Molecular evidence for the role of a ferric reductase in iron transport.

Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brush-border membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/DMT1 (divalent metal-transporter 1). The protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat.

BACKGROUND: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation. AIMS: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat. METHODS: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control. RESULTS: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin. CONCLUSIONS: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.

The ferric-reducing activity of duodenal brush-border membrane vesicles is associated with a b-type haem.

Rabbit brush-border membrane vesicles possess ferricyanide reducing activity. This activity is preferentially dependent on NADH as reductant, and can be stimulated by the addition of FMN. The latency of activity observed following vesicle solubilisation suggests that the responsible component is transmembranous, and partially sequestered on the inner-face of the vesicles prior to full solubilisation. Subsequent increases in detergent concentration (> 0.3% w/v lauryl maltoside) were found to be inhibitory. Ferricyanide reducing activity was effectively inhibited by the sulphydryl modifying reagents N-ethyl malemide and p-chloromercuribenzoate, but not by the flavin analogue diphenylene iodonium. The ferric-reducing activity co-purified with a b-type haem when applied to Sephacryl S-200 columns. The putative cytochrome was found to be immunologically distinct from neutrophil cytochrome b558.

Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in the rat.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal damage by a non-prostaglandin (PG) dependent "topical" action and by inhibiting cyclooxygenase. AIMS: To discriminate between these two effects by studying some key pathophysiological steps in NSAID enteropathy following administration of (R)- and (S)-flurbiprofen, the racemic mixture, and an uncoupler, dinitrophenol. METHODS: The effects of dinitrophenol, racemic, (R)-, and (S)-flurbiprofen on mitochondria were assessed in vitro and on key pathophysiological features of small intestinal damage in vivo (ultrastructure by electron microscopy, mucosal prostanoid concentrations, intestinal permeability, inflammation, and ulcer count) in rats. RESULTS: All the drugs uncoupled mitochondrial oxidative phosphorylation in vitro, caused mitochondrial damage in vivo, and increased intestinal permeability. Dinitrophenol and (R)-flurbiprofen caused no significant decreases in mucosal prostanoid concentrations (apart from a decrease in thromboxane (TX) B2 concentrations following (R)-flurbiprofen) while racemic and (S)- flurbiprofen reduced mucosal prostanoids significantly (PGE, TXB2, and 6-keto-PGF1alpha concentrations by 73-95%). Intestinal inflammation was significantly greater following administration of (S)-flurbiprofen and racemate than with dinitrophenol and (R)-flurbiprofen. No small intestinal ulcers were found following dinitrophenol or (R)-flurbiprofen while both racemic and (S)-flurbiprofen caused numerous ulcers. CONCLUSIONS: Dinitrophenol and (R)-flurbiprofen show similarities in their actions to uncouple mitochondrial oxidative phosphorylation in vitro, alter mitochondrial morphology in vivo, increase intestinal permeability, and cause mild inflammation without ulcers. Concurrent severe decreases in mucosal prostanoids seem to be the driving force for the development of severe inflammation and ulcers.

Effects of decyl-aurachin D and reversed electron transfer in cytochrome bd.

Decyl-aurachin D is a near-stoichiometric inhibitor of cytochrome bd from Azotobacter vinelandii. Interaction of decyl-aurachin D with the oxidase induces a redshift of the alpha-band and Soret band of a b-type cytochrome, probably b-558, suggesting close proximity of the inhibitor binding site to this haem and hence to the proposed quinol binding domain. The compound does not affect the oxygen binding site directly as judged from unchanged CO recombination kinetics to haem d in dithionite-reduced enzyme. Although in the presence of ubiquinol-1 a decyl-aurachin D containing sample generates levels of haem reduction and catalytic intermediates similar to the control, the approach to this steady state is severely inhibited. In addition to the spectral effect on b-558, decyl-aurachin D raises the midpoint potential of haem b-558, but also lowers that of haem b-595. Consistent with the shift in midpoint potentials, electron backflow from haem d to the b-type haems can be observed in decyl-aurachin D inhibited samples following photolysis of the mixed-valence CO-ligated form of the enzyme. The data show that decyl-aurachin D acts on the donor side of haem b-558 without substantially affecting internal electron transfer rates or the oxygen reduction site.

Intestinal tolerability of nitroxybutyl-flurbiprofen in rats.

BACKGROUND: Nitric oxide derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) are thought to be much less ulcerogenic than their parent compounds. AIM: To compare the effect and potency of flurbiprofen and nitroxybutyl-flurbiprofen to uncouple mitochondrial oxidative phosphorylation (an early pathogenic event in NSAID enteropathy), increase intestinal permeability (transitional stage), and cause macroscopic small intestinal damage. METHODS: In vitro uncoupling potency was assessed using isolated coupled rat liver mitochondria and in vivo by electron microscopy of rat small intestinal mucosa (two hours after the drugs). A dose-response study with flurbiprofen (single doses of 5, 10, 20, and 40 mg/kg) and equimolar doses of nitroxybutyl-flurbiprofen was performed; assessing their effect on intestinal permeability (at 18-20 hours), with 51Cr EDTA, and the number of pointed (< 5 mm) and longitudinal (> 5 mm) small intestinal ulcers at 24 hours. RESULTS: Flurbiprofen, but not nitroxybutyl-flurbiprofen, stimulated coupled respiration in vitro. Both drugs, however, uncoupled in vivo; in the case of nitroxybutyl-flurbiprofen possibly because hydrolysis of its ester bond released free flurbiprofen. Intestinal permeability was uniformly and equally increased with both drugs compared with controls. The number of small intestinal ulcers, pointed and longitudinal, was significantly reduced with nitroxybutyl-flurbiprofen apart from the number of longitudinal ulcers with the highest dose. CONCLUSIONS: These studies show that nitroxybutyl-flurbiprofen is associated with significantly less macroscopic damage in the small intestine than flurbiprofen but was associated with mitochondrial damage in vivo and caused similar increases in permeability of the small intestine, suggesting that its beneficial effect is on the later pathogenic stages of the damage.

Mitochondrial damage: a possible mechanism of the "topical" phase of NSAID induced injury to the rat intestine.

BACKGROUND: The "topical" effect of non-steroidal anti-inflammatory drugs (NSAIDs) seems to be an important cause of NSAID induced gastrointestinal damage. AIM: To examine the possible mechanism of the "topical" phase of damage in the small intestine. METHODS: Electron microscopy and subcellular organelle marker enzyme studies were done in rat small intestine after oral administration of indomethacin (doses varied between 5 and 30 mg/kg). The effect of conventional and non-acidic NSAIDs on rat liver mitochondrial respiration was measured in vitro in a Clarke-type oxygen electrode. RESULTS: The subcellular organelle marker enzymes showed mitochondrial and brush border involvement within an hour of indomethacin administration. Electron microscopy showed dose dependent mitochondrial changes following indomethacin administration consistent with uncoupling of oxidative phosphorylation (or inhibition of electron transport) which were indistinguishable from those seen with the uncoupler dinitrophenol. Parenteral indomethacin caused similar changes, but not in rats with ligated bile ducts. A range of NSAIDs, but not paracetamol or non-acidic NSAIDs which have a favourable gastrointestinal tolerability profile, uncoupled oxidative phosphorylation in vitro at micromolar concentrations and inhibited respiration at higher concentrations. In vivo studies with nabumetone and aspirin further suggested that uncoupling or inhibition of electron transport underlies the "topical" phase of NSAID induced damage. CONCLUSION: Collectively, these studies suggest that NSAID induced changes in mitochondrial energy production may be an important component of the "topical" phase of damage induction.

Nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation.

OBJECTIVE: There is a lack of correlation between cyclooxygenase (COX) inhibition and nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal (GI) damage; it has been suggested that mucosal damage may be initiated by a "topical" action of NSAIDs involving mitochondrial injury. We evaluated the effect of a range of NSAIDs and related compounds on mitochondrial function and assessed the differences between them in relation to their physicochemical properties. METHODS: Stimulation of respiration, as an indicator of mitochondrial uncoupling, was measured in isolated coupled rat liver mitochondrial preparations, using an oxygen electrode. RESULTS: Conventional NSAIDs and acidic prodrugs all had stimulatory effects on mitochondrial respiration at micromolar concentrations (0.02-2.7 microM); higher concentrations were inhibitory. The uncoupling potency was inversely correlated with drug pKa (r = -0.87, P < 0.001; n = 12). Drugs known to have good GI tolerability, including modified flurbiprofen (dimero-flurbiprofen and nitrobutyl-flurbiprofen), nabumetone (a non-acidic prodrug), and non-acidic highly selective COX-2 inhibitors, did not cause uncoupling. CONCLUSION: The ability to uncouple mitochondrial oxidative phosphorylation is a common characteristic of antiinflammatory agents with an ionizable group. Modification or absence of an ionizable moiety reduces the effect on mitochondria and could lead to improved NSAID GI safety.

Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels.

A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.

Mucosal surface ferricyanide reductase activity in mouse duodenum.

Mouse duodenum possesses mucosal surface ferricyanide reductase activity. The reducing activity, determined in vitro by measuring ferrocyanide production from ferricyanide, was found to be greater in duodenal fragments when compared with ileal fragments. Experiments with right-side out tied-off duodenal sacs show that reduction occurs mainly on the mucosal side and indicates that the reducing activity is associated with the brush border membrane. Experiments using mice with increased levels of iron absorption (hypoxic, iron-deficient) showed corresponding increases in reducing activity. The increase was present in duodenal but not ileal fragments. Inhibitor studies showed no effect of several compounds which inhibit other, more characterized, transplasma membrane reductases. In particular, doxorubicin (10 microM) and quinacrine (1mM) were without effect on duodenal mucosal transplasma membrane reducing activity. Depolarization of the membrane potential with high medium K+ inhibited reducing activity. N-ethyl malemide (1 mM) was a potent inhibitor, but iodoacetate was found to be less inhibitory. Comparison with inhibitory effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated that the effect of N-ethyl malemide on reducing activity was not secondary to GAPDH. Collectively these results indicate that mouse duodenum possesses mucosal surface transplasma membrane ferricyanide reductase activity and that the activity is correlated with the process of intestinal iron absorption. Furthermore, the reducing activity appears to be distinct from other reported transplasma membrane reductases.

A suggested mechanism for the catalytic cycle of cytochrome bd terminal oxidase based on kinetic analysis.

The apparent oxygen affinity of cytochrome bd from Escherichia coli and Azotobacter vinelandii has been measured using oxymyoglobin as a sensitive monitor of oxygen concentration. In membrane preparations, the Km(O2) and respiratory rate varied with the nature of the primary substrate used (malate, lactate, reduced nicotinamide adenine dinucleotide (NADH), or ubiquinol-1). At maximum respiratory rates, the Km(O2) for cytochrome bd from A. vinelandii was 4.1 microM, approximately 2 times higher than the corresponding value for the E. coli enzyme. There were no significant differences between the Km(O2) values for membrane-bound and purified cytochrome bd from A. vinelandii when ubiquinol-1 was used as primary substrate. The kinetic parameters Km(O2) and Vmax provide a value of 2.8 x 10(8) M-1 s-1 for the bimolecular rate constant for oxygen reaction with the enzyme, suggesting that this reaction is diffusion-controlled. Kinetic analysis indicates a mechanism involving a ternary complex. A scheme for the reaction mechanism of cytochrome bd is proposed.

Small pH gradients inhibit cytochrome c oxidase: implications for H+ entry to the binuclear center.

Respiring cytochrome c oxidase proteoliposomes generate internal alkalinity (delta pH) and a membrane potential (delta psi). Valinomycin collapses delta psi, increases delta pH, and slows steady state respiration. If delta pH is heterogeneously expressed trapped probes will underestimate it. Internal pH changes were therefore followed in COV containing two buffer systems of differing pKs. The alkalinization rate at pH 7 was unaffected by adding AMPSO (pK 9.0) to the usual internal HEPES (pK 7.5). At higher pH, AMPSO slowed the approach to steady state. delta pH inhibition is therefore not due to a large alkalinization in a small COV fraction. The O2-reducing center may move protons via a local aqueous phase that is near electrical and pH equilibrium with the phase inside the COV. The dielectric in this membrane region can put the center electrically 'inside' even though it is physically 'outside'.

Cytochrome bd oxidase from Azotobacter vinelandii. Purification and quantitation of ligand binding to the oxygen reduction site.

Cytochrome bd has been purified from Azotobacter vinelandii by a new simplified procedure. The heme and total iron content has been measured, as has the number of high affinity CO and NO binding sites. Spectral changes indicate high affinity binding of CO and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2 molecules of heme b or per 3 atoms of iron. The results clearly define a stoichiometry of one heme d per complex. Low affinity binding of CO and NO to heme b595 also occurs at higher ligand concentrations. EPR heme-nitrosyl signals are seen with NO bound to both hemes b595 and d but with no indication of spin exchange coupling. Exposure of the air-oxidized complex to alkaline pH results in removal of molecular oxygen from heme d and a change in line shape of the high spin region of the EPR spectrum. Cyanide binds to both heme d and heme b595 in the air-oxidized complex, displacing molecular oxygen from heme d. The rate of cyanide binding to heme d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to heme b595 as assessed by the loss of the high spin EPR signal. In addition, the cyanide binding rate in the presence of reductant is only 3 times that of the rate of binding to the air-oxidized enzyme, in contrast to the copper-containing oxidases where strong redox cooperativity makes these two rates differ by a factor of at least 10(6). The results do not support the idea of the presence of two strongly interacting hemes in a binuclear center.