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Baragatti B, Schwartzman ML, Angeloni D, Scebba F, Ciofini E, Sodini D, Ottaviano V, Nencioni S, Paoliechi A, Graves JP, Zeldin DC, Gotlinger K, Luin S, Coceani F. EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid. Am J Physiol Heart Circ Physiol 297: H2161-H2168, 2009. First published October 2, 2009; doi: 10.1152/ajpheart.00576.2009.--We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor(EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450(CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for [omega]-hydroxylation(CYP4A31) and 12R-hydroxylation(CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids(EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and [omega]-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-1inked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel. bradykinin; arachidonic acid epoxygenase; arachidonic acid monooxygenase; fetal and neonatal physiology doi: 10.1152/ajpheart.00576.2009

We have previously reported that adenoviral-mediated delivery of cytochrome P-450 (CYP) 4A2, which catalyzes the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), results in endothelial dysfunction and hypertension in Sprague-Dawley (SD) rats (Wang JS, Singh H, Zhang F, Ishizuka T, Deng H, Kemp R, Wolin MS, Hintze TH, Abraham NG, Nasjletti A, Laniado-Schwartzman M. Circ Res 98: 962-969, 2006). In this study, we targeted the vascular endothelium by using a lentivirus construct expressing CYP4A2 under the control of the endothelium-specific promoter VE-cadherin (VECAD-4A2) and examined the effect of long-term CYP4A2 overexpression on blood pressure and kidney function in SD rats. A bolus injection of VECAD-4A2 increased blood pressure (P < 0.001) by 26, 36, and 30 mmHg 10, 20, and 30 days postinjection, respectively. Arteries from VECAD-4A2-transduced rats produced increased levels of 20-HETE (P < 0.01), expressed lower levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) (P < 0.05), generated higher levels of superoxide anion, and displayed decreased relaxing responsiveness to acetylcholine (P < 0.05). Proteinuria increased by twofold in VECAD-4A2-transduced rats compared with controls. Treatment of VECAD-4A2-transduced rats with HET0016, an inhibitor of 20-HETE biosynthesis, not only attenuated the increase in blood pressure (P < 0.05) but also improved vascular function (acetylcholine-induced relaxations) and reduced plasma creatinine and proteinuria. HET0016 treatment decreased oxidative stress and increased the phosphorylated state of key proteins that regulate endothelial function, including eNOS, AKT, and AMPK. Collectively, these findings demonstrate that augmentation of vascular endothelial 20-HETE levels results in hypertension, endothelial dysfunction, and renal injury, which is offset by HET0016 through a reduction in vascular 20-HETE coupled with a lessening of oxidative stress and the amplification of pAKT, pAMPK, and p-eNOS levels leading to normalization of endothelial responses. CYP4A-derived 20-HETE; endothelial dysfunction; lentiviral vectors; endothelial-specific promoter; oxidative stress doi: 10.1152/ajprenal.00364.2009

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 CYP4AI. 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 [+ or -] 3 [micro]m) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 [+ or -] 4 [micro]m). 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. myogenic vasoconstriction; 20-hydroxyeicosatetraenoic acid; cytochrome P-450 oxygenases; vascular reactivity

20-Hydroxyeicosatetraenoic acid (20-HETE), which promotes renal vasoconstriction, is formed in the rat kidney primarily by cytochrome P-450 (CYP) 4A isoforms (4A1, 4A2, 4A3, 4A8). Nitric oxide (NO) has been shown to bind to the heme moiety of the CYP4A2 protein and to inhibit 20-HETE synthesis in renal arterioles of male rats. However, it is not known whether NO interacts with and affects the activity of CYP4A1 and CYP4A3, the major renal CYP4A isoforms in female rats. Incubation of recombinant CYP4A1 and 4A3 proteins with sodium nitroprusside (SNP) shifted the absorbance at 440 nm, indicating the formation of a ferric-nitrosyl-CYP4A complex. The absorbance for CYP4A3 was about twofold higher than that of CYP4A1. Incubation of SNP or peroxynitrite (PN; 0.01-1 mM) with CYP4A recombinant membranes caused a concentration-dependent inhibition of 20-HETE synthesis, with both chemicals having a greater inhibitory effect on CYP4A3-catalyzed activity. Moreover, incubation of CYP4A1 and 4A3 proteins with PN (1 mM) resulted in nitration of tyrosine residues in both proteins. In addition, PN and SNP inhibited 20-HETE synthesis in renal microvessels from female rats by 65 and 59%, respectively. We previously showed that microvessel CYP4A1/CYP4A3 expression and 20-HETE synthesis are decreased in late pregnancy. Therefore, we investigated whether such a decrease is dependent on NO, the synthesis of which has been shown to increase in late pregnancy. Administration of [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) to pregnant rats for 6 days (days 15-20 of pregnancy) caused a significant increase in systolic blood pressure, which was prevented by concurrent treatment with the CYP4A inhibitor 1-aminobenzotriazole (ABT). Urinary N[O.sub.2]/N[O.sub.3] excretion decreased by 40 and 52% in L-NAME-and L-NAME + ABT-treated groups, respectively. Interestingly, renal microvessel 20-HETE synthesis showed a marked increase following L-NAME treatment, and this increase was diminished with coadministration of ABT. These results demonstrate that NO interacts with CYP4A proteins in a distinct manner and it interferes with renal microvessel 20-HETE synthesis, which may play an important role in the regulation of blood pressure and renal function during pregnancy. cytochrome P-450 4A; [N.sup.G]-nitro-L-arginine methyl ester

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.sup.2+]-activated [K.sup.+] 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.sub.m] arachidonic acid [omega]-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4Al-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 E[C.sub.50] from 0.37 [+ or -] 0.04 [micro]M in plasmid-transfected arteries to 0.07 [+ or -] 0.01 [micro]M 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. 20-hydroxyeicosatetraenoic acid; arachidonic acid; phenylephrine; cytochrome P-450

Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries. Am J Physiol Renal Physiol 283:F60-F67, 2002. First published February 12, 2002; 10.1152/ajprenak00265. 2001.--20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor and mediator of the myogenic response, is a major arachidonic acid metabolite in the microvasculature of the rat kidney formed primarily by the cytochrome P-450 (CYP) 4A isoforms, CYP4A1, CYP4A2, and CYP4A3. We examined CYP4A isoform expression and 20-HETE synthesis in microdissected interlobar, arcuate, and interlobular arteries; mRNA for all CYP4A isoforms was identified by RT-PCR. Western blot analysis indicated that the levels of CYP4A2/ 4A3-immunoreactive protein increased with decreased arterial diameter, whereas those of CYP4Al-immunoreactive protein remained unchanged. 20-HETE synthesis was the highest in the interlobular arteries (17 [+ or -] 1.62 [nmol*[mg.sup.-1* [h.sup.-1]) and, like CYP4A2/4A3-immunoreactive protein, decreased with increasing vessel diameter (4.5 [+ or -] 1.21, 2.65 [+ or -] 0.58, and 0.81 [+ or -] 0.14 nmol*[mg.sup.-1] in the arcuate, interlobar, and segmental arteries, respectively). 20-HETE synthesis in the renal artery and the abdominal aorta was undetectable. The observed decreased immunoreactivity of NADPH-cytochrome P-450 (c) oxidoreductase with increased arterial diameter provided a possible explanation for the decreased capacity to generate 20-HETE in the large arteries. The increase in CYP4A isoform expression and 20-HETE synthesis with decreasing diameter along the preglomerular arteries and the potent biological activity of 20-HETE underscore the significance of 20-HETE as a modulator of renal hemodynamics. rat kidney; arachidonic acid; NADPH-cytochrome P-450 (c) oxidoreductase; hydroxyeicosatetraenoic acid

Renal 20-hydroxyeicosatetraenoic acid synthesis during pregnancy. Am J Physiol Regulatory Integrative Comp Physiol 282: R383-R389, 2002.--We examined whether renal 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis is altered during gestation. Renal microsomal arachidonic acid [omega]-hydroxylase activity increased by 50 and 48% in rats on days 12 and 19 of gestation, respectively. Renal microvessel 20-HETE synthesis increased by 50 and 82% in rats on days 6 and 12 of gestation, respectively, and returned to control levels at day 19 of gestation. In contrast, 20-HETE synthesis in isolated medullary thick ascending limb was unchanged from control levels on days 6 and 12 of gestation, but it increased twofold on day 19 of gestation. This increase on day 19 of gestation was associated with a twofold increase in urinary 20-HETE excretion, and it coincided with a 23-mmHg fall in blood pressure. Moreover, change in the rate of 20-HETE synthesis in microvessels was consistent with the level of expression of cytochrome P450 (CYP)4A proteins. Administration of the CYP4A inhibitor 1-aminobenzotriazole (ABT) for 2 days on day 12 of pregnancy or for 5 days starting on day 15 of pregnancy caused a transient but significant reduction in systolic blood pressure. ABT treatment also decreased urinary sodium, urinary 20-HETE, and renal and microvessel 20-HETE synthesis. This study, to our knowledge, is the first to demonstrate that 20-HETE synthesis in the kidney is altered in time- and site-specific manners during pregnancy. The localized pattern of changes suggests that there are distinct regulatory mechanisms for 20-HETE synthesis in the kidney during pregnancy. medullary thick ascending limb; renal microvessels; 1-aminobenzotriazole

A study was conducted on antisense oligonucleotides (ODNs) designed after the active cytochrome P4A1 (CYP4A1) and CYP4A2/4A3 expression in Sprague-Dawley rats. The effectiveness of the antisense ODNs in inducing isoform-specific inhibition of the in vitro and in vivo synthesis of the 20-hydroxyeicosatetraenoic acid (20-HETE), the metabolite formed after the omega-hydroxylation of arachidonic acid was also investigated. The findings imply that CYP4A1 holds the potential of regulating renal function and blood pressure based on its 20-HETE synthetisizing activity in the rat kidney.

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR. Am J Physiol Regulatory Integrative Comp Physiol 280: R255-R261, 2001.--The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low [K.sub.m] arachidonic acid [Omega]-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 [+ or -] 3 to 121 [+ or -] 4 mmHg (P [is less than] 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE syn.thesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine ([EC.sub.50] 0.69 [+ or -] 0.17 vs. 1.77 [+ or -] 0.40 [micro]M). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR. 20-hydroxyeicosatetraenoic acid; phenylephrine; myogenic response; kidney; blood pressure