P450-dependent Pathway Research Articles

The role of eicosanoids in tumor growth and metastasis.

Mobilization of esterified arachidonic acid (AA) from membrane glycerolipid pools represents the key regulatory step in cellular responses to various stimuli, such as growth factors, cytokines, chemokines and circulating hormones. Released AA is metabolized via the cyclo-oxygenase (COX 1 and COX 2), lipoxygenase (LOX) or P450-dependent epoxygenase pathways to generate eicosanoids. In addition to their normal biological activities, such as stimulation of mitogenesis and cellular motility, eicosanoids have also been postulated to contribute to tumorigenesis and to the progression of certain tumor cells. Various AA metabolites have been implicated in a variety of growth-related signaling pathways involving ras (Han et al. 1991), interferon-α, epithelial growth factor (EGF), cyclic adenosine monophosphate, protein kinase C (PKC; Hannigan and Williams 1991; Peppelenbosch et al. 1993; Tang et al. 1995a), mitogen-activated kinases (Rao et al. 1988) and fos (Danesch et al. 1994). Numerous studies have demonstrated a strong correlation between growth-factor-promoted cell proliferation and generation of various COX products, primarily prostaglandins (Nolan et al. 1988).

Multiple arachidonic acid metabolites inhibit sodium-dependent phosphate transport in OK cells

The cytochrome P450-dependent monoxygenase pathway represents a major route for the metabolism of arachidonic acid (AA) in the kidney. In turn, AA metabolites have been shown to affect renal electrolyte metabolism, including sodium transport. Specifically AA, 20-HETE and 12-HETE inhibit sodium-dependent (Na+-Pi) uptake into renal culture cells, and both 12-HETE and 14,15 EET have been shown to reduce renin release from renal cortical slices. Since the bulk of Pi transport occurs in the proximal tubule (PT), and the PT is a major site of AA metabolism, we studied the effect of AA and several of its metabolites on Na+-Pi uptake into PT-like opossum kidney (OK) cells. Incubation of OK cells in AA (10−8M) resulted in 17% inhibition of Pi uptake. Three metabolites of omega-hydroxylation of AA induced significant decreases in Pi uptake: 19R-HETE (10−8M) by 36% (P=0.008), 19 S -HETE (10−8M) by 24% (P=0.002) and 20-COOH-AA (10−8M), a metabolite of 20-HETE, by 25% (P>0.0001). 14,15 EET (10−8M), a breakdown product of AA by the epoxygenase pathway, had the greatest effect on Pi uptake in OK cells. It decreased Pi uptake by 47% (P>0.0001). Addition of the P450 inhibitor, 7-ER (10−8M), to OK cells resulted in a significant stimulation (28%) of Pi uptake (P=0.016). These results indicate that these AA metabolites have a significant inhibitory effect on Na+-Pi uptake in OK cells.

Cytochrome P450-dependent metabolic pathways and glucuronidation in trout liver slices

We investigated the capacity of trout precision-cut liver slices to metabolize xenobiotics and steroids. As a first approach, liver slices were compared with freshly isolated trout hepatocytes, using 7-ethoxycoumarin (7-EC) and testosterone as substrates. Trout liver slices and freshly isolated hepatocytes had a similar capacity for conducting cytochrome P450-dependent metabolism, as indicated by the rate of oxidative metabolism of 7-EC and testosterone, and by the metabolic profile of these substrates. A lower rate of glucuronidation in slices compared with hepatocytes was observed with testosterone (50 μM), whereas the opposite situation occurred with 7-EC used at higher concentration (100 μM). In a second step, we investigated the effect of β-naphthoflavone on 7-EC and testosterone biotransformation, using slices maintained in culture for 24 h, with or without the inducer added. The results were compared with the metabolic rates of these substrates incubated with liver slices originating from trout pretreated in vivo with β-naphthoflavone. Cytochrome P450-mediated rates of 7-EC dealkylation and testosterone hydroxylation decreased to 38 and 55% of the control value, respectively, when incubations were performed in 24-h cultured slices instead of freshly cut slices. Exposure of the slices to 50 μM β-naphthoflavone resulted in about 3 times higher deethylation rate of 7-EC. A similar value was obtained when treatment occurred in vivo. As demonstrated in rat by several authors, liver slices seem a useful and simple tool for studying the metabolic pathways of xenobiotics and steroids and for the assessment of inducers of the CYP1A1 family.

The physiological role of P450-derived arachidonic acid metabolites

Arachidonic acid is metabolized to biologically active substances by three major enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450s. The third pathway, P450-dependent pathway, includes allylic oxidation, omega-hydroxylation, and epoxidation of arachidonic acid. Of these metabolites, the physiological role of 20-hydroxyeicosatetraenoic acid (20-HETE) produced by CYP4A isoforms has been extensively studied. 20-HETE affects ion transport, constricts blood vessels and participates in tubuloglomerular feed back. Increased production of 20-HETE is a major factor in elevating blood pressure in spontaneously hypertensive rats (SHR). We have found that CYP4A2 level in SHR is much higher than that of normotensive rat. Recently, factors of endothelial origin other than nitric oxide and prostaglandins were reported. Inhibitors of P450-dependent arachidonic acid metabolism greatly reduce the vasodilator effect and this factor is speculated to be an epoxide of arachidonic acid. We have isolated CYP2C23 from rat kidney and have found that it produces arachidonic acid epoxides. We have investigated changes in the CYP2C23 levels in physiological and pathophysiological conditions. Multiple pathways of arachidonic acid metabolism by P450 have been reported and the diverse properties of these metabolites and the wide distribution of the P450 system make them prime candidates for participation in regulatory mechanisms of the circulation and transporting epithelia.

Clopidogrel: a review of its mechanism of action

The search for active antiplatelet drugs within the original chemical class of the thienopyridines, led to the discovery of clopidogrel, a novel ADP-selective agent whose antiaggregating properties are several times higher than those of ticlopidine. The antiaggregating properties of this compound are well known and, very recently, new results have clarified its mechanism of action. Clopidogrel is active only after intravenous or oral administration, and no circulating activity has been found in the plasma of treated animals or human volunteers. Experiments in rats have demonstrated that the antiaggregating activity was caused by a shortlasting metabolite generated in the liver by a cytochrome P450-dependent pathway. The antiaggregating property of clopidogrel is caused by an inhibition of the binding of ADP to its platelet receptors, and more specifically to the low affinity receptors, the high affinity binding sites being unaffected by clopidogrel. Several events in the ADP activation process, including adenylyl cyclase down-regulation, protein tyrosine phosphorylation, activation of the GPIIb-IIIa complex, fibrinogen binding, aggregation and release, were inhibited by clopidogrel and indicate their close relationship with the activation of a low affinity receptor by ADP. In contrast, binding of ADP to its high affinity binding sites (clopidogrel-resistant receptors) induced shape change, cytosolic calcium increase and phosphorylations of several other proteins, some events which were clopidogrel-sensitive. Thus, clopidogrel not only constitutes a potent antithrombotic drug in humans but also a good tool to study the effect of ADP on platelets.

Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney.

We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20-HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.

Regulation of Na-K-ATPase activity in the proximal tubule: role of the protein kinase C pathway and of eicosanoids.

To evaluate further the signal transduction mechanisms involved in the short-term modulation of Na-K-ATPase activity in the mammalian kidney, we examined the role of phospholipase C-protein kinase C (PLC-PKC) pathway and of various eicosanoids in this process, using microdissected rat proximal convoluted tubules. Dopamine (DA) and parathyroid hormone (either synthetic PTH1-34 or PTH3-34) inhibited Na-K-ATPase activity in dose-dependent manner; this effect was reproduced by PKC530-558 fragment and blocked by the specific PKC inhibitor calphostin C, as well as by the PLC inhibitors neomycin and U-73122. Pump inhibition by DA, PTH, or arachidonic acid, and by PKC activators phorbol dibutyrate (PDBu) or dioctanoyl glycerol (DiC8) was abolished by ethoxyresorufin, an inhibitor of the cytochrome P450-dependent monooxygenase pathway, but was unaffected by indomethacin or nordihydroguaiaretic acid, inhibitors of the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, respectively. Furthermore, each of the three monooxygenase products tested (20-HETE, 12(R)-HETE, or 11,12-DHT) caused a dose-dependent inhibition of the pump. The effect of DA, PTH, PDBu or DiC8, as well as that of 20-HETE was not altered when sodium entry was blocked with the amiloride analog ethylisopropyl amiloride or increased with nystatin. We conclude that short-term regulation of proximal tubule Na-K-ATPase activity by dopamine and parathyroid hormone occurs via the PLC-PKC signal transduction pathway and is mediated by cytochrome P450-dependent monooxygenase products of arachidonic acid metabolism, which may interact with the pump rather than alter sodium access to it.

The metabolism of testosterone by the periwinkle ( Littorina littorea) in vitro and in vivo: Effects of tributyl tin

Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/liter has been associated with disrupted sexual physiology (imposex) in 40–50 species of marine gastropod. Imposex is a state of pseudohemaphrodism in which females exhibit nonfunctional secondary male characteristics. It has been suggested that the mechanism underlying imposex involves disrupted metabolism of endogenous sex hormones and in particular inhibition of cytochrome P450-dependent aromatization of androgens to estrogens. In the current study, the effects of TBT on the ability of the Periwinkle ( Littorina littorea) to metabolize the androgenic sex steroid testosterone was examined in vitro and in vivo. Microsomes were prepared from ‘digestive gland’ (visceral complex including the gonads) and combined kidney and gill fractions. CO-ligated reduced difference spectra contained peaks at 459 nm and calculated P450 contents of 0.3 and 0.05 nmol/mg. Digestive gland microsomes were found to be capable of oxidative metabolism of testosterone in either the presence or absence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6β-, 6α- and 15β-hydroxytestosterone, 17β-estradiol and an unidentified metabolite possibly 3α-androstene-17β-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the products of the NADPH-dependent steroid 5α-reductase-3α(β)hydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3α(β)androstane-17β-diols (DHT diols). TBT, even at high concentrations up to 100 μM, had only modest effect on P450-dependent testosterone metabolism in vitro, producing increases in androstenedione formation and only 30–40% inhibition of aromatase. In vivo, [ 14C] testosterone was injected directly into the cephalopedal sinus of adult snails and animals maintained in sea water at 15 °C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost completely metabolized during this time, predominantly to water-soluble sulfur conjugates of testosterone, the 5α-reduced products and 6α-hydroxytestosterone. However, with increasing concentrations of TBT, more radioactivity was retained within the animal and was increasingly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DHT and DHT-diols. Thus it appears that, in vivo, TBT inhibits sulfur conjugation of testosterone and its phase I metabolites and their excretion resulting in a build-up of pharmacologically active androgens in the tissues. This data are consistent with the hypothesis that TBT-induced imposex in sensitive gastropods, such as stenoglossans, may arise from peturbations in sex steroid metabolism. However, the major biochemical targets of TBT appear to be steroid conjugation and excretory transport mechanisms rather than P450-dependent oxidative pathways such as aromatase.

A dapsone-induced blood dyscrasia in the mouse: evidence for the role of an active metabolite.

In the female mouse, dapsone (50-500 mg kg-1, p.o.) caused a dose-related methaemoglobinaemia which peaked at 0.5-1 h with recovery to baseline values occurring by 4 h. Cimetidine (100 mg kg-1, p.o.), a known inhibitor of several hepatic P450 isozymes administered 1 h before dapsone, prevented the methaemoglobinaemia. In-vitro, dapsone required activation by mouse hepatic microsomes to cause methaemoglobin formation in mouse erythrocytes and cytotoxicity to human mononuclear leucocytes. In both instances, the toxic effects were markedly reduced by cimetidine. Daily dosing of mice with dapsone (50 mg kg-1, p.o.) for 3 weeks induced a blood dyscrasia, characterized by a fall of platelet and white blood cell counts, which was inhibited by cimetidine (100 mg kg-1, p.o. daily). It is concluded that an active metabolite of dapsone arising from a P450-dependent pathway is involved in the genesis not only of the methaemoglobinaemia but also the blood dyscrasia arising from repeated administration of the drug in this species.

Intracellular signaling in the regulation of renal Na-K-ATPase. II. Role of eicosanoids.

We recently reported a novel intracellular mechanism of renal Na-K-ATPase regulation by agents that increase cell cAMP, which involves protein kinase A-phospholipase A2 and is mediated by one or more arachidonic acid metabolites (Satoh, T., H. T. Cohen, and A. I. Katz. 1992. J. Clin. Invest. 89:1496). The present studies were, therefore, designed to assess the role of eicosanoids in the modulation of Na-K-ATPase activity in the rat cortical collecting duct. The effect of various cAMP agonists (dopamine, fenoldopam, vasopressin, forskolin, and dibutyryl cAMP), which inhibited the pump to a similar extent (approximately 50%), was independent of altered Na entry as it was elicited in the presence of amiloride or nystatin, or when NaCl was replaced with choline Cl. This effect was completely blocked by SKF 525A or ethoxyresorufin, two inhibitors of the cytochrome P450-dependent monooxygenase pathway, or by pretreating the animals with CoCl2, which depletes cytochrome P450. Equimolar concentrations (10(-7) M) of the cyclooxygenase inhibitors indomethacin or meclofenamate caused only a partial inhibition of the cAMP agonists' effect on the pump, whereas nordihydroguaiaretic acid or A 63162, two inhibitors of the lipoxygenase pathway, were without effect. Furthermore, two products of this pathway, leukotriene B4 and leukotriene D4, had no effect on Na-K-ATPase activity, and ICI 198615, a leukotriene receptor antagonist, did not alter pump inhibition by cAMP agonists. Several P450 monoxygenase arachidonic acid metabolites (5,6-epoxyeicosatrienoic acid; 11,12-epoxyeicosatrienoic acid; 11,12-dihydroxyeicosatrienoic acid; and 12(R)-hydroxyeicosatetraenoic acid) as well as PGE2 inhibited the Na:K pump in dose-dependent manner, but the effect of PGE2 was blocked when Na availability was altered, whereas that of 12(R)-HETE remained unchanged. We conclude that the cytochrome P450-monooxygenase pathway of the arachidonic acid cascade plays a major role in the modulation of Na:K pump activity by eicosanoids in the rat cortical collecting duct, and that products of the cyclooxygenase pathway may contribute to pump inhibition indirectly, by decreasing intracellular Na.

Immunohistochemical Demonstration of Ethanol-Inducible

The microsomal ethanol-oxidizing system (MEOS) is a P450-dependent pathway for ethanol oxidation in hepatic microsomes. The bulk of MEOS activity is catalyzed by P450 2E1 (an ethanol-inducible isozyme of P450) in animal livers treated with ethanol. Rat brains also metabolize certain drugs, and it is theorized that a mechanism for drug metabolism exists within the brain which acts on P450. We compared immunohistochemical localization of P450 2E1 between ethanol-treated rats and pair-fed control rats. In control rats, immunoreactive P450 2E1 was detected in minute amounts in both basal ganglia and cerebellar cortices. After ethanol treatment, the content of P450 2E1 increased in the basal ganglia, and the enzyme was also induced in the cerebellar cortices, substantia nigra and hippocampus. We speculate that the rat brain metabolizes ethanol by P450 2E1.

Biomechanisms of cocaine-induced hepatocyte injury mediated by the formation of reactive metabolites

Cocaine is an intrinsic hepatotoxin in laboratory animals, and there is growing evidence that high doses of cocaine can precipitate hepatic necrosis in humans. The rodent model of cocaine hepatotoxicity is commensurate with the concept that a multistep mainly cytochrome P-450 dependent N-oxidative pathway is responsible for the expression of hepatocellular injury. Among the possible biomechanisms by which cocaine exerts its cytotoxic effects, direct oxidative damage by reactive oxygen species generated by redox cycling during the metabolic cascade seems most important. The role of the ensuing lipid peroxidation and protein thiol oxidation is less clear. Similarly, the functional role of irreversible (covalent) binding of a not yet defined electrophilic cocaine intermediate to hepatocellular proteins remains enigmatic so long as the critical molecular targets have not been identified. Finally, glutathione plays a pivotal protective role against cocaine-induced hepatic injury. Interactions with ethanol or inducers of the expression of the cytochrome P-450IIB subfamily can potentiate cocaine hepatotoxicity. Thus, the net amount of the ultimate reactive species seems to determine the severity of the hepatic lesions and to be responsible for the marked interspecies, interstrain, and sex differences. Recent advances in culture techniques of hepatocytes and precision-cut liver slices from various species including man have made it possible to correlate cocaine biotransformation with cytotoxicity and to selectively study the putative cellular mechanisms. Clearly, more studies are necessary to further illuminate our understanding of the role of the biochemical and molecular events precipitating hepatic necrosis during cocaine-mediated hepatotoxicity.

Arachidonic acid metabolites by cytochrome P-450 dependent monooxygenase pathway in bovine adrenal fasciculata cells

[1- 14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min at 37°C. The metabolites were separated and purified by reverse phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15-DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 μM.

Eicosanoids and heart disease

The discovery of prostaglandins over 5 decades ago heralded a new era in the study of mediators or factors involved in physiological and pathophysiological processes. Prostaglandins, however, do not represent the sole products derived from arachidonic acid or other fatty acid precursors; instead these fatty acids can undergo metabolism to numerous bioactive compounds derived from cyclooxygenase, lipoxygenase, as well as the recently discovered epoxygenase cytochrome P-450 dependent pathways. Collectively, these products are commonly referred to as eicosanoids and have been implicated in numerous biological phenomena. It has long been hypothesized that the prostaglandins play an important role in cardiovascular regulation. For instance, the concept of a balance between the vasoconstrictor, prothrombotic properties of some arachidonic acid derived products and the opposing actions of others led to the development of drug therapies against thromboembolic disorders. Aspirin, for example, a well-known cyclooxygenase inhibitor, has been shown to be efficacious against myocardial infarction and stroke of embolic origin. Moreover, cyclooxygenase- and lipoxygenase-derived products have been implicated in various types of cardiac dysfunction including coronary constriction, arrhythmogenesis, anaphylactic reactions, or ischemic and reperfusion injury.In view of the evolving complexity of arachidonic acid metabolism and increasing evidence that eicosanoids, either alone, or through interaction with other substances, represent important mediators in either the development of heart disease or the myocardial response to injury, we organized this symposium entitled Eicosanoids and Heart Disease concurrent with the 31st Annual Meeting of the Canadian Federation of Biological Societies. A one-half day symposium precludes the possibility of detailed coverage of the many areas in cardiovascular disease in which eicosanoid participation could be implicated and which ideally one would hope to cover. The organizers' aim was to address, in the form of research presentations or reviews, current areas of investigation dealing with selected topics in heart disease.The success of this symposium was made possible by the participation of several distinguished scientists. We would also like to thank the Pharmacological Society of Canada, the Canadian Heart Foundation, Upjohn Canada, Sterling Drug, and Ciba-Geigy U.S. A. for financial support. The organizers thank Dr. J. Burka for serving as Guest Editor and Mrs. L. Hendrickson of this Journal for coordinating the submission of the manuscripts.

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 ± 2.5 °C with control microsomes. Energies of activation (about 15–17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.

Studies on the in vitro biological N-oxidation of trimethoprim.

In vivo studies on the metabolism of trimethoprim have shown that N-oxidation to isomeric N-oxides occurs, with considerable inter-species variation in the proportion of each N-oxide excreted. No in vitro studies on the metabolism of trimethoprim have been reported to date. We now report a sensitive and specific HPLC method for the simultaneous determination of the isomeric N-oxides of trimethoprim. In vitro metabolic studies with hepatic microsomes from several animal species have been conducted. Optimum incubation conditions have been established and the use of potential metabolic inducers, activators and inhibitors has demonstrated that the N-oxidation of trimethoprim to its isomeric N-oxides is mediated via a cytochrome P450 dependent pathway.

Studies on the effect of different inhibitors of arachidonic acid metabolism on glyceryltrinitrate-induced relaxation and cGMP elevation in bovine vascular tissue.

This study was performed in order to investigate the possible involvement of arachidonic acid metabolites in the mediation of glyceryltrinitrate (GTN)-induced relaxation in isolated bovine mesenteric artery (BMA) and vein (BMV) and in bovine coronary artery (BCA). Concentration-effect curves for GTN were established on the different types of vessels, precontracted with 2.5 microM phenylephrine (BMA) or K+-depolarization (BMV and BCA), in the presence or absence of different inhibitors of the arachidonic acid metabolism. The used inhibitors of arachidonic acid metabolism were: 10 microM quinacrine (a phospholipase A2 inhibitor), 100 microM acetylsalicylic acid, 20 microM indomethacin (cyclooxygenase inhibitors), 3 mM tranylcypromine (inhibitor of PGI2 synthesis), 50 microM nordihydroguairetic acid and 40 microM BW 755C (lipoxygenase inhibitors). In addition, SKF 525A (10 microM) was tested on BMA; this agent is considered to block the cytochrome P450-dependent monooxygenase pathway. The effect on the endogenous levels of cyclic nucleotides after treatment with some of the inhibitors were also measured. Quinacrine and acetylsalicylic acid had no statistical significant effect (P greater than 0.05) on the pD2-value for GTN-induced relaxation in BMA, BMV and BCA. The results obtained with indomethacin were very variable. This drug was almost completely without effect on the GTN induced relaxation in BCA. In BMA a significant potentiation of the relaxant response was obtained (P = 0.002), while in BMV a significant inhibition was seen (P = 0.049).(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon activation of the thiol:Protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P 50 for glucagon of 7.8 × 10 −10 M which was only observed at microsomal protein concentrations of less than 100 ug/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase. There is an active interest in elucidating the mechanism of the transduction of hormonal action. Among the mechanisms which have been most actively studied have been those schemes which involve changes in the phosphorylation status of critical enzymes in various metabolic pathways (1). Recently Ziegler (2,3) has suggested an alternative mechanism in which the regulation of the activity of these pathways is mediated by changes in the oxidation-reduction state of various enzymes in the particular metabolic pathway. Even though to date there have been no published reports presenting data directly supporting this mechanism (3), a recent study from our laboratory would suggest that changes in the ratio of protein disulfides to sulfhydryl groups may be important in hormone action (4). In this studies we observed that in isolated, hepatic microsomes, the ATP-dependent calsium pump is inactivated through direct oxidation by a NADPH-dependent system (5). This inhibition appears to be catalyzed by a cytochrome P-450 dependent pathway. Further, the pump activity can be partially restored by reduced glutathione, presumably through a microsomal, thiol:protein disulfide oxidoreductase (5, MS submited). Finally we have found that physiological concentrations of norepinephrine (10 −8 M), when added to the isolated microsomal preparation, can significantly increase this NADPH-dependent inhibition of the calcium pump (4). Together these observations are consistent with the hypothesis that the hormonal action of norepinephrine in the hepatocyte may be mediated by inactivation of the calcium pump and that this inactivation is due to the reversible oxidation of critical sulfhydryls in one or more components of the calcium pump system. Since our previous studies suggested that the modulation of oxidative processes could mediate the transduction of the action of norepinephrine, it is possible that the activities of reductive enzymes are also modulated by hormones (3). In the current study, we have therefore examined in incubations of hepatic microsomes, the effect of various concentrations of glucagon on the activity of the GSH dependent microsomal enzyme, thiol:protein disulfide oxidoreductase (EC 1.8.4.2). This enzymes catalyzes the GSH reduction of protein disulfides to sulfhydryls. We find that in this preparation the addition of physiological concentrations the hormone (10 −9) stimulated from 2.3–6.4 fold the GSH reduction of endogenous and exogenous protein disulfides.

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [ 14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methylviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxy-ethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450

In order to define the site of bioactivation of CCl 4, CHCl 3 and CBrCl 3 in the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomes, the 14C-labeled hepatotoxins were incubated in vitro with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O 2 (8:2) and addition of a cytochrome c reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CCl 4, CHCl 3, and CBrCl 3 was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N 2 enhanced the binding of CCl 4, inhibited the binding of CHCl 3 and did not influence the binding of CBrCl 3. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome c reductase and that CCl 4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl 3 activation proceeds by cytochrome P-450 dependent oxidative pathways.