Houli Jiang

Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors

Rat renal interlobar arteries express heme oxygenase 2 (HO-2) and manufacture carbon monoxide (CO), which is released into the headspace gas. CO release falls to 30% and 54% of control, respectively, after inhibition of HO activity with chromium mesoporphyrin (CrMP) or of HO-2 expression with antisense oligodeoxynucleotides (HO-2 AS-ODN). Patch-clamp studies revealed that CrMP decreases the open probability of a tetraethylammonium-sensitive (TEA-sensitive) 105 pS K channel in interlobar artery smooth muscle cells, and that this effect of CrMP is reversed by CO. Assessment of phenylephrine-induced tension development revealed reduction of the EC(50) in vessels treated with HO-2 AS-ODN, CrMP, or TEA. Exogenous CO greatly minimized the sensitizing effect on agonist-induced contractions of agents that decrease vascular CO production, but not the sensitizing effect of K channel blockade with TEA. Collectively, these data suggest that vascular CO serves as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a TEA-sensitive K channel.

Stimulation of rat erythrocyte P2X7 receptor induces the release of epoxyeicosatrienoic acids

Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators—epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte‐derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation.

Altered Release of Cytochrome P450 Metabolites of Arachidonic Acid in Renovascular Disease

The aim of the present cross-sectional study was to investigate whether activation of the renin-angiotensin system in renovascular disease affects the cytochrome P450 &ohgr;/&ohgr;-1 hydroxylase (20-hydroxyeicosatetraenoic acid [20-HETE]) and epoxygenase (epoxyeicosatrienoic acids [EETs]) pathways of arachidonic acid metabolism in vivo, each of which interacts with angiotensin II. Plasma concentration and urinary excretion of 20-HETE and EETs and their metabolites, dihydroxyeicosatrienoic acids, were measured in urine and plasma by mass spectrometry in 10 subjects with renovascular disease, 10 with essential hypertension, and 10 healthy normotensive subjects (control subjects), pair-matched for gender and age. Vascular and renal function were evaluated in all of the subjects. Plasma 20-HETE was highest in subjects with renovascular disease (median: 1.20 ng/mL; range: 0.42 to 1.92 ng/mL) compared with subjects with essential hypertension (median: 0.90 ng/mL; range: 0.40 to 2.17 ng/mL) and control subjects (median: 0.45 ng/mL; range: 0.14 to 1.70 ng/mL; P<0.05). Plasma 20-HETE significantly correlated with plasma renin activity in renovascular disease (rs=0.67; n=10; P<0.05). The urinary excretion of 20-HETE was significantly lower in subjects with renovascular disease (median: 12.9 &mgr;g/g of creatinine; range: 4.4 to 24.9 &mgr;g/g of creatinine) than in control subjects (median: 31.0 &mgr;g/g of creatinine; range: 11.9 to 102.8 &mgr;g/g of creatinine; P<0.01) and essential hypertensive subjects (median: 35.9 &mgr;g/g of creatinine; range: 14.0 to 72.5 &mgr;g/g of creatinine; P<0.05). Total plasma EETs were lowest, as was the ratio of plasma EETs to plasma dihydroxyeicosatrienoic acids, an index of epoxide hydrolase activity, in renovascular disease (ratio: 2.4; range: 1.2 to 6.1) compared with essential hypertension (ratio: 3.4; range: 1.5 to 5.6) and control subjects (ratio: 6.8; range: 1.4 to 18.8; P<0.01). In conclusion, circulating levels of 20-HETE are increased and those of EETs are decreased in renovascular disease, whereas the urinary excretion of 20-HETE is reduced. Altered cytochrome P450 arachidonic acid metabolism may contribute to the vascular and tubular abnormalities of renovascular disease.

Epoxyeicosatrienoic acids mediate adenosine‐induced vasodilation in rat preglomerular microvessels (PGMV) via A2A receptors

Activation of rat adenosine 2A receptors (A2A R) dilates preglomerular microvessels (PGMV), an effect mediated by epoxyeicosatrienoic acids (EETs). Incubation of PGMV with a selective A2A R agonist, 2‐p‐(2‐carboxyethyl) phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 100 μM), increased isolated PGMV EET levels to 7.57±1.53 ng mg−1 protein from 1.06±0.22 ng mg−1 protein in controls (P<0.05), without affecting hydroxyeicosatetraenoic acid (HETE) levels (10.8±0.69 vs 11.02±0.74 ng mg−1 protein). CGS 21680‐stimulated EETs was abolished by preincubation with an A2A R antagonist, 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM241385) (100 μM). A selective epoxygenase inhibitor, methylsulfonyl‐propargyloxyphenylhexanamide (MS‐PPOH; 12 μM) prevented CGS 21680‐induced increase in EETs, indicating inhibition of de novo synthesis of EETs. In pressurized (80 mmHg) renal arcuate arteries (110–130 μm) preconstricted with phenylephrine (20 nM), superfusion with CGS 21680 (0.01–10 μM) increased the internal diameter (i.d.) concentration‐dependently; vasodilation was independent of nitric oxide and cyclooxygenase activity. CGS 21680 (10 μM) increased i.d. by 32±6 μm; vasodilation was prevented by inhibition of EET synthesis with MS‐PPOH. Addition of 3 nM 5,6‐EET, 8,9‐EET and 11,12‐EET increased i.d. by 53±9, 17±4 and 53±5 μm, respectively, whereas 14,15‐EET was inactive. The responses to 5,6‐EET were, however, significantly inhibited by indomethacin. We conclude that 11,12‐EET is the likely mediator of A2A R‐induced dilation of rat PGMV. Activation of A2A R coupled to de novo EET stimulation may represent an important mechanism in regulating preglomerular microvascular tone.

Estrogen elicits cytochrome P450--mediated flow-induced dilation of arterioles in NO deficiency: role of PI3K-Akt phosphorylation in genomic regulation.

Abstract— This study investigated the mechanisms responsible for the estrogen-dependent, cytochrome P450 (CYP)-mediated dilator responses to shear stress in arterioles of NO-deficient female rats and mice. Flow-induced dilation (FID) was assessed in isolated arterioles from NG-nitro-l-arginine methyl ester (L-NAME)-treated male and ovariectomized female rats before and after overnight incubation with 17&bgr;-estradiol (17&bgr;-E2, 10−9 mol/L). In control conditions, prostaglandins (PGs) mediated FID, because indomethacin (INDO) abolished the responses. After incubation of the vessels with 17&bgr;-E2, the basal tone of arterioles was significantly reduced and FID was augmented. INDO did not affect the dilation of the vessels incubated with 17&bgr;-E2. Dilations of these vessels, however, were eliminated by PPOH and miconazole, inhibitors of CYP/epoxygenase. Simultaneous incubation of the vessels with 17&bgr;-E2 plus ICI, 182,780, an estrogen receptor antagonist, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation or the transcriptional inhibitor DRB, prevented the reduced arteriolar tone and the enhanced CYP-mediated FID caused by incubation of vessels with17&bgr;-E2. Western blot analysis indicated a significantly increased phospho-Akt level in arterioles incubated with 17&bgr;-E2 compared with those without 17&bgr;-E2. The enhanced phospho-Akt in response to 17&bgr;-E2 was localized, by immunohistochemistry, to arteriolar endothelial cells. Moreover, GC-MS analysis indicated a significantly increased production of epoxyeicosatrienoic acids, vasodilator metabolites of CYP/epoxygenase, in arterioles incubated with 17&bgr;-E2, a response that was prevented by ICI 182780 and wortmannin, respectively. Thus, estrogen, via a receptor-dependent, PI3K/Akt-mediated pathway, transcriptionally upregulates CYP activity, leading to an enhanced arteriolar response to shear stress.

Functional expression of human heme oxygenase‐1 (HO‐1) driven by HO‐1 promoter in vitro and in vivo

We developed a retrovirus‐mediated human heme oxygenase‐1 (HO‐1) gene expression system and assessed the impact of heme on the inducibility of the HO‐1 gene in rat lung microvessel (RLMV) endothelial cells and in newborn Sprague‐Dawley (SD) rats. Overexpression of the HO‐1 gene driven by HO‐1 promoter (HOP) resulted in an increase in HO‐1 protein and HO activity by 4.8‐ and 1.3‐fold, respectively, compared to the viral LTR promoter. The increased HO‐1 gene expression was associated with the enhancement of CO production. In cells transduced by HOP‐driven HO‐1 gene, there was a decrease in basal cyclooxygenase (COX) activity as measured by PGE2. The degree of HO‐1 expression and, consequently, the levels of cellular heme were directly related to COX activity. Supplementation with heme markedly increased PGE2 and cGMP synthesis. In all (6/6) of newborn SD rats injected with retrovirus LSN‐HOP‐HO‐1, both HO‐1 and neor transcripts were expressed in tissues. We hypothesize that degree of HO‐1 gene expression resulted in a differential rate of cellular heme‐dependent enzyme gene expression, which may play a vital role in maintaining cellular homeostasis. J. Cell. Biochem. 85: 410–421, 2002.

Hydrolysis of cis- and trans-Epoxyeicosatrienoic Acids by Rat Red Blood Cells

Erythrocytes serve as reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs). Incubation of rat red blood cells (RBCs) with cis- and trans-EETs produces threo- and erythro-dihydroxyeicosatrienoic acids, respectively. The Vmax of EET hydrolysis by rat intact RBCs (2.35 ± 0.24 pmol/min/108 RBCs for 14,15-trans-EET) decreased by approximately 2 to 3-fold sequentially from 14,15-, 11,12- to 8,9-EETs for both cis- and trans-isomers. The Vmax of trans-EET hydrolysis by RBCs is approximately 2 to 3 times that of the corresponding cis-EETs. Incubation of EETs with recombinant murine soluble epoxide hydrolase (sEH) yielded the same geometric and regio preferences of EET hydrolysis as with rat intact RBCs. The principal epoxide hydrolase activity for EET hydrolysis (approximately 90%) is present in the erythrocyte cytosol. Western blots of sEH suggested a concentration of sEH protein to be approximately 2 μg/mg protein or 0.4 μg/109 RBCs. The apparent Km values of EETs were between 1 and 2 μM, close to the Km for purified sEH as reported. Erythrocyte hydration of cis- and trans-EETs was blocked by sEH inhibitors, 1,3-dicyclohexylurea and 4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid. Erythrocyte sEH activity was inhibited more than 80% by 0.2% bovine serum albumin in the buffer. Preferred hydrolysis of 14,15-EETs and trans-epoxides characterizes sEH activity in RBCs that regulates the hydrolysis and release of cis- and trans-EETs in the circulation. Inhibition of sEH has produced antihypertensive and antiinflammatory effects. Because plasma trans-EETs would increase more than cis-EETs with sEH inhibition, the potential roles of trans-EETs and erythrocyte sEH in terms of circulatory regulation deserve attention.

Increases in plasma trans-EETs and blood pressure reduction in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) are vasodilator, natriuretic, and antiinflammatory lipid mediators. Both cis- and trans-EETs are stored in phospholipids and in red blood cells (RBCs) in the circulation; the maximal velocity (V(max)) of trans-EET hydrolysis by soluble epoxide hydrolase (sEH) is threefold that of cis-EETs. Because RBCs of the spontaneously hypertensive rat (SHR) exhibit increased sEH activity, a deficiency of trans-EETs in the SHR was hypothesized to increase blood pressure (BP). This prediction was fulfilled, since sEH inhibition with cis-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (AUCB; 2 mg·kg(-1)·day(-1) for 7 days) in the SHR reduced mean BP from 176 ± 8 to 153 ± 5 mmHg (P < 0.05), whereas BP in the control Wistar-Kyoto rat (WKY) was unaffected. Plasma levels of EETs in the SHR were lower than in the age-matched control WKY (16.4 ± 1.6 vs. 26.1 ± 1.8 ng/ml; P < 0.05). The decrease in BP in the SHR treated with AUCB was associated with an increase in plasma EETs, which was mostly accounted for by increasing trans-EET from 4.1 ± 0.2 to 7.9 ± 1.5 ng/ml (P < 0.05). Consistent with the effect of increased plasma trans-EETs and reduced BP in the SHR, the 14,15-trans-EET was more potent (ED(50) 10(-10) M; maximum dilation 59 ± 15 μm) than the cis-isomer (ED(50) 10(-9) M; maximum dilation 30 ± 11 μm) in relaxing rat preconstricted arcuate arteries. The 11,12-EET cis- and trans-isomers were equipotent dilators as were the 8,9-EET isomers. In summary, inhibition of sEH resulted in a twofold increase in plasma trans-EETs and reduced mean BP in the SHR. The greater vasodilator potency of trans- vs. cis-EETs may contribute to the antihypertensive effects of sEH inhibitors.

Maternal and fetal epoxyeicosatrienoic acids in normotensive and preeclamptic pregnancies.

BACKGROUND Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 metabolites of arachidonic acid posited to act in the circulatory adaptation to pregnancy and the development of preeclampsia. Red blood cells (RBCs) may function as major contributors of cis- and trans-EETs. METHODS We performed paired analyses of EETs, dihydroxyeicosatrienoic acids (DHETs), and 20-HETE in RBCs, plasma, and urine from preeclamptic and normotensive pregnant and nonpregnant women. Blood from fetal and maternal circulation was collected. EETs, DHETs, and 20-HETE were analyzed by gas chromatography and liquid chromatography mass spectrometry. Vascular function and inflammation indices were analyzed. RESULTS Plasma EET is higher in normotensive (median, range; 9.9, 6.3-25.2ng/mL n = 29) and preeclamptic (10.9, 6.0-48.0ng/mL, n = 19) women than in nonpregnant controls (7.3, 3.7-10.2ng/mL, n = 19) and correlate with RBC EETs, C-reactive protein, and arterial stiffness. Renal production of EETs, measured as urinary DHETs, was reduced in preeclamptic (4.5, 1.6-24.5ng/mg creatinine) compared to normotensive (11.4, 1.6-44.5ng/mg creatinine) pregnancies. EETs are 3- to 5-fold greater in fetoplacental than in maternal circulation (RBCs 36.6, 13.1-69.4 vs. 12.5, 6.4-12.0ng/10(9) cells; plasma 31.6, 8.5-192.6 vs. 12.0, 6.8-48.0ng/mL). Both cis- and trans-EETs are present in fetal RBCs. CONCLUSIONS RBCs contribute to elevated levels of EETs in the fetoplacental circulation. EETs may modulate systemic and fetoplacental hemodynamics in normal and preeclamptic pregnancies. Decreased renal EET generation may be associated with the development of maternal renal dysfunction and hypertension in preeclampsia.

High Potassium Intake Enhances the Inhibitory Effect of 11,12-EET on ENaC

High dietary potassium stimulates the renal expression of cytochrome P450 (CYP) epoxygenase 2C23, which metabolizes arachidonic acid (AA). Because the AA metabolite 11,12-epoxyeicosatrienoic acid (11,12-EET) can inhibit the epithelial sodium channel (ENaC) in the cortical collecting duct, we tested whether dietary potassium modulates ENaC function. High dietary potassium increased 11,12-EET in the isolated cortical collecting duct, an effect mimicked by inhibiting the angiotensin II type I receptor with valsartan. In patch-clamp experiments, a high potassium intake or treatment with valsartan enhanced AA-induced inhibition of ENaC, an effect mediated by a CYP-epoxygenase-dependent pathway. Moreover, high dietary potassium and valsartan each augmented the inhibitory effect of 11,12-EET on ENaC. Liquid chromatography/mass spectrometry showed that the rate of EET conversion to dihydroxyeicosatrienoic acids (DHET) was lower in renal tissue obtained from rats on a high-potassium diet than from those on a control diet, but this was not a result of altered expression of soluble epoxide hydrolase (sEH). Instead, suppression of sEH activity seemed to be responsible for the 11,12-EET-mediated enhanced inhibition of ENaC in animals on a high-potassium diet. Patch-clamp experiments demonstrated that 11,12-DHET was a weak inhibitor of ENaC compared with 11,12-EET, whereas 8,9- and 14,15-DHET were not. Furthermore, inhibition of sEH enhanced the 11,12-EET-induced inhibition of ENaC similar to high dietary potassium. In conclusion, high dietary potassium enhances the inhibitory effect of AA and 11,12-EET on ENaC by increasing CYP epoxygenase activity and decreasing sEH activity, respectively.