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PubMed2005

Theoretical studies of manganese and iron superoxide dismutases: superoxide binding and superoxide oxidation.

Density-functional calculations indicate that the second sphere of coordination around the metal centers of manganese and iron superoxide dismutases (MnSODs and FeSODs) plays an important role in the binding of O2(-). In these systems, O2(-) prefers to bind to Mn or Fe in end-on configurations. For human and E. coli MnSODs, the bound O2(-) forms hydrogen bonds with the tyrosine and glutamine amino acid residues in the second sphere of coordination. In the cases of E. coli and T. elongates FeSODs, hydrogen bonding occurs between the bound O2(-) and the tyrosine amino acid only because the glutamine is too far away for an effective bonding interaction. The manner in which the O2(-) binds to the metal center in MnSODs and FeSODs can affect the rate of subsequent protonation and determine the mechanism for the formation of H2O2. Both Mn- and Fe-containing superoxide dismutases contain a metal-bound solvent molecule that has been suggested to be involved in the uptake of a H+ upon reduction of the metal center [Bull, C.; Fee, J. A. J. Am. Chem. Soc. 1985, 107, 3295; Miller, A.-F.; Padmakumar, K.; Sorkin, D. L.; Karapetian, A.; Vance, C. K. J. Inorg. Biochem. 2003, 93, 71]. Using density-functional theory, we confirm this suggestion and show the involvement of the second sphere of coordination in the process. We show that the oxidation of superoxide by Mn- or Fe-containing superoxide dismutases is facilitated by a cooperative effect between superoxide binding, protonation of the OH- bound to the metal, and electron transfer from the superoxide molecule to the oxidized metal. In particular, proton transfer through tyrosine-34 on the absence of a bound superoxide is uphill while, once superoxide is bound, the energetic barrier is lowered. It is this barrier that likely keeps the resting state (Mn(III)SOD) of the enzyme with a bound hydroxide, instead of a water. This work provides a model for the mechanism of reaction of superoxide with the oxidized form of the metal within Mn- and FeSODs.

Hydrogen Bonding↗

PubMed1988

Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of Rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin.

Superoxide dismutase, which has been shown to be present in a number of tissues, exhibits cyclic changes during the reproductive cycle of rats. An inverse correlation is seen between the levels of superoxide dismutase and superoxide radical. In immature, pseudopregnant rats, primed with human Chorionic Gonadotropin, lutropin seemed to induce ovarian superoxide dismutase, which could be blocked significantly by the introduction of anti-LH serum. These results point out the specific induction of superoxide dismutase by lutropin. It is reasonable to postulate that during luteal functioning, luteinizing hormone induces superoxide dismutase which in turn seems to play a central role generating hydrogen peroxide from superoxide anion radicals. Hydrogen peroxide, thus formed, drives the peroxidase-ascorbate system, responsible for production of progesterone.

Animals↗

PubMed1998

CuZn-superoxide dismutase, extracellular superoxide dismutase, and glutathione peroxidase in blood from individuals homozygous for Asp90Ala CuZu-superoxide dismutase mutation.

The Asp90Ala CuZn-superoxide dismutase mutation is associated with amyotrophic lateral sclerosis (ALS) in both homo- and heterozygous form. We analyzed antioxidant enzymes in blood from 44 individuals homozygous and 114 individuals heterozygous for the Asp90Ala mutation as well as 66 blood relatives carrying the wild-type allele only. Erythrocyte CuZn-superoxide dismutase activity was reduced by 9% in the homozygous individuals, confirming previous findings on a smaller cohort. The specific activity of Asp90Ala mutant CuZn-superoxide dismutase in erythrocytes was equal to that of isolated mutant enzyme and slightly higher than that of isolated wild-type enzyme. There was no evidence for the presence of inactive mutant molecules in erythrocytes, and the lower activity is due to the occurrence of fewer active molecules. There were no significant differences between the groups in plasma extracellular superoxide dismutase content, and the erythrocyte glutathione peroxidase activities were virtually identical. Also, there were no differences in these parameters between homozygous individuals with or without ALS. There was no evidence for any association with ALS of a polymorphic extracellular superoxide dismutase mutation, Arg213Gly. The absence of response of the blood antioxidant enzymes to the Asp90Ala CuZn-superoxide dismutase mutation does not support the theory that the ALS-linked CuZn-superoxide dismutase mutations cause disease by increased oxidant stress.

Alanine↗

PubMed1988

Effect of superoxide dismutase on the autoxidation of various hydroquinones--a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase.

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.

Hydroquinones↗

PubMed2000

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase.

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.

Ferricyanides↗

PubMed1984

Isolation of the Escherichia coli iron superoxide dismutase gene: evidence that intracellular superoxide concentration does not regulate oxygen-dependent synthesis of the manganese superoxide dismutase.

A mixed-sequence synthetic oligodeoxynucleotide probe was used to identify clones within the Escherichia coli genomic library of Clarke and Carbon having an extrachromosomal copy of iron superoxide dismutase. Plasmids pLC13-47 and pLC18-11 were shown to contain the structural gene of the iron superoxide dismutase and to overproduce this protein under conditions of chloramphenicol amplification of plasmid copy number. The activities of both manganese and iron proteins were measured in extracts of host cells and plasmid-bearing cells grown over a wide range of oxygenation. The results confirm previous demonstrations that the manganese protein is repressed under anaerobic conditions and induced in the presence of oxygen. Induction of the manganese protein with increasing oxygenation was quantitatively similar in cells differing approximately equal to 7-fold in iron superoxide dismutase, suggesting that the intracellular concentration of superoxide might not be responsible for regulating synthesis of the manganese-containing superoxide dismutase.

Aerobiosis↗

PubMed1991

Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus.

Superoxide anion radical, a radical whose toxicity is well documented, is present in the uterus of albino rat. The cyclic changes that it undergoes during the reproductive cycle are also illustrated. An inverse correlation is seen between the levels of superoxide radical and the enzyme dismutating it, i.e., superoxide dismutase (EC 1.15.1.1), which is also reported to be present in the uterus of albino rat and is shown to exhibit cyclic changes during its oestrous cycle. The high levels of superoxide radical at pro-oestrus enable us to hypothesize that superoxide may be involved in regulating the oedema and cell proliferation of the uterus during proestrus.

Animals↗

PubMed1994

Effects of oxidative stress on expression of extracellular superoxide dismutase, CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts.

To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.

Cell Line↗

PubMed1998

Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase in the rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1alpha (IL-1alpha), IL-beta, IL-6, and tumor necrosis factor alpha. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,Zn-SOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

Animals↗

PubMed2006

Superoxide auto-augments superoxide formation and upregulates gp91(phox) expression in porcine pulmonary artery endothelial cells: inhibition by iloprost.

Central to the aetiology of Acute Respiratory Distress Syndrome (ARDS) is superoxide, the principal source of which is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). To test whether superoxide may influence NADPH oxidase expression directly, the effect of incubation of superoxide with porcine pulmonary arterial endothelial cells on the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) and superoxide formation was investigated. Since iloprost has been purported to be potentially effective in treating ARDS, the effect of iloprost on superoxide-mediated effects was also studied. Pulmonary artery endothelial cells were incubated with xanthine/xanthine oxidase which generates superoxide, or tumour necrosis factor alpha (TNFalpha) or thromboxane A(2) analogue, U46619 (+/- superoxide dismutase [SOD] or catalase or iloprost) for 16 h. Cells were then washed and superoxide formation assessed spectrophometrically and gp91(phox) expression using Western blotting. The role of NADPH oxidase was also studied in the above settings using apocynin, an NADPH oxidase inhibitor. Superoxide, TNFalpha and U46619 elicited an increase in the formation of superoxide and induced gp91(phox) expression in pulmonary artery endothelial cells following a 16 h incubation an effect blocked by the continual presence of SOD and apocynin but not catalase. Apocynin completely inhibited superoxide formation induced with xanthine/xanthine oxidase after the 16 h incubation. Rotenone and allopurinol were without effect. Iloprost inhibited the formation of superoxide and gp91(phox) expression. These data demonstrate that superoxide upregulates gp91(phox) expression in pulmonary artery endothelial cells and thus augments superoxide formation, an effect blocked by iloprost. This constitutes a novel mechanism by which vascular superoxide creates a self-perpetuating cascade that may be of importance to the etiology of ARDS and other vasculopathies.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2004

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry.

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.

Ascorbic Acid↗

PubMed1984

Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.

Animals↗

PubMed1980

Distribution of CuZn superoxide dismutase and Mn superoxide dismutase in human tissues and extracellular fluids.

Among various human tissues liver had the highest content of both CuZn superoxide dismutase and Mn superoxide dismutase. There were comparatively small differences in CuZn superoxide dismutase among other organs. The Mn superoxide dismutase contents were roughly half as large as the CuZn superoxide dismutase contents. The total amount of CuZn superoxide dismutase in the body was estimated to be 3900 mg. The superoxide dismutase activities of serum and cerebrospinal fluid corresponded to between 0.2-0.3 mg CuZn superoxide dismutase per liter. CuZn superoxide dismutase was apparently the source of the superoxide dismutase activity in cerebrospinal fluid, whereas this enzyme only accounted for about 15% of the serum activity. The superoxide dismutase activities of ceruloplasmin, transferrin and ferritin were very low, 40 000, 350 000 and 330 000 times lower than that of human CuZn superoxide dismutase. These factors contributed very little to serum superoxide dismutase activity. The activity of lymph was more than twice that of serum. Sera from patients with severely impaired renal function or bilaterally nephrectomized had a very high superoxide dismutase activity.

Female↗

PubMed1978

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.

Amitrole↗

PubMed1986

Ceruloplasmin, extracellular-superoxide dismutase, and scavenging of superoxide anion radicals.

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 X 10(4) M-1 s-1 per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 X 10(5) M-1 s-1 per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.

Ceruloplasmin↗

PubMed1994

Effects of recombinant superoxide dismutase on manganese superoxide dismutase gene expression in gerbil hippocampus after ischemia.

BACKGROUND AND PURPOSE: We reported that recombinant human superoxide dismutase ameliorates delayed neuronal death in the postischemic gerbil hippocampus. Since postischemic induction of copper-zinc superoxide dismutase messenger RNA was abolished by this treatment, oxygen radicals generated on reperfusion may induce the expression of this gene. In the present study we examined whether oxygen radicals also induce the expression of manganese superoxide dismutase messenger RNA in the postischemic brain. METHODS: We induced transient cerebral ischemia by occluding the bilateral common carotid arteries of gerbils. Recombinant human superoxide dismutase (8 x 10(5) U/kg) or apo-superoxide dismutase was administered intravenously 1 minute before a 5-minute occlusion of the carotid arteries. We analyzed both copper-zinc and manganese superoxide dismutase RNA by in situ hybridization histochemistry and by Northern and dot blot analyses using radioisotope-labeled oligonucleotide probes. RESULTS: Hybridization with the manganese superoxide dismutase messenger RNA occurred at the limit of detection in normal CA1 neurons. We observed striking increases in the labeling of CA1 up to 24 hours after 5 minutes of ischemia. The hybridization occurred anew in glial cells of the CA1 layer during 3 to 7 days. Pretreatment with recombinant human superoxide dismutase had no effect on the postischemic induction of manganese superoxide dismutase messenger RNA, whereas the same treatment significantly attenuated (P < .01) the increase in copper-zinc superoxide dismutase messenger RNA. CONCLUSIONS: Our results demonstrated temporal postischemic induction of manganese superoxide dismutase messenger RNA. The inducer may not be superoxide radicals but may be other chemical mediators such as cytokines.

Amino Acid Sequence↗

PubMed1981

Copper- and zinc-containing superoxide dismutase and manganese-containing superoxide dismutase in human tissues and human malignant tumors.

Superoxide dismutases might conceivably protect against both ionizing radiation and free radical-producing antibiotic antitumor drugs. Copper- and zinc-containing superoxide dismutase (CuZn superoxide dismutase) and manganese-containing superoxide dismutase (Mn superoxide dismutase) were specifically assayed in human malignant tumors and for comparison in human tissues. The tumors possessed less CuZn superoxide dismutase than did the more metabolically active tissues, but there was a large overlap between the tissue and the tumor levels. Mn superoxide dismutase was found in all tumors, and the ratio between the activities of CuZn superoxide dismutase and Mn superoxide dismutase was not different from that of the normal tissues. Human tumors are thus different from tumors from other species which have been reported to be deficient or very low in Mn superoxide dismutase. There was no obvious relation between sensitivity to ionizing radiation and content of the enzymes among the tumors and the tissues, nor did tumor types known to be responsive to radical-producing drugs possess less CuZn superoxide dismutase or Mn superoxide dismutase than other tumors.

Adult↗

PubMed2006

Simultaneous measurement of superoxide generation and intracellular Ca2+ concentration reveals the effect of extracellular Ca2+ on rapid and transient contents of superoxide generation in differentiated THP-1 cells.

We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca2+ concentration ([Ca2+]i) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated superoxide response occurred without change in [Ca2+]i. The cells treated with TMB-8, an intracellular Ca2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca2+]i elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca2+]i elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca2+ influx from the extracellular domain. Verapamil, voltage-dependent Ca2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca2+ release or extracellular Ca2+ influx, respectively. Taking these results together, the characteristic extracellular Ca2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca2+]i.

Calcium↗