Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel.

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.     

Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes

To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.     

Superoxide generation is inhibited by phospholipase A2 inhibitors. Role for phospholipase A2 in the activation of the NADPH oxidase.

The stimulation of O2.- generation by phorbol 12-myristate 13-acetate (PMA) in human neutrophil-derived cytoplasts was inhibited by a variety of phospholipase A2 inhibitors in a concentration-dependent manner. Inhibition was found to be independent of the order of addition of the inhibitor and PMA. The most potent inhibitor, RO 31-4639, inhibited O2.- generation with an IC50 value (concentration causing 50% inhibition) of 1.5 microM. The addition of either arachidonic acid or SDS, in the presence of the inhibitors, was able to restore O2.- generation. The results suggest that arachidonic acid, released by phospholipase A2, is necessary for both the activation and the maintenance of O2.- generation by the NADPH oxidase.     

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex

The Na+-motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated delta psi (inside positive) and delta pH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an electrochemical potential of Na+ by the influx of Na+.     

Proton translocation by cytochrome oxidase in (antimycin + myxothiazol)-treated rat liver mitochondria using ferrocyanide or hexammineruthenium as electron donor.

When O2 was injected into an anaerobic suspension of valinomycin-treated rat liver mitochondria inhibited with rotenone, antimycin, and myxothiazol, a small amount of O2 (0.23-0.33 ng-atom of O/mg of protein) was reduced extremely rapidly (within the 2 s time-resolution of the oxygen electrode). The subsequent steady-state rate of flow of electrons to oxygen was very low [less than 3 nequiv. X s-1 X (g of mitochondrial protein)-1]. In the presence of valinomycin there was a rapid ejection of protons synchronous with the rapid phase of O2 consumption corresponding to 0.38-0.61 nequiv. of H+ X (mg of mitochondrial protein)-1. When valinomycin was replaced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) there was a rapid alkalification of the medium corresponding to 0.20-0.42 nequiv. of H+ X (mg of mitochondrial protein)-1. When 2 mM-Fe(CN)6(4-) was present to re-reduce endogenous cytochrome c, O2 consumption was still biphasic but the second phase of O2 consumption was very much more rapid [600 nequiv. X s-1 X (g of protein)-1], and resulted in the virtually complete consumption of the O2 in the pulse within 4 s. With 60 microM-Ru(NH3)6(2+) as reductant, O2 consumption was even faster [1200 nequiv. X s-1 X (g of protein)-1]. In a medium containing 150 mM-choline chloride with Ru(NH3)6(2+) as reductant, the proton per reducing equivalent stoichiometry (delta H+O/e-) was +0.95 in the presence of valinomycin and -0.94 in the presence of FCCP. In choline chloride medium containing Ru(NH3)6(2+) and valinomycin, there was an uptake of K+ ions corresponding to 1.86 K+/e-. It is concluded that nearly 1 proton is translocated outwards through cytochrome oxidase per oxidizing equivalent injected in this medium. In low ionic strength sucrose-based medium, with Ru(NH3)6(2+) as reductant, delta H+O/e- was 1.05 in the presence of valinomycin, and -0.71 in the presence of FCCP. It is concluded that the translocation of protons is accompanied by net acid production in this medium.     

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.     

Detection of a sodium pump in the terminal segment of the bacterial respiratory chain

An alkalo- and halotolerant aerobic microorganism has been isolated which, according to microbiological data and the ribosomal 5S-RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism termed as Bacillus FTU proved to be resistant to the protonophorous uncoupler CCCP. The fast growth of Bacillus FTU in the presence of CCCP was shown to require high Na+ concentrations in the medium. A procedure has been developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only upon addition of an exogenous respiratory substrate. The exhausted cells were found to oxidize ascorbate in the presence of TMPD in a cyanide-sensitive fashion. Ascorbate oxidation was coupled to the uphill Na+ extrusion stimulated by CCCP and a penetrating weak base, diethylamine (DEA), as well as by valinomycin with or without DEA. The operation of the Bacillus FTU terminal oxidase resulted in the generation of delta psi which, in a Na+ medium, was slightly decreased by CCCP and strongly by CCCP + DEA. In a K+ medium CCCP discharged delta psi even without DEA. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium. CCCP + DEA were inhibitory in both media. The data obtained indicate that there is a Na+-motive terminal oxidase in Bacillus FTU. It is suggested that delta microNa formed by the oxidase can be utilized by an Na+-driven ATP-synthase.     

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.     

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.     

Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.     

NMR evaluation of ammonium ion movement within a unimolecular G-quadruplex in solution

d[G4(T4G4)3] has been folded into a unimolecular G-quadruplex in the presence of 15NH4+ ions. NMR spectroscopy confirmed that its topology is the same as the solution state structure determined earlier by Wang and Patel (J. Mol. Biol., 1995; 251: 76-94) in the presence of Na+ ions. The d[G4(T4G4)3] G-quadruplex exhibits four G-quartets with three 15NH4+-ion-binding sites (O1, I and O2). Quantitative analysis utilizing 15NH4+ ions as a NMR probe clearly demonstrates that there is no unidirectional 15NH4+ ion movement through the central cavity of the G-quadruplex. 15NH4+ ions move back and forth between the binding sites within the G-quadruplex and exchange with ions in bulk solution. 15NH4+ ion movement is controlled by the thermodynamic preferences of individual binding sites, steric restraints of the G-quartets for 15NH4+ ion passage and diagonal versus edge-type arrangement of the T4 loops. The movement of 15NH4+ ions from the interior of the G-quadruplex to bulk solution is faster than exchange within the G-quadruplex. The structural details of the G-quadruplex define stiffness of individual G-quartets that intimately affects 15NH4+ ion movement. The stiffness of G-quartets and steric hindrance imposed by thymine residues in the loops contribute to the 5-fold difference in the exchange rate constants through the outer G-quartets.     

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes

Direct stretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl(-) SAC resemble those of the volume-sensitive Cl(-) current, I(Cl,swell). Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), an AT1R competitive antagonist, blocked Cl(-) SAC but did not significantly alter the background Cl(-) current in the absence of integrin stretch. AT1R signaling is mediated largely by H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 microM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl(-) SAC elicited by stretch and the background Cl(-) current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl(-) SAC as well as a large fraction of the background Cl(-) current. With continuing integrin stretch, Cl(-) SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl(-) current that was rapidly and completely blocked by DPI (60 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), the eventual product of NADPH oxidase activity, also activated Cl(-) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 microM) or AEBSF (0.5 mM) did not fully reactivate Cl(-) SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl(-) SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl(-) SAC, providing a second regulatory pathway.     

Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein.

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.     

Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule. Effect of oxygen

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital-treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro-light-guides. Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r = 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased. Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH4Cl infusion in each region, measured with micro-light-guides and the rate of urea synthesis by the whole organ. When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 +/- 31 mumol g-1 h-1 and 61 +/- 24 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 +/- 23 mumol g-1 h-1 in periportal areas and 152 +/- 19 mumol g-1 h-1 in pericentral regions. During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 +/- 76 mumol g-1 h-1 and 72 +/- 34 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 +/- 17 mumol g-1 h-1 and 387 +/- 99 mumol g-1 h-1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 mumol g-1 h-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force.

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Reversible activation of NADPH oxidase in membranes of HL-60 human leukemic cells

NADPH oxidase in membranes of undifferentiated and dimethylsulphoxide-differentiated HL-60 cells was activated by arachidonic acid (AA) in the presence of Mg2+ and a cytosolic cofactor (CF) found in differentiated HL-60 cells. Basal superoxide (O2-) formation was enhanced several-fold by addition of the stable GTP-analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), prior to AA and was completely prevented by that of GDP. Basal and GTP gamma S-stimulated O2- formation was terminated by GDP. In the presence of Mg2+ or EDTA, basal O2- formation ceased after 25 or 10 min, respectively, and was reinitiated by GTP gamma S or GTP gamma S plus Mg2+. Albumin terminated O2- formation, which was reactivated by AA in the presence of GTP gamma S. Our results show that (1) activation of NADPH oxidase in HL-60 membranes is dependent on endogenous GTP, Mg2+, AA and CF, which is induced during myeloid differentiation, and that (2) NADPH oxidase activation is a reversible process modulated by exogenous guanine nucleotides at various stages of activity of NADPH oxidase. We suggest crucial roles of guanine nucleotide-binding proteins in the activation, deactivation and reactivation of the enzyme.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.