Dienoic Acid Research Articles

Kinetic parameters of oxidation of individual fatty acids with ozone

Using an original automated analyzer of double bonds we determined the rate constants for oxidation of saturated and unsaturated mono- and dienoic fatty acids (in vivo substrates for beta-oxidation in the mitochondria) by the ozone titration method. The rate constant for O(3) oxidation is maximum for oleic monoenoic acid, lower for dienoic linoleic, and very low for saturated palmitic acid. The rate constant for oxidation of oleic fatty acid, which by one order of magnitude surpasses that for oxidation of essential arachidonic acid, indicates that oleic acid is a leading in vivo acceptor of active O(2) species. By the rate of trapping active oxygen species and in the quantitative aspect, endogenously produced oleic acid can be regarded as the main biological antioxidant.

Identification of unusual fatty acids of four alpine plant species from the Pamirs

Fatty acid composition and structure in total lipids from the green above-ground parts of four alpine plants, Oxygraphis glacialis, Primula macrophylla, Rhodiola pamiroalaica, and Swertia marginata, were established by GC and GC–MS. A total of 55 fatty acids was detected, and 48 of them were identified. Ubiquitous palmitate, linoleate, and linolenate predominated in the lipids accounting for about 72–90% of the total fatty acids. At the same time, the latter contained numerous species, which were unusual for higher plants and included saturated odd-numbered n-acids (six C 15–C 25 species, 0.26–1.40%), saturated even-numbered very-long-chain n-acids (six C 20–C 30 species, 1.00–2.49%), iso-acids (nine C 15–C 26 species, 0.64–1.53%), anteiso-acids (four C 15–C 20 species, 0.08–1.57%), certain uncommon mono- and dienoic acids, as well as 16:3ω3, 18:3ω6, and 18:4ω3 acids that are absent from the most higher plants. Nine fatty acids were found here for the first time in higher plants and two may be new to science. The evidence on the unusual fatty acids is discussed with respect to their distribution in living organisms, pathways of biosynthesis, and chemotaxonomic role.

Transfection of CYP4A1 cDNA decreases diameter and increases responsiveness of gracilis muscle arterioles to constrictor stimuli.

Cytochrome P-450-4A1 (CYP4A1) is an omega-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 +/- 3 microm) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 +/- 4 microm). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40-100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.

Differential temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms overexpressed in Saccharomyces cerevisiae

AbstractThe oxygen‐independent temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms has been investigated using Saccharomyces cerevisiae cells expressing each FAD2 gene. Yeast cells transformed with the FAD2‐1 gene showed the highest percentage of dienoic acids when they were grown at 10–15 °C. In contrast, the maximal level of dienoic acids for S. cerevisiae cells expressing the FAD2‐2 and FAD2‐3 genes were obtained at 30 and 35 °C, respectively. Temperature shifts in the phase of exponential growth, from 30 to 15 °C or from 15 to 30 °C, produced changes in the final percentage of dienoic acids, mostly in yeast cells transformed with the FAD2‐1 gene, to reach the content corresponding to the new temperature. Low temperature (15 °C) increased the amount of neutral lipids in all transformed yeast cells, mainly because it favored triacylglycerol accumulation. In addition, the FAD2‐expressing yeast cells showed a higher polar lipid content than those transformed with the empty vector. Dienoic acids were present in all lipids, although high temperature (30 °C) favored their accumulation in neutral lipids. As the main conclusion, the low thermal stability observed for the major and seed specific isoform (FAD2‐1) is the key factor controlling the direct temperature regulation in sunflower seeds.

Oxidative Stability of Soybean and Sesame Oil Mixture during Frying of Flour Dough

ABSTRACT: Effects of roasted sesame seed oil on the oxidative stability of soybean oil during frying of flour dough at 160 °C were studied by determining fatty acid composition and conjugated dienoic acid (CDA), p‐anisidine (PA), and free fatty acid (FFA) values. Concentration of sesame oil in frying oil was 0%, 10%, 20%, or 30% (v/v). Tocopherols and lignan compounds in the frying oil were also determined by high‐performance liquid chromatography. As the number of fryings performed by the oil increased, linolenic acid content in frying oil decreased, and the decreasing rate was lower in frying oil containing sesame oil than in the oil containing no sesame oil. CDA and FFA values of frying oil increased during frying and their relative values to the initial value were lower in frying oil containing sesame oil than in the oil containing no sesame oil. This indicates that the addition of sesame oil improved thermooxidative stability of frying oil, possibly due to the presence of lignan compounds in sesame oil. Tocopherols and lignan compounds in frying oil decreased during frying. As the amount of sesame oil in frying oil increased, degradation of tocopherols increased and lignan compounds degradation decreased. Tocopherols were suggested to protect lignan compounds in sesame oil from decomposition during frying.

P2X receptor-stimulated calcium responses in preglomerular vascular smooth muscle cells involves 20-hydroxyeicosatetraenoic acid.

The current study tested the hypothesis that endogenous 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the increase in intracellular calcium ([Ca2+]i) elicited by P2X receptor activation in renal microvascular smooth muscle cells. Vascular smooth muscle cells obtained from rats were loaded with fura-2 and studied using standard single cell fluorescence microscopy. Basal renal myocyte [Ca2+]i averaged 96 +/- 5 nM. ATP (10 and 100 microM) increased vascular smooth muscle cell [Ca2+]i by 340 +/- 88 and 555 +/- 80 nM, respectively. The cytochrome P450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), significantly attenuated the peak myocyte [Ca2+]i responses to 10 and 100 microM ATP. ATP (100 microM) increased vascular smooth muscle cell [Ca2+]i by 372 +/- 93 and 163 +/- 55 nM in the presence of DDMS or 20-HEDE, respectively. The P2X receptor agonist, alpha,beta-methylene-ATP (10 microM), increased myocyte [Ca2+]i by 78 +/- 12 nM, and this response was significantly attenuated by DDMS (40 +/- 15 nM). In contrast, the vascular smooth muscle cell [Ca2+]i evoked by the P2Y agonist, UTP (100 microM), was not altered by DDMS or 20-HEDE. The effect of 20-HETE on [Ca2+]i was also assessed, and the peak increases in [Ca2+]i averaged 62 +/- 12 and 146 +/- 70 nM at 20-HETE concentrations of 1 and 10 microM, respectively. These results demonstrate that 20-HETE plays a significant role in the renal microvascular smooth muscle cell [Ca2+]i response to P2X receptor activation.

Frying stability of high‐oleate and regular soybean oil blends

AbstractThe objective of this work was to study the frying stability of soybean oil (SBO) with reduced linoleate (18∶2) and linolenate (18∶3) and elevated oleate (18∶1) contents. High‐oleate SBO [HO SBO, 79% oleic acid (OA)] and a control (conventional SBO, 21.5% OA) were tested as is, as well as blended in different ratios to make three blended oils containing 36.9, 50.7, and 64.7% OA, abbreviated as 37%OA, 51%OA, and 65%OA, respectively. In addition, a low‐linolenate (LL) SBO containing 1.4% 18∶3 and 25.3% 18∶1 was tested. Bread cubes (8.19 cm3) were fried in each of 18 oils (6 treatments×3 replicates). We hypothesized that stability indicators would be indirectly related to the total 18∶2 plus 18∶3 percentages and/or the calculated oxidizability. In general, the results were fairly predictable based on total 18∶2 and 18∶3 concentrations. The overall frying stability of the six oil treatments, from the best to the poorest, was: 79%OA, 65%OA, 51%OA, LL≥37%OA, and the control, with respective total compositions for 18∶2 plus 18∶3 of 10.3, 23.6, 36.3, 59.6, 48.9, and 62.8%. The greatly reduced concentration of 18∶3 in the LL SBO made it more stable than the 37%OA, even though the combined composition of 18∶2 and 18∶3 of LL was greater than that of the 37%OA. Blending conventional SBO with HO SBO had a profound effect on the oxidative stability index and color of the blended oils, but the values were not linearly predictable by the percentage of control in the blended oil. Other stability indices, including calculated oxidizability, calculated iodine value, conjugated dienoic acid value, and viscosity, changed in linear response to an increased proportion of the control in the blends.

Nodulisporic acids D-F: structure, biological activities, and biogenetic relationships.

Nodulisporic acids D, E, and F are the newest members of a family of nontremorogenic indole-diterpenoids that are potent, orally bioavailable, antiflea agents derived from a fungus belonging to the genus Nodulisporium. The four members of the D series are each devoid of an isoprene residue that is present at C-26 in the three nodulisporic acids described originally (the A series). Nodulisporic acid E (11a) has a simpler structure, which lacks not only the isoprene residue at C-26 but also two that form the A/B rings. Nodulisporic acid F is the simplest of all nodulisporic acids and is devoid of all three isoprene residues of the indole unit; as such, it represents the earliest biosynthetic intermediate in this series. A biogenetic grid based on mutation studies is proposed that encompasses all the known nodulisporic acids. Structure-activity relationships of the known natural nodulisporic acids have been elucidated. Within a series the most active compound possesses a dienoic acid chain, and overall, the end product of the biogenetic grid, i.e., nodulisporic acid A, exhibits the most potent antiflea activity. Additionally, the stereochemistries of C-3' ' and C-4' ' of nodulisporic acid D(2) and therefore of nodulisporic acids A(2), B(2), and C(2) have been assigned.

Effects of Carrot Powder in Dough on the Lipid Oxidation and Carotene Content of Fried Dough during Storage in the Dark

ABSTRACT: Lipid oxidation and carotene content changes in fried dough containing carrot powder during storage at 60°C in the dark were studied. Flour dough containing carrot powder at 0%, 10%, 20%, or 30% was fried in 160°C soybean oil and stored in a 60°C incubator in the dark for 12 d. As storage time of the fried dough increased, conjugated dienoic acids and β‐anisidine values of the fried dough increased. Addition of carrot to the dough lowered CDA and PAV of fried dough during storage and improved the lipid oxidative stability of the fried dough in the dark. Carotenes in the fried dough containing carrot powder were decomposed during storage, and faster decomposition rate was found in the dough containing less carrot powder.

Effects of a 20-HETE antagonist and agonists on cerebral vascular tone

This study examined the effects of a 20-hydroxyeicosatetraenoic acid (20-HETE) antagonist, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid (WIT002) and two agonists, 4-amino- N-(20-hydroxy-eicosa-5( Z),14( Z)-dienoyl) benzenesulfonamide (ABSA) and 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid (WIT003), on the diameter of rat middle cerebral arteries in vitro and on cerebral blood flow in vivo. WIT003, ABSA and 20-HETE all had a similar effect to reduce the diameter of the middle cerebral artery by 26%. WIT003 and 20-HETE both increased intracellular Ca 2+ concentration ([Ca 2+] i) in vascular smooth muscle cells isolated from the middle cerebral artery. In contrast, WIT002 had no effect on the basal diameter of the middle cerebral artery but it attenuated the vasoconstrictor responses and the rise in [Ca 2+] i in vascular smooth muscle cells following administration of 20-HETE and 5-hydroxytryptamine (5-HT). WIT003 partially restored the vasoconstrictor response to 5-HT in the middle cerebral artery after administration of an inhibitor of the endogenous synthesis of 20-HETE. Infusion of the 20-HETE agonists, WIT003 and ABSA, into cisterna magna of rats reduced baseline cerebral blood flow by 20%, whereas administration of the 20-HETE antagonist, WIT002, had no effect. Intracisternal injection of WIT002 attenuated the fall in cerebral blood flow following injection of blood into the cisterna magna, whereas administration of the 20-HETE agonist, ABSA, potentiated this response. These findings indicate that the 20-HETE agonists, WIT003 and ABSA, increase cerebral vascular tone both in vivo and in vitro and suggest blocking the vasoconstrictor actions of 20-HETE may be useful to prevent the acute fall in cerebral blood flow following subarachnoid hemorrhage.

Recommended methods of fatty acid methylester preparation for conjugated dienes and trienes in food and biological samples.

In this study, we compared three acid-catalyzed methods and three base-catalyzed methods for the methylester preparation of conjugated dienoic fatty acids and conjugated trienoic fatty acids in food and biological samples. Among the six methods examined, the sodium methoxide/methanol (NaOCH3/MeOH) method and the tetramethylguanidine/ methanol (TMG/MeOH) method of methylester preparation from tung oil were most efficient in preventing the artificial isomerization of alpha-eleostearic acid (alpha-ESA; 9c,11t,13t-18:3) to beta-eleostearic acid (beta-ESA: 9t,11t,13t-18:3) and for avoiding the artificial generation of unknown byproducts. Hydrochloric acid/methanol (HCl/MeOH), sulfuric acid/methanol (H2SO4/MeOH) and AOCS (boron trifluoride/methanol (BF3/MeOH)) methods of methylester preparation from tung oil resulted in the breakdown of alpha-ESA due to their long reaction periods and high reaction temperatures. In addition, these three methods did not prevent the generation of beta-ESA. For the methylester preparation of tung oil free fatty acids, the BF3/MeOH method (30 min at room temperature) did not lead to artificial beta-ESA formation or byproducts, while the trimethylsilyldiazomethane (TMSN2CH3) method did form artifacts. For the methylation of conjugated linoleic acid (CLA, free fatty acid), the BF3/ MeOH and TMSN2CH3 methods completely suppressed artificial isomerization of c,t-CLA and t,c-CLA to t,t-CLA. The results indicated that the BF3/MeOH method for free fatty acids is the best method for the methylester preparation of both conjugated dienoic and trienoic fatty acids with respect to preventing artificial isomerization and the formation of byproducts. The BF3/MeOH method was applicable to both food and biological samples.

Yeast Delta 12 fatty acid desaturase: gene cloning, expression, and function.

In an effort to elucidate the molecular mechanisms of fatty acid desaturation in yeast, a complete gene encoding Delta 12 fatty acid desaturase of Saccharomyces kluyveri was cloned. The open reading frame of this gene (named Sk-FAD2) consists of 1,251 bp, encoding 416 amino acids. The deduced Sk-FAD2 protein had 37-55% identity with those from other filamentous fungi. Unlike the genes of these other fungi, S. cerevisiae expressing Sk-FAD2 was found to be capable of synthesizing the dienoic fatty acid hexadecadienoic acid as well as linoleic acid. Moreover, the Sk-FAD2-disrupted strain of S. kluyveri was unable to produce polyunsaturated fatty acids. These results suggested that Sk-FAD2 protein is a unique Delta 12 fatty acid desaturase in S. kluyveri. Analysis of transcriptional expression revealed that Sk-FAD2 was not repressed by exogenous unsaturated fatty acids but responded to low-temperature stress.

Separation of isomers of dienoic acids by electromigration techniques

AbstractDifferent capillary electromigration techniques were employed to resolve geometrical isomers of sorbic acid, decadienoic acid, and ethyl sorbate. Since these substances differ in their polarity, shape, and size, various electromigration approaches were investigated to separate the four geometrical isomers of each compound. With capillary electrophoresis (CE) modified with a cyclodextrin (β‐CD) the four isomers of sorbic acid were separated using a buffer that consists of 60 mM tetraborate and 8 mg/mL β‐CD. The separation of decadienoic acid geometrical isomers was not possible, even at elevated tetraborate and cyclodextrin concentrations. The four isomers of decadienoic acid were successfully separated using micellar electrokinetic chromatography (MEKC) with a buffer consisting of 30 mM tetraborate and 100 mM SDS and microemulsion electrokinetic chromatography (MEEKC). Ethyl sorbate is the least polar of all the studied substances and its isomers could not be separated by MEKC or MEEKC. The resolution was improved and isomers were fully separated using capillary electrochromatography (CEC) with ODS stationary phase and a mobile phase consisting of 10 mM boric acid in 50% acetonitrile. Minor differences in the polarity and the shape of isomers and high resolving power of the applied techniques were sufficient for separation of very similar compounds. We have shown that versatile electromigration techniques can be applied for separation of geometrical isomers of dienoic acids and their esters.

Inhibitors of 20-hydroxyeicosatetraenoic acid reduce renal vasoconstrictor responsiveness.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived constrictor eicosanoid produced by the preglomerular vasculature where it contributes to regulation of tone. Removal of the tonic inhibitory influence of nitric oxide (NO) has been reported to increase renal 20-HETE release. Because inhibition of NO synthesis enhances responses to vasoconstrictor agents, we examined a contribution for increased 20-HETE generation. In the rat kidney perfused with Krebs' buffer, responses to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha), a thromboxane A2 mimetic, were compared before and after 50 microM L-nitroarginine (L-NA) to inhibit NO synthase. L-NA raised perfusion pressure (PP) from 79 +/- 3 to 190 +/- 7 mm Hg and enhanced constrictor responsiveness to U46619. U46619 (10, 30, 100, and 300 ng) increased PP by 7 +/- 1, 17 +/- 2, 50 +/- 7, and 67 +/- 7 mm Hg, respectively, before L-NA and 15 +/- 1, 37 +/- 7, 68 +/- 10, and 85 +/- 11 mm Hg, respectively, after L-NA, which did not increase 20-HETE efflux from the kidney. Nonetheless, an inhibitor of omega-hydroxylase, dibromododecencyl methylsulfonimide (DDMS), which reduced 20-HETE release, normalized the enhanced responsiveness to U46619. When PP was elevated with phenylephrine, vasoconstrictor responses to U46619 were similarly enhanced, an effect that was also prevented by DDMS. DDMS and an antagonist of 20-HETE, 20-HEDE [20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid], also reduced vasoconstrictor responses to U46619 in the absence of elevation of PP. Because 20-HETE inhibits K+ channels, we examined the effects of K+ channel inhibitors on vasoconstrictor responses and showed that both tetraethylammonium (TEA) and charybdotoxin enhanced renal vasoconstrictor responses to U46619. However, the inhibitory effects of 20-HEDE on vasoconstrictor responses remained after treatment with TEA. These results support a role for 20-HETE vasoconstrictor responses but suggest an action independent of K+ channels.

LIPID‐AUTOXIDATION‐LIMITED SHELF‐LIFE OF NIXTAMALIZED INSTANT CORN MASA

ABSTRACTConsumers often report the presence of off‐flavors and off‐odors characteristic of lipid oxidation in commercial dry instant corn masa, but the extent of this problem is unknown. The lipid oxidation states of two common brands of nixtamalized instant corn masa produced in Mexico were evaluated during 23‐weeks of storage at 15, 25, 35, 45, and 55C. Lipid autoxidation was evaluated by determining peroxide values (PV) and conjugated dienoic acids (CD) of extracted lipids, and sensory analyses of flours and tortillas made from stored flours. Increased storage time significantly increased lipid oxidation (P &lt; 0.001). Similarly, increased storage temperature significantly decreased the time to reach rancid flavor thresholds. The Q10 values for flours and tortillas were 1.58 and 1.75, respectively. The shelf‐lives of instant corn masa were 5.8 months when stored at 25C and only 2 months at 45C.

The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression.

Genetic analysis of the wound response pathway in tomato indicates that systemin and its precursor protein, prosystemin, are upstream components of a defensive signaling cascade that involves the synthesis and subsequent action of the octadecatrienoic acid (18:3)-derived plant hormone jasmonic acid (JA). The suppressor of prosystemin-mediated responses2 (spr2) mutation, which was isolated previously as a suppressor of (pro)systemin-mediated signaling, impairs wound-induced JA biosynthesis and the production of a long-distance signal for the expression of defensive Proteinase inhibitor genes. Using a map-based cloning approach, we demonstrate here that Spr2 encodes a chloroplast fatty acid desaturase involved in JA biosynthesis. Loss of Spr2 function reduced the 18:3 content of leaves to <10% of wild-type levels, abolished the accumulation of hexadecatrienoic acid, and caused a corresponding increase in the level of dienoic fatty acids. The effect of spr2 on the fatty acyl content of various classes of glycerolipids indicated that the Spr2 gene product catalyzes most, if not all, omega3 fatty acid desaturation within the "prokaryotic pathway" for lipid synthesis in tomato leaves. Despite the reduced levels of trienoic fatty acids, spr2 plants exhibited normal growth, development, and reproduction. However, the mutant was compromised in defense against attack by tobacco hornworm larvae. These results indicate that jasmonate synthesis from chloroplast pools of 18:3 is required for wound- and systemin-induced defense responses and support a role for systemin in the production of a transmissible signal that is derived from the octadecanoid pathway.

Stereoselective preparations of epoxy-, fluoro- and related derivatives of ricinoleic acid and 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid

Methyl ricinoleate 4 and methyl 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoate 7 were subjected to epoxidation using mCPBA, or tert-butyl hydroperoxide in the presence of vanadyl acetylacetonate or titanium (IV) isopropoxide with D-(-)- or L-(+)-diisopropyl tartrate. Epoxidations using mCPBA gave equal quantities of the disatereoisomeric epoxides 5/6 and 8/9 from 4 and 7 respectively. Other methods gave preferentially epoxides 5 or 8, except for treatment of 7 with tert-butyl hydroperoxide in the presence of titanium (IV) isopropoxide and L-(+)-diisopropyl tartrate which gave preferentially epoxide 9. Hydroxy-derivative 4, 7, 5, 6, 8 and 9 were converted into the fluoro-derivatives 16 - 21 by trimethylsilylation and treament with diethylaminosulphur trifluoride. Epoxides 6 and 9 were converted into 2-oxazolines 22 and 23 respectively by reaction with acetamide.

Glomerular autacoids stimulated by bradykinin regulate efferent arteriole tone

We have shown that when efferent arterioles are perfused retrograde to avoid the influence of vasoactive autacoids released by the glomerulus, bradykinin causes dilatation via release of cytochrome p450 (cp450) metabolites, probably epoxyeicosatrienoic acids (EETs). Here we tested the hypothesis that the glomerulus releases cyclooxygenase (COX) and cp450 metabolites. These eicosanoids, acting as vasopressor and vasodepressor autacoids, control efferent arteriole resistance downstream from the glomerulus. Rabbit efferent arterioles were perfused orthograde through the glomerulus from the end of the afferent arteriole to determine whether bradykinin induces the release of glomerular autacoids that influence efferent arteriole resistance. Efferent arterioles were preconstricted with norepinephrine, and increasing doses of bradykinin were added to the perfusate in the presence or absence of COX and cp450 inhibitors. When efferent arterioles were perfused orthograde through the glomerulus, bradykinin at 10 nmol/L caused significant and reproducible dilatation; diameter increased from 8.0 +/- 0.5 to 12.6 +/- 0.4 microm (P < 0.05). This effect was not modified by a nitric oxide synthase (NOS) inhibitor. In the presence of indomethacin, a COX inhibitor, bradykinin-induced dilatation was almost completely blocked (from 8.0 +/- 0.5 to 9.3 +/- 0.6 microm). This blockade was completely reversed by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), a specific antagonist of the vasoconstrictor cp450 metabolite 20-hydroxyeicosatetraenoic acid (20-HETE); diameter increased from 6.6 +/- 0.7 to 13.2 +/- 0.5 microm. To test the hypothesis that this dilatation was due to EETs, a specific inhibitor of EET synthesis, N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH), was added to the arteriolar perfusate. In the presence of indomethacin and 20-HEDE, bradykinin caused dilatation and this effect was completely blocked by MS-PPOH (1 microm) (from 7.6 +/- 0.6 to 7.3 +/- 0.5 microm). We concluded that in response to bradykinin, the glomerulus releases COX metabolites (probably prostaglandins) that have a vasodilator effect. When COXs are inhibited, the vasoconstrictor 20-HETE released by the glomerulus is able to oppose the vasodilator effect of bradykinin. This vasodilator effect is mediated by EETs released by the glomerulus and/or the efferent arteriole and does not involve nitric oxide. The balance between these opposing effects of various eicosanoids controls efferent arteriole resistance downstream from the glomerulus.

Purification and Characterization ofmeta-Cleavage Compound Hydrolase from a Carbazole DegraderPseudomonas resinovoransStrain CA10

2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries.

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca(2+)-activated K(+) channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-K(m) arachidonic acid omega-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC(50) from 0.37 +/- 0.04 microM in plasmid-transfected arteries to 0.07 +/- 0.01 microM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.