Cytochrome P450 Metabolites Of Arachidonic Acid Research Articles

20-HETE Activates the Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-κB Translocation and Promoter Binding.

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/tyrosine kinase/mitogen-activated protein kinase (MAPK)/inhibitor of κB kinase (IKK)β-mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor κB (NF-κB) activation prevented the 4.58-fold (±0.78; P < 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-κB-binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; P < 0.05) and 2.53-fold (± 0.24; P < 0.05), respectively. The 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKβ, and NF-κB activation. Sequence analysis demonstrated the presence of two and one putative NF-κB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-κB to each of the putative binding sites (S1, 3.43 ± 0.3-fold enrichment versus vehicle; S2, 3.72 ± 0.68-fold enrichment versus vehicle; S3, 3.20 ± 0.18-fold enrichment versus vehicle; P < 0.05). This is the first study to identify NF-κB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-κB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry

Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3–104 folds in standard solution and 5–138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.

Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p&lt;0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p&lt;0.05) and 2.53-fold (± 0.24; p&lt;0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p&lt;0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Abstract 159: Inhibition of Ace Does Not Prevent 20-hete-mediated Vascular Injury in Cyp4a12tg Mice

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite in the microcirculation, increases smooth muscle contraction and proliferation, stimulates endothelial dysfunction and activation and promotes hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells, 20-HETE increases angiotensin converting enzyme (ACE) mRNA by 3.1-fold (± 0.16; p&lt;0.05), ACE protein by 4.2-fold (±0.99; p&lt;0.05) and cellular and extracellular ACE activity by 2.2-fold (±0.09; p&lt;0.05) and 1.96-fold (±0.16; p&lt;0.05), respectively. The contribution of ACE to vascular 20-HETE-mediated microvascular remodeling in hypertension was assessed using the Cyp4a12 transgenic mouse in which the expression of the Cyp4a12-20-HETE synthase is driven by a tetracyclin (doxycycline, DOX)-sensitive promoter. Treatment of Cyp4a12tg mice with DOX increased blood pressure (133±6 vs. 107±3 mmHg; p&lt;0.05) and remodeling of renal resistance arteries (M/L, 0.15±0.01 vs. 0.07±0.01; p&lt;0.05); these increases were abrogated by co-treatment with the 20-HETE antagonist, 20-HEDGE. Renal ACE protein expression was significantly higher in DOX-treated (1.6 ± 0.17 fold; p&lt;0.05) as compared to untreated Cyp4a12tg mice; co-treatment with 20-HEDGE prevented the increase in ACE expression. Lisinopril treatment of Cyp4a12tg mice concurrently receiving DOX prevented the blood pressure increase (133±6 vs. 104±2 mmHg; p&lt;0.05) but did not interfere with renal microvascular 20-HETE production (1.05±0.18 vs 1.09±0.30 ng/mg). Co-treatment with lisinopril and DOX attenuated but did not prevent remodeling of renal microvessels (M/L, 0.07±0.0 vs. 0.15±0.01 vs. 0.11±0.01; p&lt;0.05 in water, DOX and DOX+Lisinopril, respectively). In contrast, co-treatment with 20-HEDGE prevented DOX-induced remodeling of resistance arteries of Cyp4a12tg mice. This study demonstrates that 20-HETE is a key determinant of microvascular remodeling in DOX-induced Cyp4a12tg hypertensive mice. 20-HETE’s mechanism only partially relies on activation of the RAS. Thus, the 20-HETE-driven microvascular remodeling does not fully depend on the upregulation of ACE and elevation of blood pressure.

Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells

The transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-and Mg2+ permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca2+ influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca2+]i transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 μmol/l) as well as moderate heat (<40 °C) elicited [Ca2+]i transients. Hypotonicity increased [Ca2+]i and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca2+ transients and underlying currents induced by known activators of this channel.

20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1

The cytochrome P-450 arachidonic acid metabolite 20-HETE is central to the regulation of vascular tone, renal function, and blood pressure and is synthesized in the rat kidney in response to angiotensin II (ANG II) and endothelin-1 (ET-1). There are very few studies examining the cellular synthesis of 20-HETE in humans. We aimed to measure human neutrophil and platelet 20-HETE levels under basal conditions and after ANG II, ET-1, and calcium ionophore (CaI). 20-HETE was measured in human platelets and neutrophils after saline (control), CaI (2.5 μg/ml), and ANG II or ET-1 (10 nmol/l-1 μmol/l) incubations. The effect of cells, which were preincubated with the ω-hydroxylase inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl) (HET0016, 10 nM), ANG II types 1 or 2 (AT(1) or AT(2)) receptor inhibition with irbesartan (1 μmol/l) or PD-123319 (1 μmol/l), or endothelin receptor subtypes A or B (ET(A) or ET(B)) receptor inhibition with BQ-123 or BQ-778 (100 nmol/l), was studied. Neutrophil and platelet content and release of 20-HETE was significantly increased by CaI and blocked by the ω-hydroxylase inhibitor HET0016. ANG II and ET-1 significantly increased neutrophil and platelet content and release of 20-HETE. ANG II increased 20-HETE via the AT(2) receptor. ET-1 increased 20-HETE through the ET(B) receptor in platelets and both the ET(A) and ET(B) receptors in neutrophils. These studies show that human platelets and neutrophils synthesize 20-HETE in response to ANG II and ET-1. 20-HETE synthesis in both cell types was predominantly mediated via the AT(2) and ET(B) receptors. Stimulation via these receptor pathways has generally been thought to be cardioprotective and requires further studies in clinical situations associated with low-grade inflammation or where ANG II and ET-1 are elevated to clarify the role of 20-HETE.

Endothelin-1 induced vascular smooth muscle cell proliferation is mediated by cytochrome p-450 arachidonic acid metabolites.

Endothelins (ETs) are a family of three peptides (ET-1, ET-2, ET-3) that are implicated in the physiological control of vascular smooth muscle cell (VSMC) and myocardial contractility and growth. ET-1 is vasoactive peptide that acts via ET-A receptors coupling inducing vascular smooth muscle cell contraction. ET-1 is involved in the development and maintenance of hypertension. Aim of this study was to investigate whether ET-1 can induce vascular smooth muscle cell proliferation through arachidonic acid (AA) metabolites formed via cytochrome P¬450 (CYP-450). VSMC proliferation was measured by [3H]thymidine incorporation in cultured cells treated by ET-1 (10 to l00 nmol/L) in presence of different inhibitors of CYP-450 (17-ODYA 5 μmol/L), lipoxygenase (LO) (baicalein 20 μmol/L) and cyclooxygenase (COX) (indomethacin 5 μmol/L). ET-1 (10 to 100 nmol/L) induced VSMC proliferation and this effect was attenuated by CYP-450 inhibitor (17-ODYA) and lipoxygenase (LO) inhibitor (baicalein) but not by cyclooxygenase (COX) inhibitor (indomethacin). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) and 12-HETE increased [3H]thymidine incorporation in VSMC. Inhibitors of MAP kinase (PD-98059 50 μmol/L) and cPLA2 (MAFP 50 μmol/L) attenuated ET-1 as well as 20-HETE induced VSMC proliferation. These results suggest AA metabolites via CYP-450 mediates ET-1 induce VSMC proliferation.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

A reduction in alcohol consumption is associated with reduced plasma F 2-isoprostanes and urinary 20-HETE excretion in men

There is considerable evidence that chronic moderate-to-high alcohol consumption increases blood pressure. The mechanisms by which this occurs are not clear. Alcohol consumption can induce oxidative stress and cytochrome P450 (CYP450) isoforms that are associated with oxidative stress and may influence vascular tone. To study the role of such mechanisms we examined whether reducing alcohol intake in moderate-to-heavy drinkers (40–110 g/day) resulted in changes in urinary excretion of 20-HETE, a CYP450 metabolite of arachidonic acid, and plasma and urinary F 2-isoprostanes as markers of lipid peroxidation. After a 4-week run-in period during which healthy men maintained their usual drinking pattern they were randomized to a two-way crossover intervention study. In each of the 4-week treatment periods subjects either substituted their usual alcohol intake with a 0.9% alcohol beer or maintained their usual alcohol intake. Plasma and urinary F 2-isoprostanes and urinary 20-HETE were measured by gas chromatography mass spectrometry, and serum γ-glutamyl transpeptidase (γ-GT) was measured as a biomarker of alcohol consumption, at the end of each study period. Sixteen healthy men age 51.0 ± 2.7 years and with a BMI of 26.4 ± 0.61 kg/m 2 completed the study. The reductions in alcohol intake (72.4 ± 5.0 vs 7.9 ± 1.6 g/day, p < 0.001) and serum γ-GT (geometric mean 24.4 U/L (95% CI 19.7, 30.2) vs 18.6 U/L (95% CI 15.5, 22.2, p < 0.01) were accompanied by a significant fall in blood pressure as well as urinary 20-HETE excretion (158 ± 23 vs 109 ± 19 pmol/mmol creatinine, p < 0.001) and plasma F 2-isoprostanes (3438 ± 158 vs 2929 ± 145 pmol/L, p = 0.01). A substantial reduction in alcohol consumption in healthy men lowered plasma F 2-isoprostanes and urinary 20-HETE. Increased oxidative stress and 20-HETE production may be linked, at least in part, to the pathogenesis of alcohol-related hypertension.

TRPV4 calcium entry channel: a paradigm for gating diversity

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4alpha-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca2+ entry channels.

Characterization and identification of cytochrome P450 metabolites of arachidonic acid released by human peritoneal macrophages obtained from the pouch of Douglas

Cytochrome P450 metabolism of arachidonic acid (AA) was investigated in human peritoneal macrophages which play a central role in chronic pelvic diseases in women (for example in endometriosis). The formation of eicosanoids other than prostaglandins (PGs) by these cells is still unknown. In non-activated macrophages obtained from women in the reproductive age, the main [ 3H]-AA metabolites coeluted with epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids (DHETs) and hydroxyeicosatetraenoic acids (HETEs) in reverse-phase HPLC. After zymosan activation a shift to PGs pathway was observed. Treatment with low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increased the formation of a metabolite coeluting with 5,6-DHET. By gas chromatography/mass spectrometry 5,6-DHET (after β-naphthoflavone induction), and 14,15-DHET as well as 11,12-DHET (after AA stimulation) were identified as major epoxygenase metabolites, respectively. The enantioselective formation of 12( S)-HETE was demonstrated by chiral-phase HPLC. Our findings demonstrate that non-activated peritoneal macrophages produce substantial amounts of bioactive cytochrome P450 metabolites of AA.

P-450 metabolites of arachidonic acid in the control of cardiovascular function.

Recent studies have indicated that arachidonic acid is primarily metabolized by cytochrome P-450 (CYP) enzymes in the brain, lung, kidney, and peripheral vasculature to 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) and that these compounds play critical roles in the regulation of renal, pulmonary, and cardiac function and vascular tone. EETs are endothelium-derived vasodilators that hyperpolarize vascular smooth muscle (VSM) cells by activating K(+) channels. 20-HETE is a vasoconstrictor produced in VSM cells that reduces the open-state probability of Ca(2+)-activated K(+) channels. Inhibitors of the formation of 20-HETE block the myogenic response of renal, cerebral, and skeletal muscle arterioles in vitro and autoregulation of renal and cerebral blood flow in vivo. They also block tubuloglomerular feedback responses in vivo and the vasoconstrictor response to elevations in tissue PO(2) both in vivo and in vitro. The formation of 20-HETE in VSM is stimulated by angiotensin II and endothelin and is inhibited by nitric oxide (NO) and carbon monoxide (CO). Blockade of the formation of 20-HETE attenuates the vascular responses to angiotensin II, endothelin, norepinephrine, NO, and CO. In the kidney, EETs and 20-HETE are produced in the proximal tubule and the thick ascending loop of Henle. They regulate Na(+) transport in these nephron segments. 20-HETE also contributes to the mitogenic effects of a variety of growth factors in VSM, renal epithelial, and mesangial cells. The production of EETs and 20-HETE is altered in experimental and genetic models of hypertension, diabetes, uremia, toxemia of pregnancy, and hepatorenal syndrome. Given the importance of this pathway in the control of cardiovascular function, it is likely that CYP metabolites of arachidonic acid contribute to the changes in renal function and vascular tone associated with some of these conditions and that drugs that modify the formation and/or actions of EETs and 20-HETE may have therapeutic benefits.

Angiotensin II-Induced Renal Vasodilation Mediated by Cytochrome P-450 Arachidonic Acid Metabolites

Objective: Activation of AT₂ angiotensin II receptor release cytochrome P-450 arachidonic acid metabolites (CYP-AA). The presence of the AT₂ receptor and its ability to mediate renal vasodilation through CYP-AA metabolites were evaluated. Methods: Vascular response to angiotensin II in the isolated perfused kidney of the rat was evaluated in the absence and presence of angiotensin II AT₁ and AT₂ receptor blockers. Results: Blockade of the AT₁ receptor unmasked a vasodilatory response that was inhibited by AT₂ receptor blockade and AA metabolism. Conclusion: The findings indicate that blockade of the AT₁ angiotensin II receptor, activation of AT₂ receptors, releases CYP-AA metabolites that induce renal vasodilation.

Cytochrome P450 metabolites of arachidonic acid may be important mediators in angiotensin II-induced vasoconstriction in the rat mesentery in vivo

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar–Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12,12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 ω-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize 14C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

Cytochrome P450 metabolites of arachidonic acid may be important mediators in angiotensin II-induced vasoconstriction in the rat mesentery in vivo

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar-Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12, 12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 omega-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize (14)C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

20-Hydroxyeicosatetraenoic acid and renal function in Lyon hypertensive rats

To evaluate the contribution of cytochrome P450 (CYP450) metabolites of arachidonic acid in the increased renal vascular resistance and blunted pressure–natriuresis response exhibited by Lyon hypertensive (LH) rats, the effects of an intrarenal infusion of 17-octadecynoic acid (3 μM), an inhibitor of the formation of epoxyeicosatrienoic and 20-hydroxyeicosatetraenoic acids, were compared in 8-week-old LH and low blood pressure (LL) control rats. 17-Octadecynoic acid failed to affect renal function in LL rats. In contrast, it reduced renal vascular resistance and shifted the pressure–natriuresis relationship to lower pressures in LH rats. Blockade of thromboxane–endoperoxide (TP) receptors with GR 32191B prevented the renal vasodilator response to 17-octadecynoic acid but not its natriuretic action. Miconazole (1 μM), an inhibitor of epoxygenase activity, had no effect on renal function in LH rats. These results indicate that CYP450 metabolites of arachidonic acid, likely 20-hydroxyeicosatetraenoic acid, contribute to the resetting of the pressure–natriuresis relation in LH rats and that the renal vasoconstrictor effects of 20-hydroxyeicosatetraenoic acid in LH rats may be related to activation of TP receptors.

Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung.

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient (Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 microM) or 17-octadecynoic acid (5 microM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 microM) significantly increased Kf,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation.

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

Cytochrome P-450 pathway in acetylcholine-induced canine coronary microvascular vasodilation in vivo.

In the canine coronary microcirculation, acetylcholine (ACh)-induced vasodilation of large (> or = 100 microns) epicardial arterioles (LgA), but not small (< 100 microns) epicardial arterioles (SmA), is blocked by nitric oxide (NO) synthase inhibitors in vivo. We hypothesized that the ACh-induced vasodilation of SmA is mediated by a cytochrome P-450 metabolite of arachidonic acid (AA). Epicardial coronary microvascular diameters in dogs were measured at baseline and after treatment with topically applied ACh (1, 10, and 100 microM), AA (1, 5, and 10 microM), or sodium nitroprusside (SNP; 10-100 microM). Coronary microvascular diameters were compared among control dogs (group OO); dogs pretreated with N omega-nitro-L-arginine (L-NNA; 70 microM topically) (group NO); dogs pretreated with L-NNA plus clotrimazole (Clo; 1.6 microM topically) or 17-octadecynoic acid (ODYA; 2 microM topically), cytochrome P-450 monooxygenase inhibitors (groups NC and NY, respectively); dogs pretreated with Clo alone (group OC); and dogs pretreated with L-NNA plus Clo with AA as the agonist (group AA). ACh-induced vasodilation of LgA was abolished by L-NNA alone, whereas in SmA, L-NNA was without effect. Clo alone did not inhibit ACh-induced dilation in either SmA or LgA. However, the combinations of L-NNA plus either Clo or ODYA abolished ACh- and AA-induced dilation of SmA (100 microM ACh: NC, 3 +/- 5%; NY, 8 +/- 2%; 10 microM AA: 6 +/- 3%) but did not affect responses to SNP. These results suggest that the ACh-induced vasodilation of SmA is mediated in part by cytochrome P-450 metabolites of AA and provide the first evidence that the cytochrome P-450 pathway contributes to the regulation of coronary resistance vessels in vivo.

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, juxtatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and omega-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 omega-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the omega-hydroxylase gene family revealed that mRNA levels for omega-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid omega-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with omega-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by omega-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.