Horst Honeck

Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation

AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage.

A Peroxisome Proliferator-Activated Receptor-α Activator Induces Renal CYP2C23 Activity and Protects from Angiotensin II-Induced Renal Injury

Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are involved in the regulation of renal vascular tone and salt excretion. The epoxygenation product 11,12-epoxyeicosatrienoic acid (EET) is anti-inflammatory and inhibits nuclear factor-κB activation. We tested the hypothesis that the peroxisome proliferator-activated receptor-α-activator fenofibrate (Feno) induces CYP isoforms, AA hydroxylation, and epoxygenation activity, and protects against inflammatory organ damage. Double-transgenic rats (dTGRs) overexpressing human renin and angiotensinogen genes were treated with Feno. Feno normalized blood pressure, albuminuria, reduced nuclear factor-κB activity, and renal leukocyte infiltration. Renal epoxygenase activity was lower in dTGRs compared to nontransgenic rats. Feno strongly induced renal CYP2C23 protein and AA-epoxygenase activity under pathological and nonpathological conditions. In both cases, CYP2C23 was themajor isoform responsible for 11,12-EET formation. Moreover, we describe a novel CYP2C23-dependent pathway leading to hydroxy-EETs (HEETs), which may serve as endogenous peroxisome proliferator-activated receptor-α activators. The capacity to produce HEETs via CYP2C23-dependent epoxygenation of 20-HETE and CYP4A-dependent hydroxylation of EETs was reduced in dTGR kidneys and induced by Feno. These results demonstrate that Feno protects against angiotensin II-induced renal damage and acts as inducer of CYP2C23-mediated epoxygenase activities. We propose that CYP-dependent EET/HEET production may serve as an anti-inflammatory control mechanism.

P450-Dependent Arachidonic Acid Metabolism and Angiotensin II--Induced Renal Damage

Transgenic rats overexpressing both human renin and angiotensinogen genes (dTGR) develop hypertension, inflammation, and renal failure. We tested the hypothesis that these pathological features are associated with changes in renal P450-dependent arachidonic acid (AA) metabolism. Samples were prepared from 5- and 7-week-old dTGR and from normotensive Sprague-Dawley (SD) rats, ie, before and after the dTGR developed severe hypertension and albuminuria. At both stages, dTGR showed significantly lower renal microsomal AA epoxygenase and hydroxylase activities that reached 63% and 76% of the control values at week 7. Furthermore, the protein levels of several potential AA epoxygenases (CYP2C11, CYP2C23, and CYP2J) were significantly reduced. Immunoinhibition studies identified CYP2C23 as the major AA epoxygenase, both in dTGR and SD rats. Immunohistochemistry showed that CYP2C23 was localized in cortical and outer medullary tubules that progressively lost this enzyme from week 5 to week 7 in dTGR. CYP2C11 expression occurred only in the outer medullary tubules and was markedly reduced in dTGR compared with age-matched SD rats. These findings indicate site-specific decreases in the availability of AA epoxygenase products in the kidney of dTGR. In contrast to renal microsomes, liver microsomes of dTGR and SD rats showed no change in the expression and activity of AA epoxygenases and hydroxylases. We conclude that hypertension and end-organ damage in dTGR is associated with kidney-specific downregulation of P450-dependent AA metabolism. Because the products of AA epoxygenation have anti-inflammatory properties, this alteration may contribute to uncontrolled renal inflammation, which is a major cause of renal damage in dTGR.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18‐Epoxyeicosatetraenoic acid (17,18‐EETeTr) stimulates vascular large‐conductance K+ (BK) channels. BK channels are composed of the pore‐forming BK α and auxiliary BK β1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine‐sensitive calcium‐release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole‐cell and perforated‐patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague–Dawley rats, BK β1 gene‐deficient (−/−), BK α (−/−), RyR3 (−/−) and wild‐type mice. The 17,18‐EETeTr (100 nm) increased tetraethylammonium (1 mm)‐sensitive outward K+ currents in VSMC from wild‐type rats and wild‐type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15‐epoxyeicosa‐5(Z)‐enoic acid (10 μm). BK channel currents were increased 3.5‐fold in VSMC from BK β1 (−/−) mice, whereas a 2.9‐fold stimulation was observed in VSMC from RyR3 (−/−) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild‐type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca2+, 100 nm), nor by external calcium influx. The 17,18‐EETeTr did not induce outward currents in VSMC BK α (−/−) cells. We next tested the vasodilator effects of 17,18‐EETeTr on isolated arteries of BK α‐deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK α (−/−) mice compared with that observed in wild‐type and BK β1 (−/−) arteries. We conclude that 17,18‐EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18‐EETeTr is active in small arteries lacking BK β1, the data further suggest that BK α represents the molecular target for the principal action of 17,18‐EETeTr. Finally, the action of 17,18‐EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Candida maltosa NADPH‐cytochrome P450 reductase: Cloning of a full‐length cDNA, Heterologous expression in Saccharomyces cerevisiae and function of the N‐terminal region for membrane anchoring and proliferation of the endoplasmic reticulum

A full‐length cDNA for NADPH‐cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes.

Cytochrome P450–Dependent Renal Arachidonic Acid Metabolism in Desoxycorticosterone Acetate–Salt Hypertensive Mice

Cytochrome P450 (P450)-dependent arachidonic acid metabolites may act as mediators in the regulation of vascular tone and renal function. We studied arachidonic acid hydroxylase activities in renal microsomes from normotensive NMRI mice, desoxycorticosterone acetate (DOCA)-salt hypertensive mice, and DOCA-salt mice treated with either lovastatin or bezafibrate, both of which improve hemodynamics in this model. Control renal microsomes had arachidonic acid hydroxylase activities of 175±12 pmol · min−1 · mg−1. The metabolites formed were 20- and 19-hydroxyarachidonic acid, representing ≈80% and ≈20% of the total hydroxylation. Treatment with DOCA-salt resulted in significantly decreased hydroxylase activities (to 84±4 pmol · min−1 · mg−1) of the total microsomal P450 content and a decrease in immunodetectable Cyp4a proteins. Lovastatin had no effect on these variables, whereas bezafibrate increased arachidonic acid hydroxylase activities to 163±12 pmol · min−1 · mg−1. In situ hybridization with probes for Cyp4a-10, 12, and 14 revealed that Cyp4a-14 was the P450 isoform most strongly induced by bezafibrate. The expression was concentrated in the cortical medullary junction and was localized predominantly in the proximal tubules. In conclusion, these results suggest that the capacity to produce 20-hydroxyarachidonic acid is impaired in the kidneys of DOCA-salt hypertensive mice. Furthermore, bezafibrate may ameliorate hemodynamics in this model by restoring P450-dependent arachidonic acid hydroxylase activities. Lovastatin, on the other hand, exerts its effects via P450-independent mechanisms.

Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells

P29 Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.