Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.
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In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs. (+info)
In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.
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The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats. (+info)
Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice.
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The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. 20-HETE production in the kidney has been extensively studied in rats and humans and occurs primarily via the actions of P-450 enzymes of the CYP4A and -4F families. Recent advancements in molecular genetics of the mouse have made it possible to disrupt genes in a cell-type-specific fashion. These advances could help in the creation of models that could distinguish between the vascular and tubular actions of 20-HETE. However, isoforms of the CYP4A and -4F families that may be responsible for the production of 20-HETE in the vascular and tubular segments in the kidney of the mouse are presently unknown. The goal of this study was to identify the isoforms of the CYP4A and -4F families along the nephron by RT-PCR of RNA isolated from microdissected renal blood vessels and nephron segments from 16- to 24-wk-old male and female C57BL/6J mice. CYP4A and -4F isoforms were detected in every segment analyzed, with sex differences only observed in the proximal tubule and glomeruli. In the proximal tubular segments from male mice, the 4A10 and -12 isoforms were present, whereas the 4A10 and -14 isoforms were detected in segments from female mice. In glomeruli, sex differences in the expression pattern of CYP4F isoforms were also observed, with male mice expressing the 4F13, -14, and -15 isoforms, whereas female mice expressed the 4F13, -16, and -18 isoforms. These results demonstrate that isolated nephron and renal vessel segments express multiple isoforms of the CYP4A and -4F families; therefore, elimination of a single CYP4A or -4F isoform may not decrease 20-HETE production in all nephron segments or the renal vasculature of male and female mice. However, the importance of CYP4A vs. -4F isoforms to the production of 20-HETE in each of these renal tubular and vascular segments of the mouse remains to be determined. (+info)
Renal arterial 20-hydroxyeicosatetraenoic acid levels: regulation by cyclooxygenase.
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20-HETE, a potent vasoconstrictor, is generated by cytochrome P-450 omega-hydroxylases and is the principal eicosanoid produced by preglomerular microvessels. It is released from preglomerular microvessels by ANG II and is subject to metabolism by cyclooxygenase (COX). Because low-salt (LS) intake stimulates the renin-angiotensin system and induces renal cortical COX-2 expression, we examined 20-HETE release from renal arteries (interlobar and arcuate and interlobular arteries) obtained from 6- to 7-wk-old male Sprague-Dawley rats fed either normal salt (0.4% NaCl) or LS (0.05% NaCl) diets for 10 days. With normal salt intake, the levels of 20-HETE recovered were similar in arcuate and interlobular arteries and interlobar arteries: 30.1 +/- 8.5 vs. 24.6 +/- 5.3 ng. mg protein(-1). 30 min(-1), respectively. An LS diet increased 20-HETE levels in the incubate of either arcuate and interlobular or interlobar renal arteries only when COX was inhibited. Addition of indomethacin (10 microM) to the incubate of arteries obtained from rats fed an LS diet resulted in a two- to threefold increase in 20-HETE release from arcuate and interlobular arteries, from 39.1 +/- 13.2 to 101.8 +/- 42.6 ng. mg protein(-1). 30 min(-1) (P < 0.03), and interlobar arteries, from 31.7 +/- 15.1 to 61.9 +/- 29.4 ng. mg protein(-1). 30 min(-1) (P < 0.05) compared with release of 20-HETE when COX was not inhibited. An LS diet enhanced vascular expression of cytochrome P-4504A and COX-2 in arcuate and interlobular arteries; COX-1 was unaffected. Metabolism of 20-HETE by COX is proposed to represent an important regulatory mechanism in setting preglomerular microvascular tone. (+info)
Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531.
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The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus. (+info)
CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis.
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Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle. (+info)
Differential expression of the components of the two alkane hydroxylases from Pseudomonas aeruginosa.
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Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes. (+info)
Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.
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Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. (+info)