Cytochrome P450 Products Research Articles

In Vitro Metabolism of 19-Nor-1α,25-(OH)2D2 in Cultured Cell Lines: Inducible Synthesis of Lipid- and Water-Soluble Metabolites

The active vitamin D analog, 19-nor-1α,25-dihydroxyvitamin D2 (19-nor-1α,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1α,24,25-(OH)3D2, 19-nor-1α,24,25,26-(OH)4D2, and 19-nor-1α,24,25,28-(OH)4D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1α,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1α,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1α,25-(OH)2D2 and 1α,25-(OH)2D2 that the lack of the C10–19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1α,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1α,25-(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1α,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1α,24,25-(OH)3D2. We conclude that 19-nor-1α,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.

腎移植患者の真菌症

Kidney transplantation therapy can be divided into two periods: the induction periods during which immunosuppressants are aggressively administered, and the maintenance periods during which treatment is continued at a maintenance dosage. During the induction periods, which usually lasts for several months after surgery, the host defense mechanism in the patient is so profoundly impaired that major infections, including deep-seated mycosis, occur in most cases. The incidence of mycosis as a result of superinfection was quite high in the l960s and l970s, when steroids and antimetabolites were the mainstay immunosuppressants, and antibacterial agents were used without any specific purpose for fear of post-transplantation infections. The new calcineurin inhibitor ciclosporin was developed around 1980 and has since become the primary immunosuppressant used to prevent graft dysfunction after transplantation. As a result of its advent, use of steroids and antimetabolites has declined. Later studies have shown that ciclosporin selectively inhibits lymphocytes and provides more information on the pattern of bacterial infections after transplantation, making it possible to optimize the use of antibacterial agents. As a result, the incidence of bacterial infections and mycosis has dramatically declined. Candida and Aspergillus are most frequently detected in post-transplantation mycosis. But Cryptococcus is also occasionally seen. Antifungal agents such as flucytosine (5-FC) and ketoconazole are effective for deep-seated mycosis such as pulmonary infections which, occurring at a relatively early stage after transplantation, threaten a patients life. However, the coadministration of ketoconazole must be carefully done because it increases blood concentrations of calcineurin inhibitors such as ciclosporin and tacrolimus by inhibiting the production of cytochrome P-450. Amphotericin B, which is effective but is associated with nephrotoxicity, is usually used in a mouthwash or inhalation therapy. Dermatomycosis such as tinea versicolor due to fragility of the skin structure is not necessarily rare even in patients with a quasi-normal host defense mechanism and good renal function at a late stage in the maintenance phase. This paper outlines change in post-transplantation mycosis in recent years and presents some case reports.

16(R)-Hydroxyeicosatetraenoic Acid, a Novel Cytochrome P450 Product of Arachidonic Acid, Suppresses Activation of Human Polymorphonuclear Leukocytes and Reduces Intracranial Pressure in a Rabbit Model of Thromboembolic Stroke

16(R)-Hydroxyeicosatetraenoic Acid, a Novel Cytochrome P450 Product of Arachidonic Acid, Suppresses Activation of Human Polymorphonuclear Leukocytes and Reduces Intracranial Pressure in a Rabbit Model of Thromboembolic Stroke

16(R)-Hydroxyeicosatetraenoic Acid, a Novel Cytochrome P450 Product of Arachidonic Acid, Suppresses Activation of Human Polymorphonuclear Leukocytes and Reduces Intracranial Pressure in a Rabbit Model of Thromboembolic Stroke

Activated polymorphonuclear leukocytes (PMNs) have been suggested to contribute to the development of increased intracranial pressure (ICP). We recently demonstrated that human PMNs produce a novel cytochrome P450-derived arachidonic acid metabolite, 1 6(R)-hydroxyeicosatetraenoic acid [16(R)-HETE], that modulates their function. It was thus of interest to examine this novel mediator in an acute stroke model. 16-HETE was assessed initially in a variety of human PMN and platelet in vitro assays and subsequently in an established rabbit model of thromboembolic stroke. A total of 50 rabbits completed a randomized, blinded, four-arm study, receiving 16(R)-HETE, tissue plasminogen activator, both, or neither. Experiments were completed 7 hours after autologous clot embolization. The primary end point for efficacy was the suppression of increased ICP. In in vitro assays, 16(R)-HETE selectively inhibited human PMN adhesion and aggregation and leukotriene B4 synthesis. In the thromboembolic stroke model, animals that received 16(R)-HETE demonstrated significant suppression of increased ICP (7.7 +/- 1.2 to 13.1 +/- 2.7 mm Hg, baseline versus final 7-h time point, mean +/- standard error), compared with either the vehicle-treated group (7.7 +/- 0.9 to 15.8 +/- 2.6 mm Hg) or the tissue plasminogen activator-treated group (7.6 +/- 0.6 to 13.7 +/- 2.1 mm Hg). The group that received the combination of 16(R)-HETE plus tissue plasminogen activator demonstrated no significant change in ICP for the duration of the protocol (8.6 +/- 0.6 to 11.1 +/- 1.2 mm Hg). 16(R)-HETE suppresses the development of increased ICP in a rabbit model of thromboembolic stroke and may serve as a novel therapeutic strategy in ischemic and inflammatory pathophysiological states.

16( R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[ m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16( R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108 ± 26 pg/10 8 cells) and 20-HETE (341 ± 69 pg/10 8 cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16( R)-HETE via a cytochrome P450 ω- and ω-4 oxygenase(s).

Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature.

Lipoxygenase (LO) and cytochrome P450 monooxygenase products of arachidonic acid (AA) have been implicated in a large number of vasoregulatory processes. In intact, blood-free, perfused and ventilated human lungs (n = 8), isolated during surgery for bronchial carcinoma, we analyzed leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs) by sequential sampling of the recirculating buffer fluid. For the analysis we used multistep, solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, with elution of all metabolites within one run and photodiode array detection to obtain full UV spectra of eluting compounds. We detected no LT release in a 15-min baseline period, but the admixture of the calcium ionophore A23187 with the buffer fluid provoked the rapid appearance of all LTs. Some baseline release of 15-HETE was observed, and in response to A23187, maximum buffer concentrations were noted for 5-HETE, with 8-HETE, 9-HETE, 11-HETE, and 12-HETE being detected at lower levels. Marked baseline liberation of 11,12-EET and 8,9-EET was observed. In response to A23187, high oxirane buffer concentrations were registered, which far surpassed those of LTs and HETEs. The eicosanoid release was paralleled by a limited pulmonary artery pressor response and progressive vascular leakage. We conclude that ex-vivo-perfused human lungs release EETs > LTs > HETEs into the vascular compartment in response to inflammatory challenge. The marked oxirane synthesis in the lung vasculature may have major impact on lung vasoregulation when considering the possible function of these AA epoxides as endothelium-derived hyperpolarizing factors.

Reactive oxygen species: role in the relaxation induced by bradykinin or arachidonic acid via EDHF in isolated porcine coronary arteries.

Although endothelium-derived hyperpolarizing factor (EDHF) is thought to be a cytochrome P-450 product (arachidonic acid metabolite) in some tissues, in porcine coronary arteries (PCAs) its nature remains unclear. Because phospholipase A2 and C are involved in the synthesis and/or release of EDHF in the PCA, the arachidonic acid (AA) pathway may be involved. In the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) and the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), both bradykinin (BK; 10(-9)-10(-6) M) and AA (10(-7)-10(-4) M) induced dose-dependent relaxation of PGF2alpha-contracted PCA rings, which was blocked by a high extracellular concentration of KCl (30 mM) or pretreatment with ouabain, a Na+/K+-adenosine triphosphatase (ATPase) inhibitor (5 x 10(-7) M). Eicosatetraynoic acid (ETYA; 20 microM), which inhibits all AA pathways, slightly affected the response to BK and AA; however, lipoxygenase or cytochrome P-450 inhibitors had no effect, suggesting that relaxation is independent of these enzymatic pathways. Because endothelial cells can generate reactive oxygen species (ROS) via metabolism of AA and independent of cyclooxygenase activity, we also studied (a) whether ROS can relax the PCA, as well as the mechanism(s) involved, and (b) the role of ROS in BK- and AA-induced relaxation. Xanthine (X; 100 microM) plus xanthine oxidase (XO; 0.02 U/ml) induced time-dependent relaxation of PGF2alpha-contracted PCA rings in the presence of indomethacin and L-NAME. Dilatation was not affected by superoxide dismutase (SOD; 500 U/ml) but was abolished by catalase (300 U/ml), suggesting that hydrogen peroxide (H2O2) is involved. When rings were contracted by depolarizing them with 30 mM KCl, X/XO failed to elicit relaxation. Ouabain abolished the response to X/XO, suggesting that X/XO may induce relaxation by hyperpolarizing vascular smooth muscle cells via stimulation of the Na+/K+-ATPase pump. We therefore questioned whether ROS might be involved in BK- and AA-induced relaxation. Because catalase combined with SOD had little or no effect, we concluded that in the PCA, the relaxation induced by BK via EDHF involves some mechanism independent of NO, AA metabolism, or ROS.

Endothelium-derived hyperpolarizing factor(s): species and tissue heterogeneity.

1. Endothelium-derived relaxing factor is almost universally considered to be synonymous with nitric oxide (NO); however, it is now well established that at least two other chemically distinct species (prostacyclin (PGI2) and a hyperpolarizing factor) may also contribute to endothelium-dependent relaxation. 2. Only relatively few studies have provided definitive evidence that an endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor PGI2, exists as a chemical mediator. 3. There is a lack of agreement as to the likely chemical identity of this putative factor. Some evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid product, possibly an epoxyeicosatrienoic acid (EET); conflicting evidence supports an endogenous cannabinoid as the mediator and still other studies infer an unknown mediator that is neither a cytochrome P450 nor a cannabinoid. 4. Data from our laboratory with a rabbit carotid artery 'sandwich' preparation have provided evidence that a mediator that meets the pharmacological expectations of a cytochrome P450 product is an EDHF. 5. Data from guinea-pig mesenteric arterioles suggest that EDHF is not a cytochrome P450 product, whereas in guinea-pig middle cerebral arteries, relaxation mediated by the NO/PGI2-independent mediator(s) is sensitive to cytochrome P450 inhibitors. In addition, in the rabbit middle cerebral artery, it is likely that endothelium-dependent hyperpolarization is mediated by both NO and PGI2. 6. In conclusion, these data indicate that EDHF is unlikely to be a single factor and that considerable tissue and species differences exist for the nature and cellular targets of the hyperpolarizing factors.

The involvement of K+ channels and the possible pathway of EDHF in the rabbit femoral artery.

Experiments were designed to characterize the cellular mechanisms of action of endothelium-derived vasodilator substances in the rabbit femoral artery. Acetylcholine (ACh, 10(-8)-10(-5) M) induced a concentration-dependent relaxation of isolated endothelium-intact arterial rings precontracted with norepinephrine (NE, 10(-6) M). The ACh-induced response was abolished by the removal of endothelium. NG-nitro-L-arginine (L-NAME, 10(-4) M), an inhibitor of NO synthase, partially inhibited ACh-induced endothelium-dependent relaxation, whereas indomethacin (10(-5) M) showed no effect on ACh-induced relaxation. 25 mM KCl partially inhibited ACh-induced relaxation by shifting the concentration-response curve and abolished the response when combined with L-NAME and NE. In the presence of L-NAME, ACh-induced relaxation was unaffected by glibenclamide (10(-5) M) but significantly reduced by apamin (10(-6) M), and almost completely blocked by tetraethylammonium (TEA, 10(-3) M), iberiotoxin (10(-7) M) and 4-aminopyridine (4-AP, 5 x 10(-3) M). The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10(-5) M) and miconazole (10(-5) M) also significantly inhibited ACh-induced relaxation. Ouabain (10(-6) M), an inhibitor of Na+, K(+)-ATPase, or K(+)-free solution, also significantly inhibited ACh-induced relaxation. ACh-induced relaxation was not significantly inhibited by 18-alpha-glycyrrhetinic acid (18 alpha-GA, 10(-4) M). These results of this study indicate that ACh-induced endothelium-dependent relaxation of the rabbit femoral artery occurs via a mechanism that involves activation of Na+, K(+)-ATPase and/or activation of both the voltage-gated K+ channel (Kv) and the large-conductance, Ca(2+)-activated K+ channel (BKCa). The results further suggest that EDHF released by ACh may be a cytochrome P450 product.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Induction of Cytochrome P450-Dependent Arachidonic Acid Metabolism in Mouse Liver Microsomes: Evidence for Species-Specific Differences in Responses

Arachidonic acid is biotransformed to metabolites active in signal transduction by cytochrome P450 (CYP) as well as by cyclooxygenase and lipoxygenase enzymes. Inducers of CYP1 enzymes, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and otherAhreceptor ligands, markedly increase liver microsomal CYP-dependent arachidonic acid epoxygenation in chicks but depress epoxygenation in rat liver microsomes where they elicit about twofold increases in formation of other CYP products, ω−1 to ω−4-OH arachidonic acid. These studies examined the effect of TCDD on metabolism of [1-14C]-labeled arachidonic acid by mouse liver microsomes. Mouse liver microsomes metabolized arachidonic acid exclusively by a CYP-dependent mechanism as evidenced by lack of metabolism in the absence of NADPH and by formation of specific CYP-dependent metabolites. The major constitutive products were epoxygenase products (EETs and EET-diols) and ω-OH arachidonic acid. Treatment with TCDD increased formation of ω−2- to ω−4-OH arachidonic acid products 23-fold, formation of ω−1-OH arachidonic acid about 5-fold, and formation of epoxygenase products and HETEs each about twofold. In contrast, TCDD treatment decreased formation of ω-OH arachidonic acid by over 70%. EET-diols comprised a greater fraction of total epoxygenase products in mouse liver microsomes than has been found for liver microsomes of other species. The high EET-diol formation was attributable to a non-TCDD-inducible, EET epoxide hydrolase activity in mouse liver microsomes. For comparison, the effect of TCDD on [1-14C]-labeled arachidonic acid was examined in homogenates of spleen, an immune system target of TCDD. While levels of total [1-14C]-arachidonic acid metabolism were comparable in both tissues, virtually all of the metabolism by spleen was CYP-independent, and it was unaffected by TCDD. Western blotting experiments showed that TCDD-induced mouse Cyp1a1 and 1a2 share immunologic epitopes with chick CYP1A4 and 1A5. However, in immunoinhibition studies, an antibody to CYP1A5, the chick arachidonate epoxygenase, was ineffective against TCDD-induced arachidonic acid metabolism in mouse liver microsomes, suggesting that there are differences in the catalytic sites or tertiary structures of CYP1A5 and the CYP-enzyme catalyzing the TCDD-induced arachidonic acid metabolism in mouse liver. This study shows that the effects of TCDD of the profile of CYP-dependent arachidonic acid metabolites and the amounts produced in mouse liver microsomes differ from other species. The findings suggest that species differences in CYP1A catalytic activities including the metabolism of arachidonic acid may contribute to species differences in sensitivity to TCDD toxicity.

Simultaneous Analysis of 4- and 5-Series Lipoxygenase and Cytochrome P450 Products from Different Biological Sources by Reversed-Phase High-Performance Liquid Chromatographic Technique

Quantification of lipoxygenase and cytochrome P450 products of both arachidonic acid (AA) and eicosapentaenoic acid (EPA) is of broad interest due to the multiple biological activities of these compounds. Wedeveloped a method combining (i) solid-phase extraction, (ii) isocratic reversed-phase high-performance liquid chromatographic separation, and (iii) online photodiode array detection with spectrum analysis for identification and measurement of all main 4- and 5-series eicosanoids (leukotrienes, hydroxyeicosatetraenoic acids/hydroxyeicosapentaenoic acids, epoxyeicosatrienoic acids) within one run. With these procedures, standard mixtures of AA- and EPA-derived lipid mediators were recovered from different biological liquids, like lung perfusate, human bronchoalveolar lavage fluid, and cell supernatant with linear characteristics for each compound. Recoveries of the different lipid mediators exceeded 80% showing excellent reproducibility. Application of the method to isolated, perfused, and ventilated human lungs challenged with the calcium ionophore A23187 and to human neutrophils stimulated in the presence of arachidonic acid and eicosapentaenoic acid withN-formyl-methionyl-leucyl-phenylalanine demonstrated the generation of a large array of lipoxygenase and cytochrome P450 products. Thus, convenient quantification of 4- and 5-series eicosanoids in fluids of biological interest is achieved by a technique comprising solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, and photodiodearray-based online spectrum analysis of eluting compounds.

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid.

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 microM N(omega)-nitro-L-arginine and 10 microM indomethacin. 2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H1-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 microM). 3. The cytochrome P450 inhibitors proadifen (10 microM) and clotrimazole (10 microM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 microM) and 7-ethoxyresorufin (10 microM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 microM) and LEM (1 microM)-induced hyperpolarization. 4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 microM), glibenclamide (10 microM) and BaCl2 (50 microM) had no significant effect on responses to ACh, AA and 11,12-EET. 5. IBTX (100 nM) significantly reduced both 11,12-EET (33 microM) and AA (30 microM) hyperpolarization without affecting the ACh (1 microM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations. 6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

Cytochrome P450 products and renal function in cirrhosis: the HETE is on.

Cytochrome P450 products and renal function in cirrhosis: the HETE is on.

The role of nitric oxide in hepatic metabolism.

Nitric oxide (NO) may regulate hepatic metabolism directly by causing alterations in hepatocellular (hepatocyte and Kupffer cell) metabolism and function or indirectly as a result of its vasodilator properties. Its release from the endothelium can be elicited by numerous autacoids such as histamine, vasoactive intestinal peptide, adenosine, ATP, 5-HT, substance P, bradykinin, and calcitonin gene-related peptide. In addition, NO may be released from the hepatic vascular endothelium, platelets, nerve endings, mast cells, and Kupffer cells as a response to various stimuli such as endotoxemia, ischemia-reperfusion injury, and circulatory shock. It is synthesized by nitric oxide synthase (NOS), which has three distinguishable isoforms: NOS-1 (ncNOS), a constitutive isoform originally isolated from neuronal sources; NOS-2 (iNOS), an inducible isoform that may generate large quantities of NO and may be induced in a variety of cell types throughout the body by the action of inflammatory stimuli such as tumor necrosis factor and interleukin (IL)-1 and -6; and NOS-3 (ecNOS), a constitutive isoform originally located in endothelial cells. Another basis for differentiation between the constitutive and inducible enzymes is the requirement for calcium binding to calmodulin in the former. NO is vulnerable to a plethora of biologic reactions, the most important being those involving higher nitrogen oxides (NO2-), nitrosothiol, and nitrosyl iron-cysteine complexes, the products of which (for example, peroxynitrite), are believed to be highly cytotoxic. The ability of NO to react with iron complexes renders the cytochrome P450 series of microsomal enzymes natural targets for inhibition by NO. It is believed that this mechanism provides negative feedback control of NO synthesis. In addition, NO may regulate prostaglandin synthesis because the cyclooxygenases are other hem-containing enzymes. It may also be possible that NO-induced release of IL-1 inhibits cytochrome P450 production, which ultimately renders the liver less resistant to trauma. It is believed that Kupffer cells are the main source of NO during endotoxemic shock and that selective inhibition of this stimulation may have future beneficial therapeutic implications. NO release in small quantities may be beneficial because it has been shown to decrease tumor cell growth and levels of prostaglandin E2 and F2 alpha (proinflammatory products) and to increase protein synthesis and DNA-repair enzymes in isolated hepatocytes. NO may possess both cytoprotective and cytotoxic properties depending on the amount and the isoform of NOS by which it is produced. The mechanisms by which these properties are regulated are important in the maintenance of whole body homeostasis and remain to be elucidated.

Cytochrome P-450-derived eicosanoids participate in the renal functional effects of ET-1 in the anesthetized rat.

We evaluated the contribution of cytochrome P-450 (CYP450)-dependent arachidonic acid (AA) metabolites and prostanoids to the renal hemodynamic and tubular effects of endothelin-1 (ET-1) in anesthetized rats. Either ET-1 (0.3, 1.0, and 3 pmol.kg-1.min-1) or vehicle was infused intravenously during two to three 30-min clearance experimental periods. Only high-dose ET-1 increased mean arterial pressure: control, 75 +/- 3 mmHg vs. experimental, 84 +/- 4 mmHg. A dose-dependent diuretic-natriuretic response to ET-1 occurred despite progressive declines in glomerular filtration rate (GFR) and renal blood flow. In the face of a 36% reduction in GFR in response to the highest dose of ET-1, urinary sodium excretion (UNaV) increased threefold from 0.57 +/- 0.11 to 1.6 +/- 0.10 mumol.100 g-1.min-1. Indomethacin (5 mg/kg) decreased basal GFR from 1.2 +/- 0.3 ml.100 g-1.min-1 to 0.8 +/- 0.1 ml.100 g-1.min-1 and potentiated the GFR lowering action of ET-1 associated with reductions in UNaV and urine volume. Cobalt chloride (CoCl2) and dibromododec-11-enoic acid (DBDD), which diminish CYP450-dependent AA metabolism through different mechanisms, were used to identify CYP450 products mediating the renal functional actions of ET-1. DBDD (12.5 micrograms/min) reduced urinary excretion of 20-hydroxyeicosatetraenoic acid from 3.4 +/- 0.9 (control) to 1.1 +/- 0.6 ng/h and abolished the negative effects of ET-1 on GFR while decreasing the diuretic-natriuretic action of ET-1. Similar effects were produced by CoCl2. Clotrimazole, an inhibitor of epoxygenase activity, was without effect on ET-1-induced renal functional changes. Thus the capacity of ET-1 to enhance prostaglandin production was primarily expressed in terms of positive effects on renal hemodynamics. In contrast, CYP450 products promoted sodium excretion despite negative effects on renal hemodynamics.

Distinct Endothelial Impairment in Coronary Microvessels from Hypertensive Dahl Rats

Hypertension has been linked to an impaired dilator function of the coronary microvascular endothelium in vivo. However, the profile and mechanism of this dysfunction remain obscure. Thus, this study compared diameter responses to acetylcholine (ACH), bradykinin (BKN), and substance P (SP) between coronary microvessels (i.d.=106+/-4 microm) dissected from left ventricles of normotensive and hypertensive Dahl rats (Dahl-NT and Dahl-HT, respectively). Vessels were cannulated and pressurized on glass pipettes at 80 mm Hg, and internal diameters were monitored by videomicroscopy. Coronary microvessels from Dahl-NT and Dahl-HT showed similar dilator responses to ACH (100 pmol/L to 10 micromol/L), with maximal diameter increases of 63+/-5 microm and 63+/-7 microm, respectively (n=31,17). However, only vessels from Dahl-NT showed dilator responses to SP (10 fmol/L to 1 nmol/L) and BKN (100 fmol/L to 10 nmol/L). All dilator responses persisted after N-nitro-L-arginine (10 micromol/L) or indomethacin (10 micromol/L), but were blunted after inhibition of cytochrome P450 by 10 micromol/L octadecynoic acid (n=6-8). These results suggest that: (1) coronary microvessels from Dahl-HT show a unique pattern of endothelial impairment, whereby ACH-induced relaxations persist at a time when dilator responses to SP and BKN are severely blunted, and (2) a cytochrome P450 product, rather than nitric oxide or prostacyclin, may partly mediate the vasodilator responses to ACH, SP and BKN.

Contribution of Arachidonic Acid Metabolites to Reduced Norepinephrine-Induced Contractions in Hypercholesterolemic Rabbit Aortas

Because alterations in the aortic metabolism of arachidonic acid and in vascular responsiveness occur in hypercholesterolemic rabbits, we hypothesized that an arachidonic acid metabolite may contribute to the regulation of vascular tone. Aortic contractions to norepinephrine were investigated in rabbits fed either standard chow or chow containing 2% cholesterol. In normal rabbits, norepinephrine (10(-6) M) elicited a 126 +/- 2% contraction compared with a 95 +/- 2% contraction in cholesterol-fed rabbits. The factor mediating the depressed response was endothelium-dependent because removal of the endothelium blocked the decrease in norepinephrine-induced contractions observed in the cholesterol-fed rabbits. The endothelium-derived factor was not nitric oxide, because blockade of nitric oxide synthase with nitro-L-arginine did not abolish the decreased response in the cholesterol-fed rabbits. Pretreatment of aortas with a cyclooxygenase inhibitor, indomethacin (10(-5) M) caused a slight decrease in the norepinephrine-induced contractions, suggesting that the factor could be a vasoconstrictor cyclooxygenase metabolite or a vasodilatory lipoxygenase or cytochrome P450 epoxygenase metabolite. Pretreatment with the thromboxane A2/prostaglandin H2-receptor antagonist, SQ 29458, had no effect on norepinephrine-induced contractions. Whereas the lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-5) M), caused a slight increase in the contractions to norepinephrine in cholesterol-fed rabbits compared with normal rabbits, the cytochrome P450 epoxygenase inhibitor, metyrapone (10(-4) M), produced a greater enhancement of norepinephrine-induced contractions in cholesterol-fed rabbits but had no effect on responses in the normal rabbits. Characterization of [3H]arachidonic acid metabolism in cholesterol-fed aortic tissue indicated that norepinephrine stimulated the synthesis of both lipoxygenase and epoxygenase metabolites in an endothelium-dependent manner. This study demonstrated that (a) an endothelium-derived metabolite of arachidonic acid regulates vascular tone, (b) this metabolite appears to be a lipoxygenase or cytochrome P450 product or both, and (c) the activity or synthesis of the factor is enhanced by hypercholesterolemia.

Regulation of calcium influx and catecholamine secretion in chromaffin cells by a cytochrome P450 metabolite of arachidonic acid.

These studies were designed to determine the role of arachidonic acid metabolites in catecholamine secretion from adrenal chromaffin cells. Inhibitors of the cytochrome P450-dependent metabolism of arachidonic acid were shown to interfere with stimulus-secretion coupling in cultured chromaffin cells. Ketoconazole (10 microM), clotrimazole (20 microM), and piperonyl butoxide (50 microM) inhibited carbachol-dependent catecholamine secretion by 44%, 83%, and 100%, respectively; histamine-dependent secretion by 25%, 60%, and 81%, and secretion induced by 59 mM KCl depolarization by 25%, 55%, and 89%. Uptake of 45Ca2+ into the cells in response to carbachol was inhibited 63% by ketoconazole, 86% by clotrimazole, and 95% by piperonyl butoxide; KCl-dependent uptake was inhibited 7%, 56%, and 85%, respectively. However, cytochrome P450 inhibitors did not inhibit catecholamine secretion when cells were stimulated with the calcium ionophores ionomycin or lasalocid. These results indicated the involvement of a cytochrome P450 product in controlling Ca2+ influx in response to membrane depolarization. Cells prelabeled with [3H]arachidonic acid formed a 3H-labeled metabolite which comigrated with authentic 5,6-epoxyeicosatrienoic (5,6-EET) acid on reverse phase and normal phase HPLC. Pretreatment with clotrimazole inhibited the production of this 3H-labeled metabolite. Addition of synthetic 5,6-EET (1 nM) to cells pretreated with piperonyl butoxide resulted in catecholamine secretion. These data suggest a role for a cytochrome P450 metabolite of arachidonic acid in agonist-stimulated catecholamine secretion.

Endothelium-dependent hyperpolarization. Beyond nitric oxide and cyclic GMP.

Blood flow and blood pressure are determined by an integration of reflex, humoral, and local vascular control mechanisms. Knowledge of these mechanisms has mushroomed over the past 15 years, particularly in the area of local endothelium-dependent vasomotor control. This has stemmed from the pioneering report in 1980 by Robert Furchgott1 demonstrating that the endothelium releases a vasodilator substance in response to acetylcholine. This concept has been expanded with knowledge that the endothelium releases a variety of relaxing and contracting factors that regulate the underlying smooth muscle.2 The most widely known endothelium-derived relaxing factor, nitric oxide, is released from endothelial cells in response to shear stress or stimulation of different receptors for a variety of neurohumoral mediators on the endothelial cell surface.3 The increase in endothelial cell calcium initiated by these stimuli increases the activity of a constitutively expressed enzyme, nitric oxide synthase, which converts l-arginine to nitric oxide and citrulline.4 5 6 7 Nitric oxide thus formed diffuses to and inhibits contraction of the underlying vascular smooth muscle.3 The physiological significance of the production of endothelial nitric oxide is suggested by the vasoconstriction observed in most vascular beds8 9 10 11 12 13 and the increase in systemic arterial blood pressure,14 15 16 which occurs on infusion of inhibitors of nitric oxide synthase. This observation further implies that under normal conditions, endothelial cells are locally liberating nitric oxide which effectively inhibits vasoconstriction arising by other mechanisms. Thus, normal vascular homeostasis depends in the periphery on a balance between neurally and humorally mediated vasoconstriction in skeletal muscle, mesentery, and the kidney, and local endothelium-dependent vasodilatation. In the truly vital areas of the heart, brain, and genitalia, vasodilator neural mechanisms reinforce the vasodilator influence of the endothelium.17 All is not known regarding the …

Lack of effect of extracellular matrix or 3T3 feeder layer on the maintenance of differentiation or survival time of cultured rat hepatocytes

Cultured rat hepatocytes are often used in in vitro studies. In this culture system the lack of the normal environment to which the cells would be exposed in vivo contributes to a change in differentiation. To study this differentiation problem we compared the effects of culturing primary hepatocytes on an extracellular matrix (ECM) isolated from normal rat livers and on a 3T3 (mouse fibroblast) feeder layer. The use of the two coatings did not result in changes in survival time, as measured by leakage of lactate dehydrogenase, differentiation (activities of pyruvate kinase and γ-glutamyltransferase) or specific functions (albumin production and concentration of cytochrome P-450) in comparison with cells cultured on plastic. Since no positive effects of the coatings were found it is proposed that more attention should be paid to methods of isolating and culturing hepatocytes, rather than to obtaining conditions for the hepatocytes that are completely comparable with the in vivo situation.