Shouzou Wei

Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel

Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10-/- mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10-/- mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease.

Epoxygenase Metabolites Contribute to Nitric Oxide-Independent Afferent Arteriolar Vasodilation in Response to Bradykinin

In the kidney, epoxyeicosatrienoic acids (EETs) have been suggested to be endothelium-derived hyperpolarizing factors (EDHFs). The aim of the present study was to determine the contribution of EETs to the preglomerular vasodilation elicited by bradykinin. Sprague-Dawley rats were studied utilizing an in vitro perfused juxtamedullary nephron preparation. The afferent arteriolar diameter was determined and the diameter averaged 19 ± 1 µm (n = 26) at a renal perfusion pressure of 100 mm Hg. Addition of 1, 10 and 100 nM bradykinin to the perfusate dose-dependently increased afferent arteriolar diameter by 5 ± 1, 12 ± 2 and 17 ± 2%, respectively. The nitric oxide inhibitor Nω-nitro-L-arginine reduced bradykinin-induced afferent arteriolar vasodilation by 50%, and the diameter increased by 9 ± 2% in response to 100 nM bradykinin. Epoxygenase inhibitors N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide or miconazole greatly attenuated the nitric oxide-independent component of the vasodilation elicited by bradykinin. Cyclooxygenase (COX) inhibition attenuated the nitric oxide-independent vasodilation elicited by 1 nM bradykinin but did not significantly affect the vascular response to 100 nM bradykinin. Combined inhibition of nitric oxide, COX and epoxygenase pathways completely abolished bradykinin-mediated afferent arteriolar vasodilation. In additional studies, renal microvessels were isolated and incubated with bradykinin and samples were analyzed by NICI/GC/MS. Under control conditions, renal microvascular EET levels averaged 49 ± 9 pg/mg/20 min (n = 7). In the presence of bradykinin, EET levels were significantly higher and averaged 81 ± 11 pg/mg/20 min (n = 7). These data support the concept that EETs are EDHFs and contribute to the nitric oxide-independent afferent arteriolar vasodilation elicited by bradykinin.

Arachidonic acid epoxygenase: Structural characterization and quantification of epoxyeicosatrienoates in plasma

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.

Enhanced renal microvascular reactivity to angiotensin II in hypertension is ameliorated by the sulfonimide analog of 11,12-epoxyeicosatrienoic acid

Objectives Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats. Materials and methods Male Sprague–Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography–mass spectroscopy. Results Systolic blood pressure averaged 118 ± 2 mmHg prior to pump implantation and increased to 185 ± 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 ± 1 μm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P < 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15–17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 ± 9 and 87 ± 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats. Conclusions Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.

The arachidonic acid epoxygenase is a component of the signaling mechanisms responsible for VEGF-stimulated angiogenesis.

Cultured lung endothelial cells (LEC) respond to VEGF or arachidonic acid with increases in cell proliferation, the formation of tube-like structures, and the activation of Akt and ERK1/2 mediated growth pathways. LECs express a VEGF inducible Cyp2c44 epoxygenase and its 11,12- and 14,15-EET metabolites increase cell proliferation, tubulogenic activity, and the phosphorylation states of the ERK1/2 and Akt kinases. Ketoconazole, an epoxygenase inhibitor, blocks the cellular responses to VEGF. LECs expressing a Cyp2c44 epoxygenase small interference RNA show reductions in Cyp2c44 mRNA levels, and in their VEGF-stimulated proliferative and tubulogenic capacities; effects that are associated with decreases in VEGF-induced phosphorylation of the ERK1/2 and Akt kinases. We conclude that the Cyp2c44 arachidonic acid epoxygenase is a component of the signaling pathways associated with VEGF-stimulated angiogenesis, and suggest a role for EETs in the growth factor-induced changes in the activation states of the ERK1/2 and Akt kinase pathways.

Development of Epoxyeicosatrienoic Acid Analogs with in Vivo Anti-Hypertensive Actions

Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.

Resolution of dihydroxyeicosanoates and of dihydroxyeicosatrienoates by chiral phase chromatography

A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-vic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vic-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality.

20-Hydroxyeicosatetraenoic acid (HETE)-dependent hypertension in human cytochrome P450 (CYP) 4A11 transgenic mice: Normalization of blood pressure by sodium restriction, hydrochlorothiazide, or blockade of the type 1 angiotensin II receptor

Male and female homozygous 129/Sv mice carrying four copies of the human cytochrome P450 4A11 gene (CYP4A11) under control of its native promoter (B-129/Sv-4A11+/+) develop hypertension (142 ± 8 versus 113 ± 7 mm Hg systolic blood pressure (BP)), and exhibit increased 20-hydroxyeicosatetraenoic acid (20-HETE) in kidney and urine. The hypertension is reversible by a low-sodium diet and by the CYP4A inhibitor HET0016. B-129/Sv-4A11+/+ mice display an 18% increase of plasma potassium (p < 0.02), but plasma aldosterone, angiotensin II (ANGII), and renin activities are unchanged. This phenotype resembles human genetic disorders with elevated activity of the sodium chloride co-transporter (NCC) and, accordingly, NCC abundance is increased by 50% in transgenic mice, and NCC levels are normalized by HET0016. ANGII is known to increase NCC abundance, and renal mRNA levels of its precursor angiotensinogen are increased 2-fold in B-129/Sv-4A11+/+, and blockade of the ANGII receptor type 1 with losartan normalizes BP. A pro-hypertensive role for 20-HETE was implicated by normalization of BP and reversal of renal angiotensin mRNA increases by administration of the 20-HETE antagonists 2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)acetate or (S)-2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)succinate. SGK1 expression is also increased in B-129/Sv-4A11+/+ mice and paralleled increases seen for NCC. Losartan, HET0016, and 20-HETE antagonists each normalized SGK1 mRNA expression. These results point to a potential 20-HETE dependence of intrarenal angiotensinogen production and ANGII receptor type 1 activation that are associated with increases in NCC and SGK1 and identify elevated P450 4A11 activity and 20-HETE as potential risk factors for salt-sensitive human hypertension by perturbation of the renal renin-angiotensin axis.

Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction

Background: Human CYP2C8 is involved in the metabolism of >20% of drugs on the market. Results: Both full-length (WT) CYP2C8 and splice variant 3 are bimodally targeted to mitochondria. Conclusion: Mitochondrial CYP2C8 metabolizes paclitaxel and arachidonic acid (ω-hydroxylation). Significance: Mitochondrial CYP2C8 is likely to play role in ischemic injury and oxidative stress. In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.

Selective changes in cholesterol metabolite levels in plasma of breast cancer patients after tumor removal

Introduction Oxygenated metabolites of cholesterol (“oxysterols”) can influence carcinogenesis and contribute to resistance to endocrine therapy, an effect mostly described in vitro. Objectives We sought to establish a method for screening plasma levels of oxysterols in breast cancer patients, estimate their individual variability and detection limits, and provide basic information about their roles in tumor biology. Method Liquid-chromatography coupled with tandem mass spectrometry was used for determination of levels of 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, and 7-ketocholesterol in plasma sample pairs from patients before and 12–24 months after surgical removal of tumors (n=24). Deuterated standards of all oxysterols were used for method validation. Result All oxysterols were successfully detected in patient plasma samples. A significant increase in the level of 7-ketocholesterol was observed in the samples following tumor removal and the start of therapy compared to the sampling before (p=0.002). This increase was unrelated to personal characteristics of patients, expression of estrogen receptor, or to adjuvant therapy type. Conclusion This study shows, for the first time, that circulating levels of oxysterols, especially 7-ketocholesterol, may reflect the presence of tumor cells in patients.