Proximal tubular-targeted overexpression of the Cyp4a12-20-HETE synthase promotes salt-sensitive hypertension in male mice.

20-Hydroxyeicosatetraenoic acid (20-HETE) has been linked to blood pressure (BP) regulation via actions on the renal microvasculature and tubules. We assessed tubular 20-HETE contribution to hypertension by generating transgenic mice overexpressing the CYP4A12-20-HETE synthase (PT-4a12 mice) under the control of the proximal tubule (PT)-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). 20-HETE levels in the kidney cortex of male (967±210 vs. 249±69 pg/mg protein), but not female (121±15 vs. 92±11 pg/mg protein) PT-4a12 mice, showed a 2.5-fold increase compared to WT. Renal cortical Cyp4a12 mRNA and CYP4A12 protein in male, but not female PT-4a12 mice increased by 2-3-fold compared to WT. Male PT-4a12 mice displayed elevated BP (142±1 vs. 111±4 mmHg, p<0.0001), whereas BP in females PT-4a12 mice was not significantly different from WT (118±2 vs. 117±2 mmHg; p=0.98). In male PT-4a12 mice, BP decreased when transitioned from a control salt (0.4%) to a low-salt diet (0.075%) from 135±4 to 120±6 mmHg (p<0.01) and increased to 153±5 mmHg (p<0.05) when placed on a high-salt diet (4%). Female PT-4a12 mice did not show changes in BP on either low- or high-salt diet. In conclusion, the expression of Cyp4a12 driven by the PEPCK promoter is sex-specific probably due to its X-linkage. The salt-sensitive hypertension seen in PT-4a12 male mice suggests a potential anti-natriuretic activity of 20-HETE that needs to be further explored.

The Blood Pressure-Lowering Effect of 20-HETE Blockade in Cyp4a14(-/-) Mice Is Associated with Natriuresis.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has been linked to pro-hypertensive and anti-hypertensive actions through its ability to promote vasoconstriction and inhibit Na transport in the ascending limb of the loop of Henle, respectively. In this study, we assessed the effects of 20-HETE blockade on blood pressure, renal hemodynamics, and urinary sodium excretion in Cyp4a14(-/-) male mice, which display androgen-driven 20-HETE-dependent hypertension. Administration of 2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate (20-SOLA), a water-soluble 20-HETE antagonist, in the drinking water normalized the blood pressure of male Cyp4a14(-/-) hypertensive mice (±124 vs. ±153 mmHg) while having no effect on age-matched normotensive wild-type (WT) male mice. Hypertension in Cyp4a14(-/-) male mice was accompanied by decreased renal perfusion and reduced glomerular filtration rates, which were corrected by treatment with 20-SOLA. Interestingly, Cyp4a14(-/-) male mice treated with 20-SOLA displayed increased urinary sodium excretion that was paralleled by the reduction of blood pressure suggestive of an antinatriuretic activity of endogenous 20-HETE in the hypertensive mice. This interpretation is in line with the observation that the natriuretic response to acute isotonic saline loading in hypertensive Cyp4a14(-/-) male mice was significantly impaired relative to that in WT mice; this impairment was corrected by 20-SOLA treatment. Hence, endogenous 20-HETE appears to promote sodium conservation in hypertensive Cyp4a14(-/-) male mice, presumably, as a result of associated changes in renal hemodynamics and/or direct stimulatory action on tubular sodium reabsorption.

Dexamethasone promotes hypertension by allele-specific regulation of the human angiotensinogen gene.

The human angiotensinogen (hAGT) gene has polymorphisms in its 2.5-kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, and -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension, whereas Hap -6G/-217G reduces cardiovascular risk. Hap -6A/-217A has increased promoter activity with enhanced transcription factor binding, including to the glucocorticoid receptor (GR). Glucocorticoid therapy frequently causes hypertension, the mechanisms for which are incompletely understood. We have engineered double transgenic (TG) mice containing the human renin gene with either Hap of the hAGT gene and examined the physiological significance of glucocorticoid-mediated allele-specific regulation of the hAGT gene. We have also studied the consequential effects on the renin angiotensin system and blood pressure. TG mice with Hap -6A and -6G were treated with and without a low dose of a GR agonist, dexamethasone (2.5 µg/ml), for 72 h. We found greater chromatin-GR binding with increased GR agonist-induced hAGT expression in liver and renal tissues of Hap -6A mice. Additionally, dexamethasone treatment increased circulating hAGT and angiotensin II levels in Hap -6A mice, as compared with -6G mice. Importantly, GR agonist significantly increased blood pressure and redox markers in TG mice with Hap-6A of the hAGT gene. Taken together, our results show, for the first time, that glucocorticoids affect hAGT expression in a haplotype-dependent fashion with SNPs in Hap -6A favoring agonist-induced GR binding. This leads to increased expression of the hAGT, up-regulation of the renin angiotensin system, and increased blood pressure and oxidative stress in Hap -6A mice.

Antioxidants condition pleiotropic vascular responses to exogenous H(2)O(2): role of modulation of vascular TP receptors and the heme oxygenase system.

AIMS: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants. RESULTS: Exogenous H(2)O(2) (0.1-10.0 µM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p<0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation. INNOVATIONS: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 µM), thromboxane (TP) synthase (CGS13080-10 µM), and TP receptor antagonist (SQ29548-1 µM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats. CONCLUSION: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2).

Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors: exchange of lysosomal pool.

Although therapeutic effect of adoptive transfer of endothelial progenitor cells (EPC) has been well-substantiated, the actual engraftment is relatively low compared to a robust functional improvement of vasculopathy. Cellular mechanisms governing this action remain elusive. A recently discovered cell-cell communication via tunneling nanotube (TNT) formation is capable of transferring mitochondria and lysosomes between the cells - "organellar diakinesis". Based on the previous demonstration of lysosomal dysfunction in endothelial cells exposed to AGE-modified collagen I, we inquired whether TNT mechanism may be involved in EPC-mediated repair of stressed endothelial cells. Here we demonstrate that EPC selectively and multiplicatively establish TNT communication with stressed endothelia. The guidance cues for the selectivity are provided by exofacially exposed phosphatidylserine moieties. Lysosomal transfer is associated with the preservation of lysosomal pH gradient, functionally reconstituting lysosomal pool of stressed cells and improving endothelial cell viability, reducing premature senescence and apoptosis. In vivo, adoptive transfer of EPC to streptozotocin-diabetic mice results in a TNT-dependent reduction of senescent endothelial cells and correction of endothelium-dependent vasorelaxation. Collectively, these data establish a selective multiplicative effect of TNT between EPC and stressed endothelia, reconstitution of the lysosomal pool, and improved viability and function of stressed endothelia.

Dual pathways of carbon monoxide-mediated vasoregulation: modulation by redox mechanisms.

RATIONALE: Vascular tissues produce carbon monoxide (CO) via HO-dependent and HO-independent mechanisms; the former in tandem with biliverdin and iron and the latter as a lone product. CO has been shown to function as both a vasoconstrictor and vasodilator; however, factors that dictate the vasoregulatory phenotype of this gas are unknown. OBJECTIVE: We investigated whether CO-mediated vasoconstriction is mechanistically linked to enhanced reactive oxygen species production that masks vasodilatory pathways. METHODS AND RESULTS: Sprague-Dawley rat interlobar and interlobular arteries were examined in terms of superoxide (O2*-) generation and vascular reactivity in the absence and presence of antioxidants. Both authentic CO and the CO-releasing molecule (CORM)-3 constricted renal arteries and increased O2*- production in a dose-dependent manner. The antioxidants tempol, ebselen, and deferoxamine inhibited CO-induced O2*- production and converted CO from constrictor to dilator. CO-induced O2*- generation was found to involve the activity of multiple oxidases including nitric oxide synthase, NADPH oxidase, xanthine oxidase, and complex IV of the mitochondrial electron chain. Furthermore, inhibition of these enzymes converted CO from constrictor to dilator. Similarly, biliverdin and bilirubin inhibited CO-induced O2*- production and vasoconstriction, allowing for a vasodilatory response to CO to be expressed. CO-induced vasoconstriction was dependent on a non-thromboxane agonist of the thromboxane receptor, whereas vasodilatory mechanisms of CO relied on the activation of soluble guanylate cyclase and calcium-gated potassium channels. CONCLUSIONS: CO-induced vasoconstriction involves the generation of reactive oxygen species, which, when negated, allows for the expression of vasodilatory pathways which are masked by the primary oxidative stress response to this gas.

Adoptive transfer of syngeneic bone marrow-derived cells in mice with obesity-induced diabetes: selenoorganic antioxidant ebselen restores stem cell competence.

There are conflicting data regarding the effects of transplantation of bone marrow-derived cells (BMDCs) on the severity of diabetes. We therefore inquired whether the competence of BMDCs is preserved on adoptive transfer into diabetic (db/db) mice and how the adoptive transfer of BMDCs affects vascular and metabolic abnormalities in these mice. Recipient db/db mice received infusions of BMDCs prepared from either db/db or non-diabetic heterozygout mice (db/m) mice and effects on endothelium-dependent relaxation, insulin sensitivity, and renal function were evaluated. Recipients of BMDCs from db/m, but not db/db donors showed better glucose control, exhibited striking improvement in endothelium-dependent relaxation in response to acetylcholine, and had partially restored renal function. Improved glucose control was due to enhanced insulin sensitivity, most likely secondary to improved vascular function. Enhanced apoptosis of endothelial progenitor cells under oxidative stress, as well as decreased endothelial progenitor cell numbers were responsible for the apparent functional incompetence of BMDCs from db/db donors. Treatment of db/db mice with Ebselen restored the resistance of both BMDCs and endothelial progenitor cells to oxidative stress, improved acetylcholine-induced vasorelaxation, and reduced proteinuria in db/db recipients of BMDC transplantation. In conclusion, infusion of BMDCs obtained from db/m donors to db/db recipient mice benefited macrovascular function, insulin sensitivity, and nephropathy. BMDCs obtained from db/db mice were functionally incompetent secondary to the increased proportion of apoptotic cells on oxidative stress challenge; their competence was restored by Ebselen therapy.

The Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction: proteomic approach.

Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N(G)-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward "metabolic hypoxia" through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

The T8590C polymorphism of CYP4A11 and 20-hydroxyeicosatetraenoic acid in essential hypertension.

A role for a deficit in transport actions of 20-hydroxyeicosatetraenoic acid (20-HETE) in hypertension is supported by the following: (1) diminished renal 20-HETE in Dahl-S rats; (2) altered salt- and furosemide-induced 20-HETE responses in salt-sensitive hypertensive subjects; and (3) increased population risk for hypertension in C allele carriers of the T8590C polymorphism of CYP4A11, which encodes an enzyme with reduced catalytic activity. We determined T8590C genotypes in 32 hypertensive subjects, 25 of whom were phenotyped for salt sensitivity of blood pressure and insulin sensitivity. Urine 20-HETE was lowest in insulin-resistant, salt-sensitive subjects (F=5.56; P<0.02). Genotypes were 13 TT, 2 CC, and 17 CT. C allele frequency was 32.8% (blacks: 38.9%; whites: 25.0%). C carriers (CC+CT) and TT subjects were similarly distributed among salt- and insulin-sensitivity phenotypes. C carriers had higher diastolic blood pressures and aldosterone:renin and waist:hip ratios but lower furosemide-induced fractional excretions of Na and K than TT. The T8590C genotype did not relate to sodium balance or pressure natriuresis. However, C carriers, compared with TT, had diminished 20-HETE responses to salt loading after adjustment for serum insulin concentration and resetting of the negative relationship between serum insulin and urine 20-HETE to a 1-microg/h lower level of 20-HETE. The effect of C was insulin independent and equipotent to 18 microU/mL of insulin (Delta20-HETE= 2.84-0.054xinsulin-0.98xC; r(2)=0.53; F=11.1; P<0.001). Hence, genetic (T8590C) and environmental (insulin) factors impair 20-HETE responses to salt in human hypertension. We propose that genotype analyses with sufficient homozygous CC will establish definitive relationships among 20-HETE, salt sensitivity of blood pressure, and insulin resistance.

Carbon monoxide stimulates the Ca2(+)-activated big conductance k channels in cultured human endothelial cells.

We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca(2+)-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N(G)-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N(G)-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

Vascular cytochrome P450 4A expression and 20-hydroxyeicosatetraenoic acid synthesis contribute to endothelial dysfunction in androgen-induced hypertension.

Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5alpha-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats. We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3+/-1.5 versus 1.5+/-0.5 ng/mg of protein per hour; P<0.05), superoxide anion (246+/-47 versus 31+/-8 cpm/microg of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrine-preconstricted renal interlobar arteries from DHT-treated rats (42.8+/-4.8%) significantly (P<0.05) increased in the presence of HET0016 (81.5+/-10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146+/-2 mm Hg in DHT-treated rats to 130+/-1 mm Hg (P<0.05). The results strongly implicate vascular cytochrome P450 4A-derived 20-hydroxyeicosatetraenoic acid in the development of androgen-induced endothelial dysfunction and hypertension.

Low Na intake suppresses expression of CYP2C23 and arachidonic acid-induced inhibition of ENaC.

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 microM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.

Endothelial dysfunction and hypertension in rats transduced with CYP4A2 adenovirus.

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114+/-1 to 133+/-1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2-transduced rats produced more 20-HETE (129+/-10 versus 97+/-7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2+/-1.6 versus 8.4+/-1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.

Decreased levels of cytochrome P450 2E1-derived eicosanoids sensitize renal arteries to constrictor agonists in spontaneously hypertensive rats.

We compared renal interlobar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in terms of cytochrome P450 (CYP) 4A and CYP2E1 protein expression; levels of 20-HETE, 19-HETE, and 18-HETE; and responsiveness to phenylephrine in the absence and presence of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 mumol/L), a CYP4A inhibitor. Relative to data in WKY, arteries of SHR exhibited diminished (P<0.05) CYP2E1 and levels of 19-HETE (66.7+/-6.0 versus 44.9+/-2.8 pmol/mg) and 18-HETE (13.8+/-1.6 versus 7.9+/-0.5 pmol/mg), whereas CYP4A and 20-HETE levels (99.3+/-9.1 versus 98.9+/-12.8 pmol/mg) were unchanged. Phenylephrine contracted vascular rings of SHR and WKY; the R(max) was similar in both strains, but SHR vessels were more sensitive as denoted by the lower (P<0.05) EC50 (0.28+/-0.07 versus 0.71+/-0.12 mumol/L). DDMS decreased 20-HETE and, to a lesser extent, 19-HETE, while increasing (P<0.05) the EC50 for phenylephrine by 475% and 54% in vessels of SHR and WKY, respectively. The desensitizing effect of DDMS was reversed by 20-HETE. Notably, the minimal concentration of 20-HETE that decreased the EC50 for phenylephrine in DDMS-treated vessels was smaller in SHR (0.1 micromol/L) than WKY (10 micromol/L), and the sensitizing effect of 20-HETE was blunted (P<0.05) by the (R) stereoisomers of 19-HETE and 18-HETE. We conclude that the increased sensitivity to phenylephrine in arteries of SHR is attributable to a vasoregulatory imbalance produced by a deficit in vascular CYP2E1-derived products, most likely 19(R)-HETE and 18(R)-HETE, which condition amplification of the sensitizing action of 20-HETE.

Endothelial dysfunction as a modifier of angiogenic response in Zucker diabetic fat rat: amelioration with Ebselen.

BACKGROUND: Progression of nephropathy in metabolic syndrome is associated with microvasculopathy and vascular dropout. METHODS: Eight- and 22-week-old Zucker diabetic fat (ZDF) and Zucker lean (ZL) rats were studied to characterize the progression of nephropathy, and to test the effect of a peroxynitrite scavenger, Ebselen, on renal microvasculature and angiogenic competence. RESULTS: Capillary density was increased, both in the cortex (P < 0.05) and in the inner medulla (P < 0.001) by the age of 8 weeks, but significantly decreased (P < 0.01 and P < 0.001) by the age of 22 weeks in ZDF compared to ZL rats. Similarly, the angiogenic competence of cortical and medullary renal explants was increased in 8-week-old ZDF (P < 0.01), but decreased at 22 weeks (P < 0.001). Alterations of angiogenic competence in ZDF rats were associated with altered expression of vascular endothelial growth factor (VEGF), reduced expression of Flk-1, and neuropilin. Acetylcholine-induced relaxation of microdissected interlobar arteries from 8-week-old ZDF rats was unimpaired, but significantly attenuated in 22-week-old ZDF rats (P < 0.001). Treatment with Ebselen partially prevented the decrease in capillary density and angiogenic competence of renal explants, and restored acetylcholine-induced vasorelaxation in 22-week-old ZDF rats. CONCLUSION: The progression of nephropathy in ZDF rats is associated with decreased angiogenic competence both ex vivo and in vivo. This is accompanied by a altered expression of VEGF system components and endothelial dysfunction, and scavenging peroxynitrite with Ebselen ameliorates the progression of microvasculopathy and partially restores angiogenesis. These findings reveal the complex mechanism of microvascular dropout in experimental metabolic syndrome.

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways.

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.

Transfection and functional expression of CYP4A1 and CYP4A2 using bicistronic vectors in vascular cells and tissues.

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 omega-hydroxylation to 20-HETE at rates of 0.85 +/- 0.29 and 0.27 +/- 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 EC50 to phenylephrine of 0.24 +/- 0.07 and 0.11 +/- 0.03 microM, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 +/- 0.21 microM; n=6, p <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.

Vascular CO counterbalances the sensitizing influence of 20-HETE on agonist-induced vasoconstriction.

We examined the influence of interactions between CO and 20-hydroxyeicosatetraenoic acid (20-HETE) on vascular reactivity to phenylephrine and vasopressin. Renal interlobar arteries incubated in Krebs buffer released CO at a rate that is decreased (from 125.0+/-15.2 to 46.3+/-8.8 pmol/mg protein per hour, P<0.05) by the heme oxygenase inhibitor chromium mesoporphyrin (CrMP; 30 micromol/L). The level of 20-HETE in vessels was not affected by CrMP (74.3+/-6.1 versus 72.5+/-16.2 pmol/mg protein), but was decreased (P<0.05) by CO (1 micromol/L; 33.2+/-7.9 pmol/mg protein) or the cytochrome P450-4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 micromol/L; 11.4+/-3.3 pmol/mg protein). Phenylephrine elicited development of isometric tension in vascular rings mounted on a wire-myograph (EC(50), 0.29+/-0.02 micromol/L; R(max), 3.78+/-0.19 mN/mm). The sensitivity to phenylephrine was decreased (P<0.05) by CO (1 micromol/L; EC(50), 0.60+/-0.04 micromol/L) or DDMS (EC(50), 0.71+/-0.12 micromol/L) and increased (P<0.05) by 20-HETE (10 micromol/L; EC(50), 0.08+/-0.02 micromol/L) or CrMP (EC(50), 0.11+/-0.02 micromol/L). Notably, neither CO nor CrMP changed the sensitivity to phenylephrine in vessels treated with DDMS. Refractoriness to CO and CrMP in such a setting was eliminated by inclusion of 20-HETE (1 micromol/L) in the bathing buffer. The aforementioned interventions affected the vascular reactivity to vasopressin in a similar manner. These data indicate that the reactivity of renal arteries to phenylephrine and vasopressin is reciprocally influenced by CO and 20-HETE of vascular origin and that CO desensitizes the vascular smooth muscle to constrictor agonists by interfering with the sensitizing influence of 20-HETE.

Angiotensin II induces carbon monoxide production in the perfused kidney: relationship to protein kinase C activation.

Heme oxygenase (HO)-derived carbon monoxide (CO) attenuates vascular reactivity to constrictor stimuli. ANG II produces vasoconstriction and induces HO-1 isoform expression. However, direct evidence that ANG II promotes HO product generation is lacking. Therefore, we examined the effects of ANG II on CO release and HO isoform expression in isolated rat kidneys. Kidneys were perfused with oxygenated Krebs buffer. ANG II (1 micromol/l) increased (P < 0.05) perfusion pressure from 97 +/- 9 to 150 +/- 14 mmHg; it also increased (P < 0.05) the concentration of CO in the venous effluent (from 27.1 +/- 11.9 to 45.6 +/- 11.7, 62.5 +/- 16.7, 94.8 +/- 20.7, and 101.9 +/- 13.1 nmol/l after 30, 60, 90, and 120 min, respectively). The pressor effect of ANG II was blunted (P < 0.05) in kidneys perfused with buffer containing losartan (10 micromol/l) or PKC inhibitors staurosporine (0.1 micromol/l) or calphostin C (1 micromol/l). Kidneys perfused with buffer containing ANG II for 120 min also displayed increased (P < 0.05) HO-1 expression. Stannous mesoporphyrin (30 micromol/l) decreased CO release (P < 0.05) in preparations perfused with and without ANG II; the HO inhibitor also increased (P < 0.05) perfusion pressure, more so in kidneys perfused with that without ANG II. We conclude that ANG II stimulates CO production and release in isolated, perfused rat kidneys. This action of ANG II is linked to the activation of AT(1) receptors and involves PKC activation and upregulation of renal HO-1 but not of HO-2 protein expression. The study suggests upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction.