Subcellular fractionation of pig coronary artery smooth muscle.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.

Expression of the internal calcium pump in pregnant rat uterus.

Uterine contraction to agents such as oxytocin during labour may utilize Ca2+ sequestered by Ca2+ pump into the sarcoplasmic reticulum (SR). Uterus expresses the SR Ca2+ pump gene SERCA2 as the mRNA splice which encodes the protein SERCA2b. The expression of SERCA2 mRNA and protein was monitored in uteri of day 15 pregnant and delivering rats. The ratio of SERCA2 mRNA to 28S RNA increased by 20% from day 15 to delivery. SERCA2 protein examined by two antibodies increased by 54-55%. Ca2+ dependent acylphosphate intermediate of 115 kD corresponding to SERCA2 also increased 72% during this period. Thus, there is an increase in the SERCA2 expression from day 15 to delivery.

Catalase activity in coronary artery endothelium protects smooth muscle against peroxide damage.

Cyclopiazonic acid contracts pig coronary artery de-endothelialized rings, and pretreating the rings with hydrogen peroxide (H(2)O(2)) inhibits this contraction (IC(50)=0.097+/-0.013 mM). We used the cyclopiazonic acid contraction to test the novel hypothesis that endothelium can protect underlying smooth muscle against luminal H(2)O(2). We perfused the arteries with Krebs' solution containing 0. 3 or 1 mM H(2)O(2), removing endothelium from the arteries either before or after the perfusion. We then cut rings from them to monitor their contraction to 10 microM cyclopiazonic acid in a H(2)O(2)-free solution. The inhibition of the cyclopiazonic acid contraction by perfusion with H(2)O(2) was significantly less when endothelium was removed after the perfusion than when it was removed before it. The specific activity of catalase in post-nuclear supernatants from freshly isolated endothelium (14.1+/-2.7 micromol/min/mg protein) was 17+/-3-fold greater than in those from smooth muscle (0.83+/-0.22 micromol/min/mg protein). Thus endothelium contained high catalase activity and protected the underlying smooth muscle against luminal peroxide.

Effects of hydrogen peroxide on pig coronary artery endothelium.

Peroxides and other reactive oxygen species damage arteries during ischemia-reperfusion. Here, we report on the effects of H(2)O(2) on contractility of pig coronary artery. We either treated 3-mm coronary artery rings with 0 to 0.5 mM H(2)O(2) in organ baths or we perfused the arteries with H(2)O(2) and then cut them into rings. In each instance, we monitored the force of contraction of 3-mm rings in H(2)O(2)-free solution with 30 mM KCl and then we determined the A23187 induced endothelium dependent relaxation as a percent of this contraction. Treatment with H(2)O(2) in the organ bath caused a decrease in the contraction but it did not affect the percent relaxation. Treating arteries with H(2)O(2) by luminal perfusion did not affect the contraction but it decreased the percent relaxation. Perfusion alone decreased the amount of endothelium remaining in the arteries and perfusing with H(2)O(2) decreased it further. The percent relaxation with A23187 correlated well with the endothelium remaining in the arteries. We propose that H(2)O(2) and shear stress can cause a loss of endothelium and that endothelium can also protect the underlying smooth muscle against luminal H(2)O(2).

Target size of Ca-pumps in pig coronary artery smooth muscle.

The target sizes of the oxalate-independent Ca uptake by the plasma membrane enriched fraction F2, and the oxalate-stimulated Ca uptake by a fraction F3 slightly enriched in the endoplasmic reticulum were determined by radiation inactivation. The oxalate-independent Ca uptake was inactivated with a D37 value of 1.96 +/- 0.30 Mrad but the oxalate-stimulated Ca uptake had a D37 value of 0.45 +/- 0.07 Mrad. Thus, in the smooth muscle the oxalate-stimulated Ca uptake appeared to be due to a structure 3 to 6 times larger than was the oxalate-independent Ca uptake. The subcellular site of the ATP-dependent azide insensitive Ca uptake in the smooth muscle has been disputed in the past. It has been suggested to be plasma membrane (PM) by several workers, and endoplasmic reticulum (ER) by others. Recently, however, there has been substantial evidence to support the hypothesis that one Ca uptake system, unaffected by oxalate, resides in the PM and another, stimulated by oxalate, is located in the ER of the smooth muscle. The evidence has been reviewed recently. Here, we show that the two modes of Ca uptake differ in their target sizes as well. To our knowledge, this is the first report on the use of radiation inactivation to distinguish between the two modes of Ca uptake in any tissue.

Coronary artery contractility, Na(+)-pump and oxygen radicals.

Oxygen radicals accumulated during ischemia and reperfusion may affect coronary contractility by endothelium dependent and independent pathways one of which may involve Na(+)-pump. Here we report a contractility assay for Na(+)-pump in pig coronary artery and use it to examine the effects of hydrogen peroxide and superoxide. Coronary artery rings contracted in a K(+)-free Krebs solution and relaxed upon subsequent exposure to K+. The relaxation approximated a single exponential decay whose rate constant depended on [K+]2. This K(+)-induced relaxation was abolished by ouabain and was attributed to Na(+)-pump. In tissues pretreated with peroxide, the rate of relaxation of the K(+)-free contracted arteries decreased with an IC50 = 1.6 +/- 0.6 mmol/l for peroxide. Another set of tissues was pretreated with the superoxide generating system containing 0.3 mmol/l xanthine + varying concentrations of xanthine oxidase (XO) and precontracted in K(+)-free Krebs solution. The rate of the K(+)-induced relaxation decreased with IC50 = 24 +/- 8 mU/ml for XO. Thus, using the relaxation assay we conclude that exposing coronary arteries to oxygen radicals can damage Na(+)-pumps.

Coronary artery acidosis: pH and calcium pump stability.

To study the effects of prolonged exposure to different pH, pig coronary artery smooth muscle subcellular fraction F3 enriched in sarcoplasmic reticulum was preincubated at 0 or 37 degrees C and pH 6.4-7.8 and then used for monitoring the rate of the oxalate-stimulated component of the ATP-dependent azide-insensitive Ca2+ uptake at constant pH (6.8) and temperature (37 degrees C). Ca2+ uptake by F3 decreased with the increasing preincubation pH. The loss of Ca2+ uptake was more rapid upon preincubation at 37 degrees C than at 0 degrees C. Dithiothreitol (DTT), when included in the preincubation solution, protected against the loss. Sucrose, KCl, ATP, and ATP plus CaCl2 protected slightly, and glutathione, catalase, superoxide dismutase, azide, ascorbate, CaCl2, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, mannitol, mercaptopropionylglycine, and FeSO4 had only marginal or no effects. Efflux of accumulated Ca2+ was more rapid from membranes that had been preincubated at 37 degrees C than from those preincubated at 0 degrees C, but it was not affected by the preincubation pH or by DTT. The loss of Ca2+ uptake due to incubation at pH 7.8 accompanied a decrease in the 115-kDa Ca(2+)-dependent acylphosphate formation due to the Ca2+ pump. The presence of DTT in the preincubation mixture increased the acylphosphate level in the control and protected against its loss at the preincubation pH 6.8 or 7.8. Thus, in the membranes isolated from coronary artery, acidosis may protect against damage to the sarcoplasmic reticulum Ca2+ pump by protonation of a key sulfhydryl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of superoxide radical on Ca2+ pumps of coronary artery.

The effect of superoxide radical on the azide-insensitive ATP-dependent Ca2+-transport by a plasma membrane (PM)-enriched fraction (F2) and an endoplasmic reticulum (ER)-enriched fraction (F3) isolated from pig coronary artery was examined using xanthine oxidase plus xanthine to generate superoxide ions. A preincubation with xanthine oxidase plus xanthine at 37 degrees C preferentially inactivated the oxalate-stimulated Ca2+ uptake by the F3 fraction rather than the phosphate-stimulated uptake by the F2 fraction, indicating that the Ca2+ pump in the ER was more susceptible to this free radical. The inactivation of the Ca2+ uptake depended on the concentrations of xanthine oxidase and xanthine in the preincubation mixture as well as on the preincubation time. Furthermore, the inclusion of superoxide dismutase in the preincubation mixture prevented the inactivation. Thus the inactivation was caused by superoxide radical. Preincubation with xanthine oxidase plus xanthine, however, altered the half-life of efflux of Ca2+ from these vesicles only marginally. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the F3 fraction showed formation of a Ca2+-dependent acid stable phosphoenzyme at 0 degree C predominantly at a protein band corresponding to 100 kDa. The level of the 100-kDa acylphosphate intermediate was inhibited in parallel with the inhibition of the Ca2+ uptake by preincubation with xanthine oxidase plus xanthine. We conclude that superoxide radical inactivates the ER Ca2+ transport by lowering the level of the phosphoenzyme.

Pig coronary artery smooth muscle: substrate and pH dependence of the two calcium pumps.

The properties of the pig coronary artery smooth muscle Ca transport mechanisms and the effects of pH were examined. The Ca pump in the plasma membrane requires less than 50 microM Mg-ATP2- for half-maximal activity, but the pump in the endoplasmic reticulum requires greater than 700 microM Mg-ATP2-. The Ca uptake by the various subcellular organelles occurs with the following affinity characteristics towards Ca2+: plasma membrane, Km = 0.91 +/- 0.06 microM, and Hill coefficient = 1.31 +/- 0.09; endoplasmic reticulum, Km = 0.58 +/- 0.05 microM, and Hill coefficient = 2.48 +/- 0.34; and mitochondria, Km = 7.1 +/- 1.16, and Hill coefficient = 0.93 +/- 0.10. The active Ca transport systems showed similar pH dependence in that the Ca uptake at pH 6.8 was greater than at 7.6. However, the pH effects were much larger on the Ca uptake by the endoplasmic reticulum and mitochondrial membranes than by the plasma membrane. The plasma membrane also showed a small amount of pH-dependent, high-affinity Ca binding. Ca efflux from passively loaded plasma membrane vesicles was similar at pH 6.4 and 7.4. From these data, it is concluded that the endoplasmic reticulum Ca pump may play a larger role in the vasodilation of large coronary artery, but the role of the plasma membrane may be more in maintenance of relaxation mechanism.

Impurities in commercial xanthine oxidase inhibit Ca pump and interfere in contractility of pig coronary artery.

Commercial, but not pure, preparations of xanthine oxidase in the absence of an aldehyde or xanthine were observed to inhibit Ca-uptake by the subcellular membranes isolated from the smooth muscle of the pig coronary artery. This inhibition was not due to xanthine oxidase but a contaminant in the preparation. The commercial preparation caused a greater relaxation of the PGF2 alpha contracted coronary artery than the pure enzyme. The tissues treated with the commercial xanthine oxidase partially lost the ability to contract subsequently to PGF2 alpha.

Protection of Ca pump of coronary artery against inactivation by superoxide radical.

Superoxide radicals inactivate endoplasmic reticular (ER) Ca2+ pump in membranes isolated from smooth muscle of pig right coronary artery [Am. J. Physiol. 255 (Cell Physiol. 24): C297-C303, 1988]. We report on protective mechanisms against such inactivation. This tissue contained superoxide dismutase (SOD) and catalase. SOD was distributed primarily in cytosolic fraction, was cyanide sensitive, and was also present in mitochondrial fraction, and approximately 25% of this was cyanide insensitive. Catalase was distributed mainly in mitochondrial fraction and did not protect against inactivation of ER Ca2+ pump by superoxide radicals generated using xanthine plus xanthine oxidase. However, cytosolic fraction protected against this inactivation by two mechanisms: 1) DTT carried over from homogenization medium and 2) its intrinsic SOD content. Soluble fraction was concentrated, dialyzed to remove 1,4-dithiothreitol (DTT), lyophilized, and suspended in a small volume of DTT-free buffer. It still protected against superoxide inactivation of Ca2+ pump. On Sephacryl-300 gel chromatography, protecting activity comigrated with SOD. DTT protected against inactivation, but glutathione and cysteine protected only partially. Neither sulfhydryl agents nor SOD could reverse the inactivation process. Ca2+ pump activity was abolished by dithionitrobenzoate and p-chloromercuric benzoate. Superoxide may inactivate ER Ca2+ pump by irreversibly modifying key sulfhydryl group(s) on pump molecule and SOD in coronary artery smooth muscle may partially protect against this inactivation.

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

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 microM 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 microM, but up to 500 microM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 microM 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.

Angiotensin II contractions in coronary artery. Nature of receptors and calcium pools.

Pig coronary artery rings denuded of endothelium contract to the vasoactive hormone angiotensin II (Ang II). The nature of Ang II receptors and their Ca(2+)-pool utilization were examined for contraction of the artery rings and for increase in ultracellular [Ca2+] ([Ca2+]i) in smooth muscle cells cultured from them. Ang II contracted the arteries (EC50 = 7 +/- 4 nM) but with a lower maximal force (1.4 +/- 0.25 N/g tissue) than the contraction with 60 mM K+ (6.11 +/- 0.63 N/g tissue). In the cultured cells it caused a transient increase in [Ca2+]i with an EC50 value of 11 +/- 4 nM. The cells bound Ang II with a dissociation constant (Kd) of 7 +/- 2 nM. Based on the effects of the Ang II antagonists saralasin, DuPont 753, dithiothreitol and PD123319, the Ang II receptors responsible for contraction, increase in [Ca2+]i and Ang II binding to coronary artery smooth muscle were of type AT1. The contraction to Ang II was abolished by EGTA but not by nitrendipine. The sarcoplasmic Ca2+ pump inhibitors cyclopiazonic acid (10 microM CPA) and thapsigargin (1 microM) produced contractions of 4.35 +/- 0.73 and 2.07 +/- 0.54 N/g, respectively. Ang II contractions in the control arteries were nearly abolished upon pretreatment with CPA and thapsigargin. CPA and thapsigargin induced contractions were abolished by exposure to EGTA for 1 h but short exposure of the cells to EGTA only modulated the CPA or thapsigargin induced increase in [Ca2+]i; Ang II induced increase in [Ca2+]i was not inhibited by 1 microM nitrendipine but was reduced significantly by a 30-60 sec exposure to EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxide inactivates calcium pumps in pig coronary artery.

To study the effects of hydrogen peroxide, pig coronary artery smooth muscle subcellular fractions enriched in plasma membrane (F2) or sarcoplasmic reticulum (F3) were incubated in various concentrations of peroxide and 5 mM azide. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake was determined for F3 and phosphate-stimulated uptake for F2. Only 1.5-5 microM hydrogen peroxide was required for 50% inhibition of the Ca2+ uptake by F3, but the corresponding concentration for F2 was 10-50 microM. This effect was not prevented by superoxide dismutase. Hydrogen peroxide inhibited the Ca(2+)-dependent formation of a 115-kDa acylphosphate band in F3 and 140- and 115-kDa bands in F2. The inhibition of Ca2+ uptake in F3, however, exceeded the inhibition of the acylphosphate formation. Efflux of Ca2+ from F2 and F3 was enhanced by hydrogen peroxide but F3 was more sensitive than F2. We conclude that hydrogen peroxide has dual effect on Ca2+ dynamics in the coronary artery smooth muscle, i.e., it inactivates the Ca2+ pumps and increases membrane permeability to Ca2+. The effect is more pronounced on sarcoplasmic reticulum than on plasma membrane. Intrinsic catalase may, however, provide partial protection against such damage.

Adenosine metabolism in small coronary arteries of pig.

Adenosine preferentially relaxes small coronary arteries over large ones, and small bovine coronary arteries are also known to have a higher density of adenosine receptors. Here we report a possible role of adenosine metabolism in this process. Subcellular fractions, from right coronary artery (lumen diameter of 2-3 mm) of pig designated as large coronary artery and its subsequent branches (lumen diameter of 0.5-1 mm) as small coronary arteries, were prepared and characterized. In comparison to the various large artery subcellular fractions, the corresponding small artery fractions were richer in 5'-nucleotidase but poorer in adenosine deaminase. Thus a cascade of events may promote adenosine relaxation in small coronary arteries: higher activity of 5'-nucleotidase leads to production of more adenosine, larger number of receptors allows greater reactivity to adenosine, and lower adenosine deaminase level promotes prolonged action of adenosine.

Sarcoplasmic reticulum Ca(2+)-pump density is higher in distal than in proximal segments of porcine left coronary artery.

Densities of sarcoplasmic reticulum (SR) Ca(2+)-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca(2+)-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca(2+)-pump acylphosphate was 7.1 +/- 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca(2+)-pump antibody was 5.3 +/- 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca(2+)-pump inhibitors Cyclopiazonic acid (CPA, EC50 = 0.19 +/- 0.06 microM) and thapsigargin (EC50 = 0.0095 +/- 0.0035 microM). With 10 microM CPA, the force of contraction per tissue wet weight was 4.2 +/- 0.5 -fold greater in distal than in proximal rings, and with 1 microM thapsigargin it was 4.0 +/- 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca(2+)-pump than the proximal segments.

SR Ca2+ pump heterogeneity in coronary artery: free radicals and IP3-sensitive and -insensitive pools.

Reactive oxygen species are known to decrease the action of agents that mobilize Ca2+ from sarcoplasmic reticulum (SR) in pig coronary artery smooth muscle. Potentially, this may be due to damage to the SR Ca2+ pump or to the myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release channels. Here we report on the effects of peroxide and superoxide on the SR Ca2+ pump and the subsequent IP3-induced Ca2+ release. Smooth muscle cells cultured from pig left coronary arteries were permeabilized using saponin and then loaded with 45Ca2+ in the presence of an ATP-regenerating system and the mitochondrial Ca2+ uptake inhibitor sodium azide. IP3 caused a release of up to 65% of the loaded 45Ca2+, whereas the Ca2+ ionophore A-23187 caused a release of > 95%. The nature of the IP3-insensitive component of the Ca2+ uptake is not known. The IP3-induced Ca2+ release occurred at 0 or 37 degrees C and was complete in < 30 s. The 50% effective concentration for IP3 was 2.7 +/- 1.0 microM at pH 6.8 and 37 degrees C. At pH 7.4 the IP3-induced Ca2+ release was slightly lower than at pH 6.4-6.8. The IP3-induced release was also inhibited by Ca2+ concentration in the release medium. To investigate the effects of peroxide or superoxide, the cells were treated with these agents, washed, skinned, and then used to examine the IP3-sensitive and -insensitive Ca2+ pools under the conditions in which the IP3-sensitive pool was 60-65% of the total. Peroxide pretreatment was equipotent in inhibiting loading into the IP3-sensitive and -insensitive Ca2+ pools. In contrast, superoxide pretreatment inhibited loading into the IP3-sensitive pool but not into the IP3-insensitive pool. These data are consistent with a model in which the SR Ca2+ pumps are heterogeneous: those required to pump Ca2+ into the IP3-sensitive pool are inhibited by peroxide and superoxide, but those loading the IP3-insensitive pool are inhibited by peroxide only.

Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b.

Endo/sarcoplasmic reticulum (ER) Ca2+-pumps are important for cell survival and communication but they are inactivated by reactive oxygen species (ROS). We have previously reported that the Ca2+-pump isoform SERCA3a is more resistant than SERCA2b to damage by peroxide. Since peroxide and superoxide differ in their redox potentials, we now report the effects of superoxide on the two Ca2+-pump isoforms. We isolated microsomes from HEK293 cells transiently transfected with SERCA2b or SERCA3a cDNA. We exposed these microsomes to superoxide which was generated using xanthine plus xanthine oxidase and catalase to prevent accumulation of peroxide due to superoxide dismutation. Superoxide damaged the Ca2+-transport activity of both isoforms but SERCA3a was damaged at higher concentrations of superoxide and upon longer periods of exposures than was SERCA2b. Thus the SERCA3a isoform is more resistant than SERCA2b to inactivation by both superoxide and peroxide.

Sarco(endo)plasmic reticulum Ca2+ pump isoform SERCA3 is more resistant than SERCA2b to peroxide.

Sarco(endo)plasmic reticulum Ca2+ pumps are encoded by genes SERCA1, SERCA2, and SERCA3. Most tissues express SERCA2 Ca2+ pumps (splice SERCA2b) which are inactivated by reactive oxygen. In contrast, SERCA3 is expressed in tissues such as tracheal epithelium, mast cells, lymphoid cells, and aortic endothelium, which are frequently exposed to oxidative stress. Therefore, we compared SERCA3 and SERCA2b proteins for their sensitivity to oxidation. We isolated microsomes from HEK-293 cells overexpressing SERCA3 or SERCA2b. We incubated the microsomes with different concentrations of hydrogen peroxide and then determined Ca2+ pump activities in them in the following three assay systems: ATP-dependent oxalate-stimulated azide-insensitive 45Ca2+ uptake by the microsomal vesicles, Ca(2+)-Mg(2+)-ATPase, and Ca(2+)-dependent acylphosphate formation. Peroxide inhibited the pump activities in microsomes with half-maximal inhibitory concentration (IC50) values of 69 +/- 14, 66 +/- 13, and 84 +/- 15 microM for the 45Ca2+ uptake, Ca(2+)-Mg(2+)-ATPase, and the acylphosphate formation reactions, respectively. However, for microsomes from SERCA3-expressing cells, the corresponding values of IC50 for peroxide were 274 +/- 47, 857 +/- 110, and 746 +/- 40 microM. Thus, in each assay system, the resistance to inactivation by peroxide was significantly (P < 0.05) higher for the SERCA3 protein than for SERCA2b. The SERCA3 resistance to oxidants may aid the cells expressing it to function during exposure to oxidative stress.

Effects of peroxide on endothelial nitric oxide synthase in coronary arteries.

Reactive oxygen species (ROS) are produced in ischemia and reperfusion. Since endothelial nitric oxide synthase (eNOS) is key to the endothelium-dependent vasodilation, we examined the effects of peroxide on this enzyme. We treated cells cultured from pig coronary artery endothelium with different concentrations of hydrogen peroxide, washed them, solubilized them and measured NOS activity by arginine to citrulline conversion. Hydrogen peroxide inhibited the eNOS activity with an IC50 value of 0.85 +/- 0.39 mM. In another experiment, we perfused arteries with solutions containing 0 or 1 mM hydrogen peroxide, washed them, removed the endothelium using a cotton swab, centrifuged and solubilized the endothelium and monitored its NOS activity. Hydrogen peroxide (1 mM) did not affect the NOS activity significantly (p > 0.05) in this assay. We conclude that the inactivation of eNOS by hydrogen peroxide does not play a major role in the ischemia-reperfusion damage because the peroxide concentrations attained during ischemia-perfusion are much lower than those affecting the eNOS activity.