These results suggest that HO-2 is constitutively expressed in the rat kidney mainly within tubular and arteriolar structures, and its activity may modulate physiological function under basal conditions. On the other hand, the basal levels of expression of HO-1 in the rat kidney are relatively low, and its contribution to HO activity and the regulation of hemoproteins such as cytochrome P450 become apparent only under pathophysiological conditions causing HO induction.

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt−1 ⋅ day−1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

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(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 +/- 3 to 121 +/- 4 mmHg (P < 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 synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC(50) 0.69 +/- 0.17 vs. 1.77 +/- 0.40 microM). 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.

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 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 NO2/NO3 excretion decreased by 40 and 52% in L-NAMEand L-NAME ϩ 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 G -nitro-L-arginine methyl ester 20-HYDROXYEICOSATETRAENOIC acid (20-HETE), the -hydroxylation product of arachidonic acid (AA), is a principal eicosanoid in vascular and tubular structures of the rat kidney whose synthesis is catalyzed primarily by isoforms of the cytochrome P-450 (CYP) 4A gene family (34). In the rat, four CYP4A isoforms, CYP4A1, 4A2, 4A3, and 4A8, have been identified. The renal expression of CYP4A isoforms has been shown to be tissue specific as well as age and sex dependent (9,12,13,19,20). For example, Sundseth and Waxman (37) demonstrated that hepatic and renal CYP4A1 and 4A3 expressions are similar in male and female rats, whereas CYP4A2 expression is undetectable in female rats. Castration of male rats decreased the levels of CYP4A2, and treatment of cast...

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.

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.

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A-derived arachidonic acid metabolite, is a potent vasoconstrictor and a modulator of vascular reactivity. We have shown that CYP4A1 and CYP4A2 are the major CYP4A isoforms expressed in the rat renal microcirculation. In the present study, we constructed two bicistronic vectors, pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2, and examined their functional efficacy in COS-1 and vascular smooth muscle (A7r5) cells and in microdissected rat interlobar arteries. Immunocytochemistry coupled with fluorescence microscopy of pIRES2-EGFP-4A1-or pIRES2-EGFP-4A2-transfected COS-1 and A7r5 cells indicated that both enhanced green fluorescence protein (EGFP) and CYP4A1/4A2 were expressed in 80 to 90% of the cells. Western blot analysis showed a 3-to 5-fold increase of CYP4A1 and CYP4A2 proteins in pIRES2-EGFP-4A1-and pIRES2-EGFP-4A2-transfected cells as compared with control pIRES2-transfected cells. Cells transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 catalyzed arachidonic acid -hydroxylation to 20-HETE at rates of 0.85 Ϯ 0.29 and 0.27 Ϯ 0.04 nmol/10 7 cells/h, respectively. Transfection of interlobar arteries with either plasmid yielded EGFP immunofluorescence that was localized to the intima, media, and adventitia. Arteries transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 showed increased vasoreactivity displaying EC 50 to phenylephrine of 0.24 Ϯ 0.07 and 0.11 Ϯ 0.03 M, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 Ϯ 0.21 M; n ϭ 6, p Ͻ 0.05). The increased vasoreactivity to phenylephrine was inhibited by N-methylsulfonyl-12,12-dibromododec-11-enamide, an inhibitor of CYP4A-catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. Removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1 cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin. 20-Hydroxyeicosatetraenoic acid (20-HETE), the -hydroxylation metabolite of arachidonic acid, is a major eicosanoid in the renal microvasculature (Ma et al., 1993;Wang et al., 1998b). When added to isolated preparations of renal interlobular and afferent arterioles, 20-HETE causes vasoconstriction (Imig et al., 1996). In isolated interlobar arteries, increased synthesis of 20-HETE or addition of 20-HETE increased responsiveness to constrictor agonists such as phenylephrine (Kaide et al., 2003). Recent studies indicated that 20-HETE promotes Ca 2ϩ entry by depolarizing smooth muscle cells secondary to blockade of K Ca channels and by increasing conductance of L-type Ca 2ϩ channels (Imig et al., 1996;Zou et al., 1996;Gebremedhin et al., 1998). These findings suggest that 20-HETE serves as an intracellular regulator of ion channel activity rather than a direct vasoconstrictor. Studies demonstrating the effect of 20-HETE on signaling proteins including protein k...

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