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 2ϩ -activated K ϩ 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-Km arachidonic acid -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 EC50 from 0.37 Ϯ 0.04 M in plasmid-transfected arteries to 0.07 Ϯ 0.01 M in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by , 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 SMALL ARTERIAL VESSELS MANUFACTURE 20-HETE, a product of arachidonic acid metabolism by cytochrome P-450 enzymes of the 4A family (CYP4A1) (8,12,17,26). Exogenous 20-HETE was reported to contract or relax vascular smooth muscle, depending on the animal species, type of vessels, and experimental conditions. Cyclooxygenase-dependent and -independent mechanisms have been implicated in 20-HETE-induced vascular contraction (6, 16, 17) and vascular relaxation (2, 4).Renal preglomerular vessels express CYP4A proteins, produce 20-HETE, and are constricted by exogenous 20-HETE. The constrictor action of exogenous 20-HETE in pressurized canine arcuate arteries, rat renal interlobular arteries, and rat renal afferent arterioles is independent of cyclooxygenase activity and has been attributed to inhibition of the opening of Ca 2ϩ -activated K ϩ channels in vascular smooth muscle cells, leading to cellular depolarization and increased Ca 2ϩ entry (8,12,14,23,28). Recent studies support the notion that endogenous 20-HETE subserves vasconstrictor mechanisms in the rat kidney. For example, inhibitors of 20-HETE synthesis were shown to increase blood flow to the kidney (29), to increase the diameter of preconstricted interlobular arteries denuded of endothelium (11), and to attenuate vasconstrictor responses elicited by endothelin (10, 22), angiotensin II (5), or increases in transmural pressure (14). 20-HETE produced by the smooth muscle isolated from renal preglomerular vessels was also shown to exert a tonic inhibitory influence on the activity of large-conductance C...

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.

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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...

The study was aimed to evaluate the potential of existing genotypes of Jerusalem artichoke (Helianthus tuberosus L.) as biomass feedstock for ethanol production. We investigated the biomass productivity and chemical composition of twenty-six Jerusalem artichoke clones grown in a semi-arid region of China. Jerusalem artichoke was demonstrated to be a sustainable feedstock for bioethanol production. All structural and non-structural carbohydrates in whole plant of Jerusalem artichoke could be 5000 L/ha. The above-ground biomass of Jerusalem artichoke could be a promising feedstock for cellulosic ethanol. The ethanol potential yield from cellulose and hemicellulose in aboveground biomass were 1821 to 5930 L/ha, contributing 29.8-66.4% of the total ethanol yield, which could be as high as that from switchgrass and sweet sorghum stem. Large variation among the investigated genotypes for carbohydrates makes it possible to select suitable clones to be used in bioethanol production in semiarid regions. Clones HB-3, HEN-3, IM-1, SC-1, SHX-3, SX-2 and ZJ-2 yielded tuber total soluble sugar higher than 4.0 t/ha. Clones BJ-4, HUB-2, HUN-2, QH-1, SD-2 and SHH-1 produced more than 5.0 t/ha cellulose and hemicellulose in above-ground biomass. Clones BJ-4 and HUB-2 have the highest ethanol potential based on structural carbohydrates. These clones were promising material if used as biofuel feedstock in this growth condition.

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