SERCA3 is more resistant to peroxide than SERCA2b. Factors other than the differences in the SERCA isoforms may also be involved. We propose a working model in which the resistance of SERCA isoforms to ROS and the ability to scavenge ROS more efficiently allow the endothelium to be better protected than the smooth muscle. Because the ROS may be generated more readily in the arterial lumen, these scavenging mechanisms may also allow the endothelium to act as a protective barrier. It has been suggested that endothelium contains mechanisms for scavenging ROS, such as catalase, superoxide dismutase, glutathione, vitamin C, and vitamin E (4, 6, 21). Whereas this observation is consistent with this working model, it must be pointed out that just about every cell contains mechanisms to scavenge ROS, and we do not know if endothelium is any better at it than smooth muscle.

    ACKNOWLEDGEMENTS

We thank Dr. E. S. Werstiuk for reviewing the manuscript and C. M. Misquitta for assistance with some of the experiments.

    FOOTNOTES

This work was supported by a Grant-in-Aid from the Heart and Stroke Foundation of Ontario.

Address for reprint requests: A. K. Grover, Dept. of Biomedical Sciences, HSC 4N41, McMaster Univ., 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.

Received 6 January 1997; accepted in final form 19 June 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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