N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.     

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophore-sis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodo-nium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopwinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.     

Xanthine oxido-reductase activity in ischemic human and rat intestine

We measured time course and extent of xanthine dehydrogenase (XD) to xanthine oxidase (XO) conversion in ischemic human and rat intestine. To model normothermic no-flow ischemia, we incubated fresh biopsies for 0, 2, 4, 8 and 16 h. At [Formula: See Text] XO was less in humans than in rats [Formula: See Text] while XD was essentially the same [Formula: See Text] After 16 h incubation at 37°C, there was no appreciable XD-to-XO conversion and no change in neither XO nor XD activity in human intestine. In contrast, the rat intestine had [Formula: See Text] ratio doubled in the first 2 h and then maintained that value until [Formula: See Text] In conclusion, no XO-to-XD conversion was appreciable after 16 h no-flow normothermic ischemia in human intestine; in contrast, XO activity in rats increased sharply after the onset of ischemia. An immunohistochemical labelling study shows that, whereas [Formula: See Text] expression in liver tissue is localised in both hepatocytes and endothelial cells, in the intestine that expression is mostly localised in epithelial cells. We conclude that XO may be considered as a major source of reactive oxygen species in rats but not in humans.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase—contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of and H₂O₂ during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.     

Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase-contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O*- and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O*- and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain)

The present study tested the hypothesis that calpain is responsible for the limited proteolytic conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). We compared the effects of various proteases on the activity and molecular weight of a purified preparation of xanthine dehydrogenase from rat liver. In agreement with previous reports, trypsin treatment produced a complete conversion of XD to XO accompanied by a limited proteolysis of XDH from an Mr of 140 kD to an Mr of 90 kD. Treatment with calpain I or calpain II did not produce a conversion from XD to XO nor did it result in partial proteolysis of the enzyme. Similarly, trypsin treatment partially degraded a reversibly oxidized form of xanthine dehydrogenase while calpain I or calpain II were ineffective. The possibility that an endogenous inhibitor prevented the proteolysis of XDH by calpain I or II was excluded by verifying that brain spectrin, a known calpain substrate, was degraded under the same incubation conditions. The results indicate that calpain is not likely to be responsible for the in vivo conversion of XD to XO under pathological conditions.     

Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) is a 300-kDa homodimer that can exist as an NAD+-dependent dehydrogenase (XD) or as an O2-dependent oxidase (XO) depending on the oxidation state of its cysteine thiols. Both XD and XO undergo limited cleavage by chymotrypsin and trypsin. Trypsin selectively cleaved both enzyme forms at Lys184, while chymotrypsin cleaved XD primarily at Met181 but cleaved XO at Met181 and at Phe560. Chymotrypsin, but not trypsin, cleavage also prevented the reductive conversion of XO to XD; thus the region surrounding Phe560 appears to be important in the interconversion of the two forms. Size exclusion chromatography showed that disulfide bond formation reduced the hydrodynamic volume of the enzyme, and two-dimensional gel electrophoresis of chymotrypsin-digested XO showed significant, disulfide bond-mediated, conformational heterogeneity in the N-terminal third of the enzyme but no evidence of disulfide bonds between the N-terminal and C-terminal regions or between XOR subunits. These results indicate that intrasubunit disulfide bond formation leads to a global conformational change in XOR that results in the exposure of the region surrounding Phe560. Conformational changes within this region in turn appear to play a critical role in the interconversion between the XD and XO forms of the enzyme.     

Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme

Xanthine oxidase (XO) was found to convert nitric oxide (NO* ) released from spermine-NONOate to nitroxyl (HNO), the one-electron reduction product of NO*, in the presence of its substrate hypoxanthine under anaerobic conditions. Under these conditions, XO lost its activity. Upon aerobic incubation of XO with its substrate, neither conversion of NO* to HNO nor inactivation of the enzyme was observed. Angeli's salt (an HNO generator) or synthetic peroxynitrite inactivated XO at low concentrations, whereas high concentrations of diethylamine-NONOate (an NO* donor) and SIN-1 (which generates peroxynitrite by releasing both NO* and superoxide) were required to inactivate XO. These results suggest that HNO generated by XO under anaerobic conditions inactivates XO. As both XO and NO* synthase are activated and/or induced in ischemia-reperfusion injury, HNO formed by XO may contribute to pathogenesis by exerting its potent oxidation activity against a variety of biological compounds.     

Adverse immune reactions to gold. I. Chronic treatment with an Au(I) drug sensitizes mouse T cells not to Au(I), but to Au(III) and induces autoantibody formation

Upon weekly i.m. injections of disodium gold thiomalate (Na2AuTM) 100% of A.SW mice produced IgG autoantibodies to antinuclear Ag and nucleolar Ag, respectively; about 70% of C57BL/6 mice produced IgG antinuclear Ag, whereas DBA/2 mice were resistant. Moreover, C57BL/6 mice, but not DBA/2 mice, showed increased mesangial deposits of IgG. These alterations were due not to disodium thiomalate, but to the gold ion of Na2AuTM. An assumed T cell reactivity of susceptible mouse strains to Na2AuTM was tested by means of the direct popliteal lymph node (PLN) assay. However, no distinct PLN reaction to Na2AuTM was detectable. Likewise, AuCl did not induce a PLN reaction. Both Na2AuTM and AuCl contain gold in the Au(I) state. The poor PLN responses to Au(I) contrasted with the strong PLN responses to Au(III) compounds. PLN reactions to Au(III) were dose dependent, T cell dependent, and specific. When Au(III) was reduced to Au(I) by addition of Na2TM or methionine before testing in the PLN assay its sensitizing capacity was significantly decreased. Thus, the oxidation state of gold, i.e., Au(III) vs Au(I), plays a major role for its sensitizing capacity. Therefore, we propose that the Au(I) of Na2AuTM is oxidized to Au(III) before T cells are sensitized and adverse immunologic reactions develop. Results obtained with the adoptive transfer PLN assay indicated that, indeed, repeated i.m. injections of Na2AuTM sensitized A.SW and C57BL/6 splenic T cells to Au(III).     

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.     

Inhibition of xanthine oxidase by theaflavin: Possible mechanism for anti-hyperuricaemia effect in mice

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors. Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a reversible competitive inhibitor and has a significant inhibitory effect on XO with an IC₅₀ value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1 causes the obvious fluorescence quenching of XO, which is mainly driven by hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1 interacts with dozens of amino acid residues surrounded in the active cavity of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine to enter into the site. Furthermore, the results from animal experiments demonstrate that TF1 is effective in reducing serum uric acid in mice. These findings suggest that TF1 may be a potential drug candidate for the treatment of hyperuricaemia.     

Time-dependent changes in superoxide dismutase, catalase, xanthine dehydrogenase and oxidase activities in focal cerebral ischaemia

Time-dependent changes in the activities of antioxidant enzymes and an oxidant enzyme, xanthine oxidase (XO), were detected in primary and peri-ischaemic brain regions during permanent occlusion of the middle cerebral artery (MCAO) in rats. There were no changes in superoxide dismutase (SOD) and catalase (CAT) activities after 3 h of MCAO, whereas antioxidant enzyme activities decreased significantly in ischaemic brain areas following 24 h of ischaemia. After 48 h, the enzyme activities returned to the baseline but then a further increase was observed in ischaemic brain areas by 72 h post-ischaemia. Normally, XO exists as a dehydrogenase (XD), but it is converted to XO which contributes to injury in some ischaemic tissues. The XO activity increased slightly at 3 h after ischaemia, but after 24 h of ischaemia it returned to the baseline and then remained relatively unchanged in ischaemic areas. Pretreatment with allopurinol before ischaemia prevented changes in SOD and CAT activities and attenuated brain oedema during 24 h of ischaemia. Neither XO nor XD activity changed in allopurinol-treated rats at the times of ischaemia. These results indicated that ischaemic brain tissue remained vulnerable to free radical damage for as long as 48 h after ischaemia, and XO was probably not an important source of free radicals in cerebral ischaemia.     

Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia.

Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling. Because hypoxia might promote generation of oxidative stress in vivo, we hypothesized that oxidative stress may play a role in the hypoxia-induced cardiopulmonary changes and examined the effect of treatment with the antioxidant N-acetylcysteine (NAC) in rats. NAC reduced hypoxia-induced cardiopulmonary alterations at 3 wk of hypoxia. Lung phosphatidylcholine hydroperoxide (PCOOH) increased at days 1 and 7 of the hypoxic exposure, and NAC attenuated the increase in lung PCOOH. Lung xanthine oxidase (XO) activity was elevated from day 1 through day 21, especially during the initial 3 days of the hypoxic exposure. The XO inhibitor allopurinol significantly inhibited the hypoxia-induced increase in lung PCOOH and pulmonary hypertension, and allopurinol treatment only for the initial 3 days also reduced the hypoxia-induced right ventricular hypertrophy and pulmonary vascular thickening. These results suggest that oxidative stress produced by activated XO in the induction phase of hypoxic exposure contributes to the development of chronic hypoxic pulmonary hypertension.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.     

Xanthine oxidase activates pro-matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells through non-free radical mechanisms

Reactive oxygen species (ROS) have been implicated in the regulation of matrix metalloproteinases (MMPs). The xanthine/xanthine oxidase (X/XO) reaction has been widely used as a source of exogenous ROS in studying MMPs, but commercial XO has also been known to be contaminated by proteolytic activity, and MMPs are protease sensitive substrate. We have investigated the activation of proMMP-2 by X/XO in cultured vascular smooth muscle cells (SMCs). SMCs were incubated with X/XO (unpurified or purified) or XO alone for 24h. X/XO activated proMMP-2 in a dose-dependent manner. A similar profile was observed using XO. Purified XO produced lower amounts of active MMP-2 compared to unpurified XO. EPR study showed that X/XO, not XO itself, produced superoxide anion, which was completely scavenged by SOD. However, X/XO-induced proMMP-2 activation could not be inhibited by combination of SOD and catalase. Incubation with XO either in cell-free conditioned media or in cells resulted in similar amounts of active MMP-2, suggesting that membrane-type-MMPs were not involved in proMMP-2 activation. This was further confirmed by the lack of inhibitory effect of hydroxamate MMP inhibitor, BB1101. Aprotinin blocked unpurified XO-induced proMMP-2 activation in a dose-dependent manner, demonstrating the proteolytic activity contained in XO is essential. We conclude that proteolytic activity contained in XO, rather the ROS derived from X/XO, is responsible for proMMP-2 activation in cultured SMCs. The results also suggest that caution needs to be taken when interpreting the reported results on activation of MMPs where X/XO had been used as an "authentic" source of superoxide anion.     

Ferroxidases and xanthine oxidase in plasma of healthy newborn infants

In the neonatal period, there is a high iron load, while both the level and molar oxidase activity of ceruloplasmin are low. On the other hand, the neonatal xanthine oxidase (XO) activity is higher than later in life and XO has a significant iron-oxidizing capacity. We therefore studied the physiological contribution of XO to the ferroxidase activity of the plasma in 20 full-term newborn infants. Ferroxidase activity was measured spectrophotometrically, with Fe⁺⁺ as substrate. The uric acid formed by XO was assayed by means of HPLC, with electrochemical detection.

The total ferroxidase activity in the plasma was about one-fourth of the adult level and rapidly increased doubling within 3 days after birth. About 90% of the plasma ferroxidase activity was due to ceruloplasmin, the remainder being accounted for by ferroxidase II. The XO activity underwent a 30% (statistically non-significant) elevation at 24 h, though ferroxidase activity attributable to XO was not detected at any time.

Accordingly, XO does not seem to add substantially to the total iron-oxidizing capacity of the plasma in the neonatal period. The high molar ferroxidase activity is probably of importance at the endothelial cell surface.     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

Auranofin modulated cytoplasmic free calcium in neutrophils by mobilizing intracellular calcium and inhibiting protein kinase

The effect of the lipophilic gold compound, auranofin (AUR) on the calcium homeostasis of human neutrophils treated with or without n-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. In agreement with previous reports, FMLP induced a rapid release of intracellular Ca2+ stores followed by a smaller influx of extracellular Ca2+. AUR and staurosporine enhanced while phorbol 12-myristate 13-acetate suppressed the secondary influx of Ca2+. Mn2(+)-quenching-of-fluorescence studies indicate that phorbol 12-myristate 13-acetate incubation blocked cation entry. AUR or staurosporine potentiation of FMLP effects on cytoplasmic free Ca2+ [( Ca2+]i) was attributed to suppression of negative feedback effects of protein kinase C. AUR (5-45 microM) per se induced a slow release of internal Ca2+ stores followed by a delayed influx of extracellular Ca2+. Control studies showed that AUR did not induce the formation of inositol 1,4,5-trisphosphate, lyse cells, or promote dye leakage. Dithiothreitol suppressed the AUR effect. AUR triggered biphasic but smaller increases in [Ca2+]i of neutrophil cytoplasts. Studies with permeabilized neutrophils showed that AUR directly released Ca2+ from internal stores. By comparison, gold sodium thiomalate, which had no effect on intact cells, also released Ca2+ from permeabilized cells. Present results indicate that AUR modulated [Ca2+]i directly by mobilized Ca2+ from multiple storage sites and indirectly by inhibiting protein kinase C.