Die Rolle von Cytochrom P450-Metaboliten und RGS2 bei der Regulation des Gefäßtonus in Mäusen

Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) –/– and +/+ mice. Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release. 17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large- conductance K+ (BK) channels. 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 a (–/–), RyR3 (–/–) and wild-type mice. 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 17,18-EETeTr did not induce outward currents in VSMC BK a (–/–) cells. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK a (–/–) mice compared with that observed in wild-type and BK β1 (–/–) arteries. 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. BK a represents the molecular target for the principal action of 17,18-EETeTr. The action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events. Angiotensin II (Ang II) activates signalling pathways predominantly through the G-protein-coupled Ang II type 1 receptor (AT1R). The regulator of G protein signalling 2 (RGS2) is a negative G protein regulator. We showed in this study that Ang II infusion increased BP more in RGS2 –/– than in RGS2 +/+ mice and that myogenic tone and vasoconstrictor responses to Ang II, ET-1 and PE were increased in isolated interlobular arterioles of RGS2 –/– mice. In both RGS2 –/– and RGS2 +/+ mice treated with Ang II, urinary adrenaline and noradrenaline excretion were similar and profoundly decreased. These findings suggest that Ang II-induced hypertension in RGS2 –/– mice is mediated through vascular mechanisms rather than sympathetic activation.Das Endothel setzt Stickstoffmonoxid (NO), Prostazyklin und Endothelium- derived hyperpolarizing factor (EDHF) frei. Epoxyeicosatriensäuren (EETs), K+ und Hydrogenperoxid (H2O2) sind Kandidaten für EDHF. EETs könnten die Gefäßfunktion aber auch über andere Mechanismen beeinflussen. In dieser Arbeit wird eine neuartige Interaktion zwischen EETs und endothelialer NO-Freisetzung durch Verwendung von Soluble epoxide hydrolase (sEH) –/– und +/+ Mäusen aufgedeckt. Die Ergebnisse zeigen, dass die EDHF-Antwort in Mesenterialarterien durch H2O2, jedoch nicht durch P450-Eicosanoide vermittelt wird. P450-Eicosanoide sind hauptsächlich vasodilatatorisch durch ihre Fähigkeit, endotheliale NO-Synthase (eNOS) zu aktivieren und NO freizusetzen. Es wird auch gezeigt, dass 17,18-Epoxyeicosatetraensäure (17,18-EETeTr) als endogener BK-Kanalöffner und Vasodilatator fungiert; dabei aktiviert 17,18-EETeTr die BKalpha-Untereinheit als molekulare Zielstruktur. Angiotensin II (Ang II) aktiviert zelluläre Signalwege hauptsächlich über G-Protein- gekoppelte Ang II-Typ 1-Rezeptoren (AT1R). Der Regulator of G protein signalling 2 (RGS2) ist ein negativer G-Protein-Regulator. Es wird gezeigt, dass Angiotensin II-Infusion den Blutdruck stärker in RGS2 –/– als in RGS2 +/+ Mäusen erhöht. Der myogene Gefäßtonus sowie die vasokonstriktorischen Antworten auf Angiotensin II, Endothelin-1 und Phenylephrin sind in isolierten Interlobulararteriolen von RGS2 –/– Mäusen erhöht. In mit Angiotensin II behandelten RGS2 –/– und RGS2 +/+ Mäusen sind die Urinadrenalin- und Urinnoradrenalinausscheidung gleich stark herabgesetzt. Diese Befunde zeigen, dass Ang II-induzierte Hypertonie in RGS2 –/– Mäusen eher über vaskuläre Mechanismen als über Sympathikusaktivierung vermittelt wird

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat

20-Hydroxyeicosatetraenoic acid (20-HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20-HETE-synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, −4A2 and −4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, −4A2 and −4A3 reduced 20-HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nω-L-nitromethyl arginine ester (L-NAME, 25 mg kg−1), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Δ=+18 to 26 mmHg), reduced renal blood flow (RBF, Δ= −1.8 to 2.9 ml min−1), increased renal vascular resistance (RVR, Δ=+47 to 54 mmHg ml−1 min−1), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and −4A2 but not −4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not −4A1 or −4A3 blunted the increase in MABP by L-NAME (38 ± 9 %, P < 0.05). ASODN to CYP4A1, −4A2 and −4A3 attenuated the reduction in RBF and the consequent increase in RVR by L-NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and −4A2 but not −4A3 attenuated L-NAME-induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L-NAME-induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, −4A2, and −4A3 isoforms in the renal vasculature and the nephron