Superoxide stimulates IP3-induced Ca2+ release from vascular smooth muscle sarcoplasmic reticulum YUICHIRO
J. SUZUKI
AND
GEORGE
D. FORD
Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Suzuki, Yuichiro J., and George D. Ford. Superoxide stimulatesIP3-inducedCa2’ releasefrom vascular smoothmuscle sarcoplasmicreticulum. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): Hll4-Hl16, 1992.-Reactive oxygen intermediates (ROI) have been implicated in a variety of pathophysiological conditions, and vascular smooth muscle may be a site of damagein such oxygen toxicity. Mechanisms of the effects of theseintermediateson vascular smoothmuscleat the cellular level, however, have not been well studied. We have previously shown that xanthine oxidase (X0) -generatedsuperoxideradicals (02.) inhibited the Ca2’-adenosinetriphosphatase of vascular smooth muscle sarcoplasmic reticulum (SR) through mechanismsthat do not involve H202 or hydroxyl radicals. In the present study, we report that the D-myo-inositol 1,4,5trisphosphate(IP&-induced Ca2’ releasefrom bovine aortic SR was alsoaffected by 0; . Hypoxanthine (100PM) plus X0 (10 mu/ml) in the presenceof catalase(100 U/ml) stimulated the IP3-induced Ca2+releasefrom SR monitored using arsenazo III. At 10 PM IP3, the releasewas doubled by 0;. treatment. As a consequence of usingthe higher SR protein concentrations requiredto observethe Ca2+release,this effect wasindependent of Ca2’ uptake inhibition induced by 0;~ Since the effect of 0;. wasnot seenwhen a nonhydrolyzable analogueof IP3 was usedto induce Ca2’ release,0;. may be inhibiting the degradation processesof IP3. aorta; inositol 1,4,5trisphosphate; inositol 1,4,5trisphosphorothioate; oxygen free radicals l
(ROI)havebeenimplicated in a variety of pathophysiological conditions (3). In some of these conditions, the first target of oxygen free radicals is the vascular system (10); however, the mechanisms of the effects of ROI on vascular smooth muscle at the cellular level have not been extensively studied. Recently, the Ca2+-transport activity (5) and Ca2+-adenosinetriphosphatase (ATPase) activity (13) of vascular smooth muscle sarcoplasmic reticulum (SR) have been shown to be inhibited by superoxide anion radicals (0~0) through mechanisms that do not involve hydrogen peroxide (H202) or hydroxyl radicals (HO*). In the present study, we found that another component of the excitation-contraction coupling mechanisms of vascular smooth muscle was affected by 0~0. We report 1,4,5trisphosthat 0;. stimulates the D-myo-inositol phate (IPS) -induced Ca 2+ release from the vascular smooth muscle SR through mechanisms that do not involve Hz02 or HO*. This effect is independent of the inhibition of the Ca2+ pump and any resultant alteration in the pump-leak system. However, the effect seems to be due to dysfunction of the IP3 degradation system present in vascular smooth muscle. REACTIVEOXYGENINTERMEDIATES
(X0; EC 1.1.3.22,from cow’smilk, Boehringer Mannheim) and 100PM hypoxanthine (HX; Sigma). Superoxidedismutase(100 U/ml; SOD; EC 1.15.1.1,from bovine erythrocytes, Sigma)and catalase(100U/ml; CAT; EC 1.11.1.6,from bovine liver, Sigma) were usedas scavengersof 0;. and H202, respectively. These scavengerswere addedto the assaymedia before the initiation of ROI generation. Fresh mature bovine descendingthoracic aortas in ice-cold saline were obtained from Pel-Freez (Rogers, AR). Crude SR vesicleswere prepared from this tissueby a method previously described(13). Protein concentrations were determinedby the method of Lowry et al. (8), using bovine serumalbumin (BSA) asa standard. Ca2’ releasewas measuredusing the metallochromic indicator dye, arsenazo III, and dual-wavelength spectroscopyat 650 and 725 nm as describedby Scarpa (12). For eachexperiment, SR protein (300 pg) was incubated in l-ml cuvettes containing 100 mM KCl, 30 mM imidazole (pH 7.0), 3 mM ATP, 3.1 mM MgC12,and 10 PM CaC12at 37°C for 30 min. Somecuvettes also contained 0.01 U/ml X0 and 100PM HX, whereasothers contained this generating system plus 100 U/ ml SOD and/or 100 U/ml CAT. After the incubation, 200 PM arsenazoIII was addedand a stablerecording of the difference of absorbanceat 650 and 725 nm (AA650-725) established.Then 10 PM of IP3 (Sigma) and 100 PM of A23187 (Sigma) were added sequentially. The Ca2’ releasedby these agents was measuredby noting the difference between the absorbance readingsbefore and after the addition of eachagent.The results are reported as percent Ca2+released,which is defined by the relationship: %Ca2’ released= ([ (AA650-725 before IP3 addition) - (AA650-725 after IP3 addition)]/(Ca2’ load)] X 100,where Ca2+ load was defined by the relationship: Ca2’ load = (AA650-725 before IP3 addition) - (AA650-725 after A23187 addition). Ca2’ load was taken to reflect the total amount of releasableCa2+ taken up by the SR preparation. To determine the effect of reactive oxygen speciesof the hydrolysis of IP3, in someexperiments, IP3 was replaced by 5 PM of the nonhydrolyzable analogue,inositol 1,4,5-trisphosphorothioate(DuPont) (1). Significant differencesbetweentwo groupswere determined by the Student’s t test at P < 0.05.
RESULTS AND DISCUSSION
Figure 1 shows that treatment with 100 PM HX plus 0.01 U/ml X0 stimulated the IP3-induced Ca2+ release from bovine aortic SR. This effect was blocked by SOD but not by CAT, suggesting that stimulation was caused by 020 through mechanisms that do not involve H202 or HO.. Stimulation of release might be due to several mechanisms. First, it could be a matter of how release is defined. For example, if the SR normally holds 100 pmol of Ca2+ with 35 pmol available for release by IPS, we would observe a “release” of 35% of the Ca2+ content. However, if the content was reduced 50% (by inhibition of the Ca2+ uptake process) while the releasable fraction MATERIALS AND METHODS remained the same, then we would observe a release of 0;. were generated by using 0.01 U/ml xanthine oxidase 35/5O or 70%. Another possible mechanism to stimulate
H114
0363-6135/92
$2.00 Copyright
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
0;.
STIMULATES
IP3-INDUCED
CA*+
RELEASE
T-T115 III.L”
protein and 44% (44.4 t 6.3, n = 5) at 80 pg/ml SR protein. This significant reduction in inhibition is consistent with our assumption that the high protein concentration present in the Ca2+ release studies prevented a significant 0;. -induced Ca2’ uptake inhibition. Another possible mechanism for the potentiation of IPS-induced Ca2+ release is inhibition of the processes catabolizing the IP3, thus increasing the effective IP3 concentration. One approach to test whether 020 is indeed affecting IP3 metabolism is using a nonhydrolyzHX/XO HX/XO control HX/XO able analogue of IP3, such as inositol 1,4,5-trisphosphoSOD CAT mignifkantly dlfferent from contd at P(O.05 rothioate (l), to induce the Ca2’ release from SR. When this compound was used, HX plus X0 did not have any Fig. 1. Effects of hypoxanthine (HX) plus xanthine oxidase (X0) on effect (36.9 t 2.0 % Ca2’ released for control; 34.5 t 4.9 inositol- 1,4,5triphosphate (IP:J -induced Ca”’ release from vascular smooth muscle sarcoplasmic reticulum (SR). SR protein, 300 pg; HX, % for experimental). myo-Inositol 1,4,5-trisphosphoro100 PM; X0, 0.01 U/ml; superoxide dismutase (SOD), 100 U/ml; thioate has been reported to be resistant to both kinasecatalase (CAT), 100 U/ml; temperature, 37°C; pH = 7.0. Values are catalyzed ATP-dependent phosphorylation and phosmeans t SE of 11 replicates from 3 preparations. phatase-catalyzed dephosphorylation (14). Thus our re140 sults are consistent with a hypothesis that 02. may be n=ll stimulating apparent IP3-induced Ca2’ release by inhibiting the processes degrading IP3. This hypothesis should be substantiated by more direct measurements of inositol phosphate metabolites and the Ca2+-channel activities. The present and previous (5, 13) findings strongly confirm the importance of 02. in the alteration of vascular smooth muscle function, especially to the SR. 020 is generally known as a poor oxidant in aqueous solution (2, ll), and the stronger oxidant, HO., was felt to be a 0 HX/XO HX/XO control HX/XO potentially more toxic agent. Subsequent generation of SOD CAT HO. from 0~0, however, requires more reactions, includload of vascular smooth muscle Fig. 2. Effect of HX plus X0 on Ca*’ ing a slow Fenton reaction. It also requires an iron SR. Values are means t SE of 11 replicates from 3 preparations. catalyst of which its in vivo concentration recently has Dosages, temperature, and pH are same as in Fig. 1. been questioned for biologically relevant HO formation by some researchers (6). The greater reactivity of HO. release, involving inhibition of the Ca2+-uptake activity, also presents a problem because HO., once formed, can is the presumption of a simple balanced pump-leak sysreact indiscriminately with many molecules. Thus it is tem. If the magnitude of the unidirectional fluxes in such unlikely to reach a critical macromolecular target unless a system are large, inhibition of the pump will produce a its generation occurs site specifically at the target (4). transient apparent release, i.e., a net efflux. The results 020, on the other hand, does not require any additional shown in Fig. 2 suggest that neither of the above mechreactions or iron catalysts. Since 0;. is generally a poor anisms could explain the observed stimulation of IPSreactant to many molecules, its concentration is more induced release because there was little or no change in likely to be conserved; thus it is available to react with Ca2+ load (and therefore presumably Ca2+ uptake) under any susceptible molecules. If any functional molecule is our experimental conditions. The findings of Fig. 2 may reactive to O& it can be a site of damage caused by ROI. at first seem to contradict both work from this laboratory Some biologically important molecules have been shown as well as that of others that demonstrated that 0~. to be affected by 0;. (4, 9). inhibits Ca2+ uptake by vascular smooth muscle SR (5, Recently, Katusic and Vanhoutte (7) reported that 13). This finding and our present results are not contra0~. generated by X0 plus xanthine in the presence of dictory if one examines the protein concentrations inCAT caused contraction of the vascular smooth muscle volved. Although we previously demonstrated that 0~. in endothelium-denuded canine basilar arteries. They can inhibit up to 75% of the Ca2’-ATPase present when suggested that 0~. may be an endothelium-derived conthe cuvette contains 20 pg/ml SR protein, demonstration tracting factor. It is possible that the effects of 020 on of Ca”’ release requires a much hi .gher protein concenthe SR function that we have described may relate to the tration, (300 pg/ml in the present study). Assuming no mechanisms of such 026 -induced contraction of vascular change in the rate of 0~. generation, it should take smooth muscle. Both actions, inhibition of the SR Cat’substantially longer to produce inhibition at higher tarATPase and potentiation of IP3-induced Ca2+ release, get concentrations. We tested this assumption briefly by would lead to a transient increase in cytosolic Ca2+, and examining the inhibition of Ca2+-ATPase achieved by possibly a contraction, followed by depletion of the SR treatment with 100 PM HX plus 0.01 U/ml X0 for 30 Ca2+ stores. min at both 20 and 80 pg/ml SR protein. Using the technique and assay previously described (l3), we found This study was supported in part by a grant-in-aid from American 74% (73.9 t 7.8, n = 5) inhibition with 20 pg/ml SR Heart Association/Virginia Affiliate to G. D. Ford. l
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
H116
020
Address University Received
STIMULATES
for reprint request: Y. J. Suzuki, 251 Life Science of California, Berkeley, CA 94720. 21 March
1991; accepted
in final
form
23 August
IPR-INDUCED Addition, 1991.
REFERENCES 1. Cooke, A. M., R. Gigg, and B. V. L. Potter. myo-Inositol1,4,5trisphosphorothioate: a novel analogue of a biological second messenger. J. Chem. Sot. Chem. Commun. 20: 1525-1526, 1987. 2. Fee, J. A. Superoxide, superoxide dismutases and oxygen toxicity. In: Metal Ion Activation of Dioxygen, edited by T. G. Spiro. New York: Wiley, 1980, p. 209-237. B. A., and J. D. Crapo. Biology of disease. Free 3. Freeman, radicals and tissue injury. Lab. Inuest. 47: 412-426, 1982. 4. Fridovich, I. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247: l-11,1986. 5. Grover, A. K., and S. E. Samson. Protection of Ca pump of coronary artery against inactivation by superoxide radical. Am. J. Physiol. 256 (Cell Physiol. 25): C666-C673, 1989. 6. Halliwell, B. A radical approach to human disease. In: Oxygen Radicals and Tissue Injury Symposium, edited by B. Halliwell. Bethesda, MD: Fed. Am. Sot. Exp. Biol., 1988, p. 139-143. 7. Katusic, 2. S., and P. M. Vanhoutte. Superoxide anion is an endothelium-derived contracting factor. Am. J. Physiol. 257 (Heart
CA’+
RELEASE
Circ. Physiol. 26): H33-H37, 1989. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 9. McCord, J. M., and W. J. Russell. Superoxide inactivates creatine phosphokinase during reperfusion of ischemic heart. In: Oxy-Radicals in Molecular Biology and Pathology, edited by P. A. Cerutti, I. Fridovich, and J. M. McCord. New York: Liss, 1988, p. 27-35. 1o . Rubanyi, G. M. Vascular effects of oxygen-derived free radicals. Free Radical Biol. Med. 4: 107-120, 1988. D. T., and J. S. Valentine. How super is superoxide? “a Sawyer, Act. Chem. Res. 14: 393-400,198l. A. Measurement of calcium ion concentrations with 12* Scarpa, metallochromic indicators. In: Detection and Measurement of Free Ca2+ in Cells, edited by C. C. Ashely and A. K. Campbell. Amsterdam: Elsevier, 1979, p. 85-115. Y. J., and G. D. Ford. Inhibition of the Ca2’-ATPase 13. Suzuki, of vascular smooth muscle sarcoplasmic reticulum by reactive oxygen intermediates. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H568-H574,1991. 14. Taylor, C. W., M. J. Berridge, A. M. Cooke, and B. V. L. Potter. Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium. Biochem. J. 259: 645-650, 1989.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
J. SUZUKI
AND
GEORGE
D. FORD
Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Suzuki, Yuichiro J., and George D. Ford. Superoxide stimulatesIP3-inducedCa2’ releasefrom vascular smoothmuscle sarcoplasmicreticulum. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): Hll4-Hl16, 1992.-Reactive oxygen intermediates (ROI) have been implicated in a variety of pathophysiological conditions, and vascular smooth muscle may be a site of damagein such oxygen toxicity. Mechanisms of the effects of theseintermediateson vascular smoothmuscleat the cellular level, however, have not been well studied. We have previously shown that xanthine oxidase (X0) -generatedsuperoxideradicals (02.) inhibited the Ca2’-adenosinetriphosphatase of vascular smooth muscle sarcoplasmic reticulum (SR) through mechanismsthat do not involve H202 or hydroxyl radicals. In the present study, we report that the D-myo-inositol 1,4,5trisphosphate(IP&-induced Ca2’ releasefrom bovine aortic SR was alsoaffected by 0; . Hypoxanthine (100PM) plus X0 (10 mu/ml) in the presenceof catalase(100 U/ml) stimulated the IP3-induced Ca2+releasefrom SR monitored using arsenazo III. At 10 PM IP3, the releasewas doubled by 0;. treatment. As a consequence of usingthe higher SR protein concentrations requiredto observethe Ca2+release,this effect wasindependent of Ca2’ uptake inhibition induced by 0;~ Since the effect of 0;. wasnot seenwhen a nonhydrolyzable analogueof IP3 was usedto induce Ca2’ release,0;. may be inhibiting the degradation processesof IP3. aorta; inositol 1,4,5trisphosphate; inositol 1,4,5trisphosphorothioate; oxygen free radicals l
(ROI)havebeenimplicated in a variety of pathophysiological conditions (3). In some of these conditions, the first target of oxygen free radicals is the vascular system (10); however, the mechanisms of the effects of ROI on vascular smooth muscle at the cellular level have not been extensively studied. Recently, the Ca2+-transport activity (5) and Ca2+-adenosinetriphosphatase (ATPase) activity (13) of vascular smooth muscle sarcoplasmic reticulum (SR) have been shown to be inhibited by superoxide anion radicals (0~0) through mechanisms that do not involve hydrogen peroxide (H202) or hydroxyl radicals (HO*). In the present study, we found that another component of the excitation-contraction coupling mechanisms of vascular smooth muscle was affected by 0~0. We report 1,4,5trisphosthat 0;. stimulates the D-myo-inositol phate (IPS) -induced Ca 2+ release from the vascular smooth muscle SR through mechanisms that do not involve Hz02 or HO*. This effect is independent of the inhibition of the Ca2+ pump and any resultant alteration in the pump-leak system. However, the effect seems to be due to dysfunction of the IP3 degradation system present in vascular smooth muscle. REACTIVEOXYGENINTERMEDIATES
(X0; EC 1.1.3.22,from cow’smilk, Boehringer Mannheim) and 100PM hypoxanthine (HX; Sigma). Superoxidedismutase(100 U/ml; SOD; EC 1.15.1.1,from bovine erythrocytes, Sigma)and catalase(100U/ml; CAT; EC 1.11.1.6,from bovine liver, Sigma) were usedas scavengersof 0;. and H202, respectively. These scavengerswere addedto the assaymedia before the initiation of ROI generation. Fresh mature bovine descendingthoracic aortas in ice-cold saline were obtained from Pel-Freez (Rogers, AR). Crude SR vesicleswere prepared from this tissueby a method previously described(13). Protein concentrations were determinedby the method of Lowry et al. (8), using bovine serumalbumin (BSA) asa standard. Ca2’ releasewas measuredusing the metallochromic indicator dye, arsenazo III, and dual-wavelength spectroscopyat 650 and 725 nm as describedby Scarpa (12). For eachexperiment, SR protein (300 pg) was incubated in l-ml cuvettes containing 100 mM KCl, 30 mM imidazole (pH 7.0), 3 mM ATP, 3.1 mM MgC12,and 10 PM CaC12at 37°C for 30 min. Somecuvettes also contained 0.01 U/ml X0 and 100PM HX, whereasothers contained this generating system plus 100 U/ ml SOD and/or 100 U/ml CAT. After the incubation, 200 PM arsenazoIII was addedand a stablerecording of the difference of absorbanceat 650 and 725 nm (AA650-725) established.Then 10 PM of IP3 (Sigma) and 100 PM of A23187 (Sigma) were added sequentially. The Ca2’ releasedby these agents was measuredby noting the difference between the absorbance readingsbefore and after the addition of eachagent.The results are reported as percent Ca2+released,which is defined by the relationship: %Ca2’ released= ([ (AA650-725 before IP3 addition) - (AA650-725 after IP3 addition)]/(Ca2’ load)] X 100,where Ca2+ load was defined by the relationship: Ca2’ load = (AA650-725 before IP3 addition) - (AA650-725 after A23187 addition). Ca2’ load was taken to reflect the total amount of releasableCa2+ taken up by the SR preparation. To determine the effect of reactive oxygen speciesof the hydrolysis of IP3, in someexperiments, IP3 was replaced by 5 PM of the nonhydrolyzable analogue,inositol 1,4,5-trisphosphorothioate(DuPont) (1). Significant differencesbetweentwo groupswere determined by the Student’s t test at P < 0.05.
RESULTS AND DISCUSSION
Figure 1 shows that treatment with 100 PM HX plus 0.01 U/ml X0 stimulated the IP3-induced Ca2+ release from bovine aortic SR. This effect was blocked by SOD but not by CAT, suggesting that stimulation was caused by 020 through mechanisms that do not involve H202 or HO.. Stimulation of release might be due to several mechanisms. First, it could be a matter of how release is defined. For example, if the SR normally holds 100 pmol of Ca2+ with 35 pmol available for release by IPS, we would observe a “release” of 35% of the Ca2+ content. However, if the content was reduced 50% (by inhibition of the Ca2+ uptake process) while the releasable fraction MATERIALS AND METHODS remained the same, then we would observe a release of 0;. were generated by using 0.01 U/ml xanthine oxidase 35/5O or 70%. Another possible mechanism to stimulate
H114
0363-6135/92
$2.00 Copyright
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
0;.
STIMULATES
IP3-INDUCED
CA*+
RELEASE
T-T115 III.L”
protein and 44% (44.4 t 6.3, n = 5) at 80 pg/ml SR protein. This significant reduction in inhibition is consistent with our assumption that the high protein concentration present in the Ca2+ release studies prevented a significant 0;. -induced Ca2’ uptake inhibition. Another possible mechanism for the potentiation of IPS-induced Ca2+ release is inhibition of the processes catabolizing the IP3, thus increasing the effective IP3 concentration. One approach to test whether 020 is indeed affecting IP3 metabolism is using a nonhydrolyzHX/XO HX/XO control HX/XO able analogue of IP3, such as inositol 1,4,5-trisphosphoSOD CAT mignifkantly dlfferent from contd at P(O.05 rothioate (l), to induce the Ca2’ release from SR. When this compound was used, HX plus X0 did not have any Fig. 1. Effects of hypoxanthine (HX) plus xanthine oxidase (X0) on effect (36.9 t 2.0 % Ca2’ released for control; 34.5 t 4.9 inositol- 1,4,5triphosphate (IP:J -induced Ca”’ release from vascular smooth muscle sarcoplasmic reticulum (SR). SR protein, 300 pg; HX, % for experimental). myo-Inositol 1,4,5-trisphosphoro100 PM; X0, 0.01 U/ml; superoxide dismutase (SOD), 100 U/ml; thioate has been reported to be resistant to both kinasecatalase (CAT), 100 U/ml; temperature, 37°C; pH = 7.0. Values are catalyzed ATP-dependent phosphorylation and phosmeans t SE of 11 replicates from 3 preparations. phatase-catalyzed dephosphorylation (14). Thus our re140 sults are consistent with a hypothesis that 02. may be n=ll stimulating apparent IP3-induced Ca2’ release by inhibiting the processes degrading IP3. This hypothesis should be substantiated by more direct measurements of inositol phosphate metabolites and the Ca2+-channel activities. The present and previous (5, 13) findings strongly confirm the importance of 02. in the alteration of vascular smooth muscle function, especially to the SR. 020 is generally known as a poor oxidant in aqueous solution (2, ll), and the stronger oxidant, HO., was felt to be a 0 HX/XO HX/XO control HX/XO potentially more toxic agent. Subsequent generation of SOD CAT HO. from 0~0, however, requires more reactions, includload of vascular smooth muscle Fig. 2. Effect of HX plus X0 on Ca*’ ing a slow Fenton reaction. It also requires an iron SR. Values are means t SE of 11 replicates from 3 preparations. catalyst of which its in vivo concentration recently has Dosages, temperature, and pH are same as in Fig. 1. been questioned for biologically relevant HO formation by some researchers (6). The greater reactivity of HO. release, involving inhibition of the Ca2+-uptake activity, also presents a problem because HO., once formed, can is the presumption of a simple balanced pump-leak sysreact indiscriminately with many molecules. Thus it is tem. If the magnitude of the unidirectional fluxes in such unlikely to reach a critical macromolecular target unless a system are large, inhibition of the pump will produce a its generation occurs site specifically at the target (4). transient apparent release, i.e., a net efflux. The results 020, on the other hand, does not require any additional shown in Fig. 2 suggest that neither of the above mechreactions or iron catalysts. Since 0;. is generally a poor anisms could explain the observed stimulation of IPSreactant to many molecules, its concentration is more induced release because there was little or no change in likely to be conserved; thus it is available to react with Ca2+ load (and therefore presumably Ca2+ uptake) under any susceptible molecules. If any functional molecule is our experimental conditions. The findings of Fig. 2 may reactive to O& it can be a site of damage caused by ROI. at first seem to contradict both work from this laboratory Some biologically important molecules have been shown as well as that of others that demonstrated that 0~. to be affected by 0;. (4, 9). inhibits Ca2+ uptake by vascular smooth muscle SR (5, Recently, Katusic and Vanhoutte (7) reported that 13). This finding and our present results are not contra0~. generated by X0 plus xanthine in the presence of dictory if one examines the protein concentrations inCAT caused contraction of the vascular smooth muscle volved. Although we previously demonstrated that 0~. in endothelium-denuded canine basilar arteries. They can inhibit up to 75% of the Ca2’-ATPase present when suggested that 0~. may be an endothelium-derived conthe cuvette contains 20 pg/ml SR protein, demonstration tracting factor. It is possible that the effects of 020 on of Ca”’ release requires a much hi .gher protein concenthe SR function that we have described may relate to the tration, (300 pg/ml in the present study). Assuming no mechanisms of such 026 -induced contraction of vascular change in the rate of 0~. generation, it should take smooth muscle. Both actions, inhibition of the SR Cat’substantially longer to produce inhibition at higher tarATPase and potentiation of IP3-induced Ca2+ release, get concentrations. We tested this assumption briefly by would lead to a transient increase in cytosolic Ca2+, and examining the inhibition of Ca2+-ATPase achieved by possibly a contraction, followed by depletion of the SR treatment with 100 PM HX plus 0.01 U/ml X0 for 30 Ca2+ stores. min at both 20 and 80 pg/ml SR protein. Using the technique and assay previously described (l3), we found This study was supported in part by a grant-in-aid from American 74% (73.9 t 7.8, n = 5) inhibition with 20 pg/ml SR Heart Association/Virginia Affiliate to G. D. Ford. l
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
H116
020
Address University Received
STIMULATES
for reprint request: Y. J. Suzuki, 251 Life Science of California, Berkeley, CA 94720. 21 March
1991; accepted
in final
form
23 August
IPR-INDUCED Addition, 1991.
REFERENCES 1. Cooke, A. M., R. Gigg, and B. V. L. Potter. myo-Inositol1,4,5trisphosphorothioate: a novel analogue of a biological second messenger. J. Chem. Sot. Chem. Commun. 20: 1525-1526, 1987. 2. Fee, J. A. Superoxide, superoxide dismutases and oxygen toxicity. In: Metal Ion Activation of Dioxygen, edited by T. G. Spiro. New York: Wiley, 1980, p. 209-237. B. A., and J. D. Crapo. Biology of disease. Free 3. Freeman, radicals and tissue injury. Lab. Inuest. 47: 412-426, 1982. 4. Fridovich, I. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247: l-11,1986. 5. Grover, A. K., and S. E. Samson. Protection of Ca pump of coronary artery against inactivation by superoxide radical. Am. J. Physiol. 256 (Cell Physiol. 25): C666-C673, 1989. 6. Halliwell, B. A radical approach to human disease. In: Oxygen Radicals and Tissue Injury Symposium, edited by B. Halliwell. Bethesda, MD: Fed. Am. Sot. Exp. Biol., 1988, p. 139-143. 7. Katusic, 2. S., and P. M. Vanhoutte. Superoxide anion is an endothelium-derived contracting factor. Am. J. Physiol. 257 (Heart
CA’+
RELEASE
Circ. Physiol. 26): H33-H37, 1989. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 9. McCord, J. M., and W. J. Russell. Superoxide inactivates creatine phosphokinase during reperfusion of ischemic heart. In: Oxy-Radicals in Molecular Biology and Pathology, edited by P. A. Cerutti, I. Fridovich, and J. M. McCord. New York: Liss, 1988, p. 27-35. 1o . Rubanyi, G. M. Vascular effects of oxygen-derived free radicals. Free Radical Biol. Med. 4: 107-120, 1988. D. T., and J. S. Valentine. How super is superoxide? “a Sawyer, Act. Chem. Res. 14: 393-400,198l. A. Measurement of calcium ion concentrations with 12* Scarpa, metallochromic indicators. In: Detection and Measurement of Free Ca2+ in Cells, edited by C. C. Ashely and A. K. Campbell. Amsterdam: Elsevier, 1979, p. 85-115. Y. J., and G. D. Ford. Inhibition of the Ca2’-ATPase 13. Suzuki, of vascular smooth muscle sarcoplasmic reticulum by reactive oxygen intermediates. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H568-H574,1991. 14. Taylor, C. W., M. J. Berridge, A. M. Cooke, and B. V. L. Potter. Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium. Biochem. J. 259: 645-650, 1989.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on August 14, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
