Soybean lipoxygenase-catalyzed oxidations by linoleic acid hydroperoxide: different reducing substrates and dehydrogenation of phenidone and BW 755C

Phenidone is not a substrate for dioxygenation by soybean lipoxygenase-1 (L1) but reduces L1Fe(III) into L1Fe(II), as shown by EPR spectroscopy. L1 catalyzes the oxidation of phenidone by 13-HPOD, the hydroperoxide formed by dioxygenation of linoleic acid by L1, with formation of 4,5-dehydrophenidone. Two moles of 13-HPOD are used per mole of phenidone dehydrogenated. Other pyrazoline derivatives such as BW 755C, but also, in a more general manner, different compounds containing phenol, aniline, hydrazine, hydroxylamine or hydrazide functions act as reducing substrates for decomposition of 13-HPOD by L1.     

Lipoxygenase and cyclooxygenase blockade by BW 755C enhances pulmonary hypoxic vasoconstriction

Lipoxygenase products (leukotrienes) have been proposed as the mediators of pulmonary hypoxic vasoconstriction. However, the supporting data are inconclusive because the lipoxygenase and leukotriene receptor blockers that reduce hypoxic vasoconstriction (such as diethylcarbamazine and the FPL's) have confounding effects. We investigated BW 755C, a potent inhibitor of both lipoxygenase and cyclooxygenase, in eight intact anesthetized dogs with acute left lower lobe atelectasis. We examined two manifestations of hypoxic vasoconstriction: shunt fraction, as an inverse indicator of regional constriction in response to local hypoxia, and the pulmonary pressor response to global alveolar hypoxia, as an index of general hypoxic vasoconstriction. During normoxia, shunt fraction, measured using a sulfur hexafluoride infusion, was 32.0 +/- 7.0%. The pulmonary pressor response to hypoxia, defined as the increase in pulmonary end-diastolic gradient produced by 10% O2 inhalation, averaged 4.5 +/- 1.8 mmHg. Then, during normoxia, BW 755C was administered. Shunt fraction fell in all eight dogs from the previous mean of 32% to 25.5 +/- 6.1% (t = 6.5, P less than 0.0005). The hypoxic pressor response rose in all dogs, from the previous 4.5 mmHg to 9.0 +/- 3.5 mmHg (t = 4.5, P less than 0.005). BW 755C enhances hypoxic vasoconstriction, an effect consistent with its activity as a cyclooxygenase inhibitor. These data do not support a substantive role for the lipoxygenase pathway in hypoxic vasoconstriction.     

The salvage of ischaemic myocardium by BW755C in anaesthetised dogs

BW755C, a dual inhibitor of the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism reduces the size of an infarct produced by 60 min of coronary occlusion followed by 5 hours reperfusion in anaesthetised beagles. This effect of BW755C is observed when the drug is given after the period of occlusion, and is independent of any haemodynamic effect. In contrast, indomethacin, which inhibits only the cyclo-oxygenase pathway, did not influence infarct size. It is suggested that the salvage of acutely ischaemic myocardium by BW755C is due to inhibition of lipoxygenase product formation by migrating cells which invade the damaged myocardium to produce an inflammatory response.     

Lipoxygenase and cyclooxygenase blockade by BW755C prevents endotoxin-induced hypotension but not changes in glucose metabolism

Arachidonic acid metabolites are mediators of various pathophysiologic events following endotoxin administration. However, their role in the endotoxin-induced increase in glucose metabolism has not been examined. Rats were administered either saline or BW755C (an inhibitor of both the cyclooxygenase and lipoxygenase pathways) 30 min prior to injection of E. coli endotoxin and whole body glucose kinetics assessed using a constant iv infusion of [6-3H] glucose. Treatment with BW755C prevented the endotoxin-induced hypotension and tachycardia. Endotoxin produced characteristic increases in the plasma glucose (23-70%) and lactate (2- to 9-fold) concentrations, as well as elevations in the rate of glucose appearance (34-63%) and metabolic clearance (40-92%). In contrast to the amelioration in hemodynamics, pretreatment with BW755C did not prevent these alterations in glucose metabolism normally seen after endotoxin. BW755C markedly reduced the endotoxin-induced increase in plasma catecholamine concentrations, but levels were still elevated 2- to 4-fold compared to control values. The results suggest that arachidonic acid metabolites mediate the early hypotensive response following endotoxin, but are not by themselves responsible for the elevated rates of glucose production and utilization.     

BW755C inhibits the 5-lipoxygenase in E-mast cells without affecting degranulation

The inhibitory effect of the drug BW755C on the 5-lipoxygenase pathway was analyzed for bone marrow-derived murine mast cells, termed E-mast cells. The drug prevented the formation of 5-HETE from exogenous [14C]arachidonic acid when IgE-sensitized cells were challenged by the antigen. BW755C also prevented formation of leukotriene C4 in a dose-dependent fashion when IgE-sensitized mast cells, preincubated with the drug, were activated with either the specific antigen or the ionophore. Leukotriene C4 inhibition occurred with a minimal drug preincubation period of 1 min before the cells were subjected to antigen-dependent activation. BW755C did not affect the degranulation response of these cells. Thus in an intact cell system BW755C prevents 5-lipoxygenation of arachidonic acid. Furthermore, this study reveals that even with transmembrane activation of E-mast cells through their IgE-Fc receptors, granule secretion is not dependent upon corresponding metabolites from the 5-lipoxygenase pathway.     

Lipoxygenase and cyclooxygenase blockade by BW755C does not prevent the secondary phase of septic pulmonary hypertension

The infusion of Group B beta hemolytic streptococci (GBS) in newborn animals generates a dual phase pulmonary hypertensive response. The initial, acute phase responds to cyclooxygenase or thromboxane inhibition, and appears to be thromboxane mediated. The second phase is characterized by a more moderate rise in pulmonary vascular resistance, accompanied by an increase in microvascular permeability. It has been speculated that this phase may be leukotriene mediated. In an attempt to clarify this, we have studied and compared the effects of the thromboxane synthetase inhibitor, Dazmegrel (DAZ), and the combined cyclooxygenase/lipoxygenase inhibitor, BW755C, on the cardiopulmonary hemodynamics of the secondary phase of GBS induced pulmonary hypertension in newborn piglets. Ten piglets were infused with GBS, and all animals developed a significant increase in pulmonary artery pressure (to 39 +/- 5 and 36 +/- 5 mmHg for DAZ and BW755C animals respectively). After one hour of GBS, either DAZ or BW755C was administered. Data were collected for another two hours following drug administration. GBS infusion was continued throughout. Both DAZ and BW755C were associated with transient, acute reductions in pulmonary artery pressure (to 22 +/- 5 and 22 +/- 8 mmHg, respectively). However, after 60 minutes, PAP again began to rise in both groups (PAP 30 +/- 5 and 30 +/- 11 mmHg respectively by 240 minutes). There were no differences between the groups at any time. These data do not support a significant role for lipoxygenase products in mediating the secondary phase of septic pulmonary hypertension.     

The mechanism of inactivation of lipoxygenases by acetylenic fatty acids

The inactivation of soybean lipoxygenase by 5,8,11,14-eicosatetraynoic acid was studied in detail. The inactivation was found to be time-dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme-product complex, yielded a Km value for 5,8,11,14-eicosatetraynoic acid of 1.3 microM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s-1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14-eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14-eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14-eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14-eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13LS-hydroperoxy-linoleic acid. These results, as well as the lack of covalent binding of 14C-labelled 5,8,11,14-eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D.T. Downing, D.G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218-223; K.H. Gibson (1977) Chem. Soc. Rev. 6, 489-510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.     

Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l-Lysine in Potato Tuber

The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue.     

Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine.

Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis.     

Mechanisms of inactivation of molybdoenzymes by cyanide

The reduced forms of xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase are inactivated by cyanide. Following gel filtration to remove excess of reductant and cyanide, the isolated enzymes remain inactive. Thiocyanate, a product of inactivation of the oxidized forms of the xanthine- and aldehyde-oxidizing enzymes by cyanide, is not released during inactivation of the reduced enzymes. Studies with [14C]cyanide show that, while stoichiometric binding is required for the onset of inactivation, its continued binding is not essential to maintenance of the inactivated state. Electron paramagnetic resonance and absorption spectroscopic studies on the isolated inactivated enzymes show that prosthetic groups other than molybdenum are fully oxidized but that the molybdenum centers are modified. Reactivation is accomplished by incubation with suitable oxidants. Aerobic reactivation of inactive sulfite oxidase required only 1 eq of ferricyanide/active site. However, under rigorously anaerobic conditions, 3 to 4 mol of ferricyanide/active site were reduced, indicating that the molybdenum centers in the inactive enzyme had been reduced below the levels attained by the native enzyme during catalysis.     

Induction of polyspermic fertilization in sea urchins by the leukotriene antagonist FPL−55712 and the 5−lipoxygenase inhibitor BW755C

The appearance of the nuclear abnormality “crater defect” is described in spermatozoa from the domestic horse (Equadae). Under scanning electron microscopy, the defect appears as a severe depression at any area of the sperm nucleus or as a blister or swelling at some point on the sperm nucleus. Ultrastructurally, the crater appears as a nuclear vacuole containing amorphous material similar to that described in bull and boar sperm. The craters ranged in size from 0.5 to 2 μm in diameter. Within ejaculates of stallions having this defect, the percentages of sperm with crater defects varied widely over time, being as high as 60% and as low as 15%.     

Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C.

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca(2+)-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, beta-carotene, and alpha-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a 'cage' type oxidative modification of PKC.     

Affinity chromatography of lipoxygenases.

A number of aminohexyl agarose derivatives of unsaturated fatty acids have been prepared and evaluated as materials for the affinity chromatography of soybean and pea lipoxygenases. A practical method for a one-stage purification of soybean lipoxygenase-1, with a purification factor of 16, is described, using either linolenate or docosa-4,7,10,13,16,19-hexaenoate as ligands. Results show that alleged competitive inhibitors do not cause sharp elution from the affinity column, and that there is an increasing specificity of binding and sharpness of elution as the proportion of unsaturation in the ligand is increased. These results are discussed in terms of the relative importance of the types of bonding involved in enzyme-substrate binding.     

Mechanisms of sodium channel inactivation

Rapid inactivation of sodium channels is crucial for the normal electrical activity of excitable cells. There are many different types of inactivation, including fast, slow and ultra-slow, and each of these can be modulated by cellular factors or accessory subunits. Fast inactivation occurs by a 'hinged lid' mechanism in which an inactivating particle occludes the pore, whereas slow inactivation is most likely to involve a rearrangement of the channel pore. Subtle defects in either inactivation process can lead to debilitating human diseases, including periodic paralyses in muscle, ventricular fibrillation and long QT syndrome (delayed cardiac repolarization) in the heart, and epilepsy in the CNS.     

Salvage of ischemic rabbit ear by BW-755C and steroids, but not by indomethacin

The effects of anti-inflammatory steroids and BW-755C (both of which inhibit the formation of prostaglandins and leukotrienes were studied in ischemia-induced edema and necrosis of the rabbit ear utilizing an occlusion reperfusion model. The results were compared with cyclooxygenase inhibitors which selectively block the synthesis of prostaglandins without affecting leukotriene production.Significant inhibition of the progression of necrosis was observed with both methyl prednisolone and BW-755C. The cyclooxygenase inhibitors, naproxen and low-dose indomethacin had no effect on the progression of ear necrosis. By contrast, high-dose indomethacin significantly inhibited edema but accelerated the progression of necrosis.     

Salvage of ischemic rabbit ear by BW-755C and steroids, but not by indomethacin

The effects of anti-inflammatory steroids and BW-755C (both of which inhibit the formation of prostaglandins and leukotrienes) were studied in ischemia-induced edema and necrosis of the rabbit ear utilizing an occlusion reperfusion model. The results were compared with cyclooxygenase inhibitors which selectively block the synthesis of prostaglandins without affecting leukotriene production. Significant inhibition of the progression of necrosis was observed with both methyl prednisolone and BW-755C. The cyclooxygenase inhibitors, naproxen and low-dose indomethacin had no effect on the progression of ear necrosis. By contrast, high-dose indomethacin significantly inhibited edema but accelerated the progression of necrosis.     

Effects of LTC4 and BW 755C,a blocker of its synthesis,on contraction in smooth muscle and operation of voltage-dependent calcium channels in nerve cells

The effects were investigated of LTC₄, a synthetic leukotriene, and BW 755C, a blocker of LTC₄ biosynthesis, on the operation of Ca channels at the cell membrane and on contraction of muscle fibers using intracellular dialysis and voltage clamping at the membrane of isolated nerve cells and by recording spontaneous contraction of the uterus in white rats at advanced stages of pregnancy. It was found that 1·10^–7 M LTC₄ stimulates the contraction of the uterus without altering its response to oxytocin application. The same concentration of LTC₄ was found to increase calcium conductance by 60±27%. At the same time, a 25±6 mV shift in peak current-voltage relationship along the voltage axis toward negative values was recorded for calcium current. BW 755C, a blocker of the key enzyme in the lipoxygenase metabolic pathway of arachidonic acid, exerts an action similar to leukotriene on calcium conductance, although brief contraction of the uterus is rapidly replaced by complete inhibition of this activity.Institute of Bioorganic Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 24–31, January–February, 1989.