n-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

Oxygen promotes closure of the ductus arteriosus at birth. We have previously presented a scheme for oxygen action with a cytochrome P450 (CYP450) hemoprotein and endothelin-1 (ET-1) being, respectively, sensor and effector, and a hypothetical monooxygenase product serving as a coupling link. We have also found in the vessel arachidonic acid (AA) 12(S)-lipoxygenase (12-lipoxygenase) undergoing upregulation at birth. Here, we examined the feasibility of a sensor-to-effector messenger originating from AA monooxygenase and 12-lipoxygenase pathways. The epoxygenase inhibitor, N-methylsulfonyl-6-(2-)hexanamide, suppressed the tonic contraction of ductus to oxygen. A similar effect was obtained with 12-lipoxygenase inhibitors baicalein and PD 146176. By contrast, none of the inhibitors modified the endothelin-1 contraction. Furthermore, an AA ω-hydroxylation product, 20-hydroxyeicosatetraenoic acid (20-HETE), reportedly responsible for oxygen contraction in the systemic microvasculature, had no such effect on the ductus. We conclude that AA epoxygenase and 12-lipoxygenase jointly produce a hitherto uncharacterized compound acting as oxygen messenger in the ductus.

Topics: Amides; Animals; Arachidonate 12-Lipoxygenase; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Ductus Arteriosus; Endothelin-1; Enzyme Inhibitors; Fetus; Flavanones; Fluorenes; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxygen; Prostaglandin Endoperoxides, Synthetic; Signal Transduction; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Adenosine-induced renovasodilation in Dahl rats is mediated via activation of adenosine(2A) receptors (A(2A)Rs) and stimulation of epoxyeicosatrienoic acid (EET) synthesis. Unlike Dahl salt-resistant rats, salt-sensitive rats exhibit an inability to upregulate the A(2A)R-EET pathway with salt loading; therefore, we examined the effect of in vivo inhibition of the A(2A)R-EET pathway on blood pressure and the natriuretic response to salt-loading in Dahl salt-resistant rats. N-Methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 20 mg/kg per day), an epoxygenase inhibitor, or ZM241385 (ZM; 5 mg/kg per day), an A(2A)R antagonist, was given daily as an IV bolus dose for 3 days before and after placing rats on high salt intake (2% saline). After 3 days of high salt, systolic blood pressure per 24 hours increased from 108+/-2 mm Hg to 136+/-5 mm Hg and 140+/-4 mm Hg when treated with MS-PPOH or ZM, respectively (P<0.001). Plasma levels of EETs and dihydroxyeicosatrienoic acids during salt loading and MS-PPOH (29.3+/-1.8 ng/mL) or ZM treatment (9.8+/-0.5 ng/mL) did not increase to the same extent as in vehicle-treated rats (59.4+/-1.7 ng/mL; P<0.001), and renal levels of EETs+dihydroxyeicosatrienoic acids were 2-fold lower with MS-PPOH or ZM treatment. On day 3 of the high salt intake, MS-PPOH- and ZM-treated rats exhibited a positive Na(+) balance, and plasma Na(+) levels were significantly increased (163.3+/-1.2 and 158.1+/-4.5 mEq/L, respectively) compared with vehicle-treated rats (142.1+/-1 mEq/L), reflecting a diminished natriuretic capacity. These data support a role for the A(2A)R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading.

Topics: Adrenergic alpha-2 Receptor Antagonists; Amides; Animals; Blood Pressure; Drinking; Eicosanoids; Hydroxyeicosatetraenoic Acids; Hypertension, Renal; Male; Natriuresis; Oxidoreductases; Rats; Rats, Inbred Dahl; Receptors, Adrenergic, alpha-2; Sodium Chloride, Dietary; Triazines; Triazoles

This study examined the metabolism of arachidonic acid (AA) by cytochrome P-450 enzymes in isolated glomeruli and the effects of selective inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatetraenoic acids (EETs) on glomerular permeability to albumin (P(alb)). Glomeruli avidly produced 20-HETE, EETs, dihydroxyeicosatetraenoic acids (diHETEs), and HETEs when incubated with exogenous AA. N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 microM) selectively inhibited the formation of 20-HETE by 95% and increased P(alb) from 0.00 +/- 0.08 to 0.73 +/- 0.10 (n = 43 glomeruli, 4 rats). Addition of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (20-5,14-HEDE; 1 microM) opposed the effects of HET0016 (10 microM) to increase P(alb) (0.21 +/- 0.10, n = 36 glomeruli, 4 rats). Preincubation of glomeruli with exogenous AA to increase basal production of 20-HETE had a similar effect. We also examined the effect of an epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MSPPOH; 5 microM), on P(alb). MSPPOH (5 microM) significantly increased P(alb) but had no effect on the synthesis of EETs in glomeruli incubated with AA. However, MSPPOH (5 microM) selectively reduced epoxygenase activity by 50% in glomeruli incubated without added AA. Pretreatment with 8,9-EET (100 nM) attenuated the effects of MSPPOH (5 microM) on P(alb). These results indicate that glomeruli produce 20-HETE, EETs, diHETEs, and HETEs and that endogenously formed 20-HETE and EETs play an essential role in the maintenance of the glomerular permeability barrier to albumin.

Topics: Amides; Amidines; Animals; Arachidonic Acid; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Kidney Glomerulus; Male; Permeability; Proteins; Rats; Rats, Sprague-Dawley

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.

Topics: 8,11,14-Eicosatrienoic Acid; Amides; Animals; Arachidonic Acid; Cardiotonic Agents; Coronary Circulation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dogs; Enzyme Inhibitors; Hemodynamics; Hydroxyeicosatetraenoic Acids; Mixed Function Oxygenases; Myocardial Ischemia; Myocardial Reperfusion Injury; Spectrometry, Mass, Electrospray Ionization; Sulfones

Bradykinin dilates efferent arterioles via release of efferent arteriole epoxyeicosatrienoic acids when perfused retrograde (no glomerular autacoids). However, when efferent arterioles are perfused orthograde through the glomerulus, bradykinin-induced dilatation is caused by a balance between: (1) the glomerular vasoconstrictor 20-hydroxyeicosatetraenoic acid and vasodilator prostaglandins, and (2) epoxyeicosatrienoic acids from the efferent arteriole and possibly the glomerulus. However, the role of 20-hydroxyeicosatetraenoic acid has only been studied with a cyclooxygenase inhibitor, which may artificially enhance its production by shunting arachidonic acid into the cytochrome P450 pathway. We hypothesized that in the absence of cyclooxygenase inhibition, bradykinin induces release of 20-hydroxyeicosatetraenoic acid from the glomerulus, which blunts the vasodilator effect of bradykinin; and that prostaglandins released from glomeruli in response to bradykinin are generated by cyclooxygenase-1. Rabbit efferent arterioles preconstricted with norepinephrine were perfused orthograde from the end of the afferent arteriole. Bradykinin was added to the perfusate with or without a 20-hydroxyeicosatetraenoic acid antagonist (20-HEDE), epoxyeicosatrienoic acid synthesis inhibitor (MS-PPOH), and/or cyclooxygenase-1 (SC-58560) or cyclooxygenase-2 inhibitor (NS-398). Bradykinin-dependent dilatation was enhanced by 20-HEDE but blunted by MS-PPOH. When the inhibitors were present, bradykinin-induced dilatation was abolished by blockade of cyclooxygenase-1 but not cyclooxygenase-2. We concluded that: (1) in the absence of cyclooxygenase inhibitors, bradykinin causes the release of a glomerular vasoconstrictor (20-hydroxyeicosatetraenoic acid) that antagonizes the vasodilator effect of epoxyeicosatrienoic acids released from the efferent arteriole and perhaps from the glomerulus, and (2) bradykinin-induced vasodilatation is caused by the release of epoxyeicosatrienoic acids from the efferent arteriole and glomerular metabolites of cyclooxygenase-1.

Topics: 8,11,14-Eicosatrienoic Acid; Amides; Animals; Arterioles; Bradykinin; Cyclooxygenase 1; Cytochrome P-450 Enzyme System; Epoxy Compounds; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Glomerulus; Male; Rabbits; Vascular Resistance; Vasodilation; Vasodilator Agents

We have shown that when efferent arterioles are perfused retrograde to avoid the influence of vasoactive autacoids released by the glomerulus, bradykinin causes dilatation via release of cytochrome p450 (cp450) metabolites, probably epoxyeicosatrienoic acids (EETs). Here we tested the hypothesis that the glomerulus releases cyclooxygenase (COX) and cp450 metabolites. These eicosanoids, acting as vasopressor and vasodepressor autacoids, control efferent arteriole resistance downstream from the glomerulus.. Rabbit efferent arterioles were perfused orthograde through the glomerulus from the end of the afferent arteriole to determine whether bradykinin induces the release of glomerular autacoids that influence efferent arteriole resistance. Efferent arterioles were preconstricted with norepinephrine, and increasing doses of bradykinin were added to the perfusate in the presence or absence of COX and cp450 inhibitors.. When efferent arterioles were perfused orthograde through the glomerulus, bradykinin at 10 nmol/L caused significant and reproducible dilatation; diameter increased from 8.0 +/- 0.5 to 12.6 +/- 0.4 microm (P < 0.05). This effect was not modified by a nitric oxide synthase (NOS) inhibitor. In the presence of indomethacin, a COX inhibitor, bradykinin-induced dilatation was almost completely blocked (from 8.0 +/- 0.5 to 9.3 +/- 0.6 microm). This blockade was completely reversed by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), a specific antagonist of the vasoconstrictor cp450 metabolite 20-hydroxyeicosatetraenoic acid (20-HETE); diameter increased from 6.6 +/- 0.7 to 13.2 +/- 0.5 microm. To test the hypothesis that this dilatation was due to EETs, a specific inhibitor of EET synthesis, N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH), was added to the arteriolar perfusate. In the presence of indomethacin and 20-HEDE, bradykinin caused dilatation and this effect was completely blocked by MS-PPOH (1 microm) (from 7.6 +/- 0.6 to 7.3 +/- 0.5 microm).. We concluded that in response to bradykinin, the glomerulus releases COX metabolites (probably prostaglandins) that have a vasodilator effect. When COXs are inhibited, the vasoconstrictor 20-HETE released by the glomerulus is able to oppose the vasodilator effect of bradykinin. This vasodilator effect is mediated by EETs released by the glomerulus and/or the efferent arteriole and does not involve nitric oxide. The balance between these opposing effects of various eicosanoids controls efferent arteriole resistance downstream from the glomerulus.

Topics: Amides; Animals; Arterioles; Bradykinin; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Kidney Glomerulus; Male; Nitric Oxide; Nitric Oxide Synthase; Paracrine Communication; Rabbits; Vasodilator Agents