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Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid that elicit vasodilation via activation of K+channels. They have been implicated as endothelium-derived hyperpolarizing factors (EDHFs), mediating the effect of some endothelium-dependent vasodilator agents such as bradykinin in some vascular tissues. We reasoned that an agent that increases the availability of free arachidonic acid should also elicit cytochrome P450-dependent vasodilation that is associated with increased release of EETs and attenuated by agents that inhibit the synthesis or action of EETs. Thus, we used thimerosal as an inhibitor of reacylation of arachidonic acid and determined the contribution of prostaglandins, nitric oxide, and EETs to the vasodilator effect in the isolated, perfused, preconstricted kidney of the rat. Thimerosal elicited vasodilator responses that were unaffected by inhibition of cyclooxygenase with indomethacin but were reduced by the further inhibition of nitric oxide synthesis. The vasodilator activity that remained after inhibition of cyclooxygenase and nitric oxide synthase was reduced by inhibition of K+channels with tetraethylammonium and was associated with increased release of EETs measured by gas chromatography-mass spectroscopy following hydrolysis to the corresponding diols. Inhibition of cytochrome P450 with miconazole or epoxygenase with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexamide reduced the nitric oxide- and prostaglandin-independent vasodilator effect of thimerosal and attenuated the increase in the release of EETs. We conclude that thimerosal causes vasodilation of the isolated perfused kidney via nitric oxide-dependent and -independent mechanisms. The nitric oxide-independent component of the response involves activation of K+channels and is likely mediated by EETs, possibly acting as EDHFs.

A cytochrome P450-derived metabolite of arachidonic acid, namely an epoxyeicosatrienoic acid (EET), has many of the properties of a hyperpolarizing factor that mediates endothelium-dependent, nitric oxide-independent vasodilation. As there are four EET regioisomers, we used pharmacological criteria, based on previous observations with bradykinin (BK), to evaluate which, if any, of the EETs could be considered a potential mediator of vasodilator responses to BK in the rat isolated heart treated with indomethacin and nitroarginine to eliminate prostaglandin and nitric oxide components of the response. Nifedipine, used as a probe for dilator mechanisms dependent on closure of voltage-dependent Ca++channels, almost abolished the vasodilator effect of cromakalim and attenuated those of BK and 5,6 EET. The vasodilator effects of the other EETs were not reduced and were excluded from consideration as mediators of BK-induced vasodilation. The vasodilator effect of 5,6 EET, as with that of BK, was markedly reduced by charybdotoxin but not iberiotoxin, suggesting the contribution of a similar type K+channel to the vascular response to both agents. As expected for a putative endothelium- and cytochrome P450-derived mediator, the coronary vasodilator effect of 5,6 EET was not affected by either removal of the endothelium or inhibition of cytochrome P450 with clotrimazole, interventions that virtually abolished the vasodilator activity of BK. Thus, of the four EET regioisomers, 5,6 EET is the most likely mediator of the vasodilator effect of BK in the isolated heart under these experimental conditions.

The role of K+channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca++-activated K+channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca++-activated K+channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca++-activated K+channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca++-activated K+channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.

The coronary vasodilator effect of bradykinin (BK) in the rat is independent of NO but dependent on activation of phospholipases with involvement of cytochrome P450 mono-oxygenase (P450) and stimulation of Ca++-activated K+channels, implicating an unidentified hyperpolarizing factor generated viaP450 metabolism of arachidonic acid (AA). Because P450 activity also generates free radicals, such as superoxide, which can lead to the formation of hydrogen peroxide and hydroxyl radicals, which are vasoactive, we addressed the contribution of superoxide to the vasodilator effect of BK in the rat heart. Using rat renal microsomes as a source of P450, we verified that P450-dependent metabolism of AA generated superoxide, as detected by chemiluminescence with lucigenin. The signal was almost abolished by inhibition of P450 with clotrimazole and the superoxide scavenger 4,5-dihydroxy-1,3-benzene sulfonic acid. However, base-line superoxide formation, detected by chemiluminescence, in cardiac slices and perfused hearts was unchanged in response to BK or AA. Furthermore, in perfused hearts treated with nitroarginine and indomethacin to eliminate NO and prostaglandins and elevate perfusion pressure, dose-dependent vasodilator responses to BK were unaffected by superoxide dismutase plus catalase, a combination that abolished dilator responses to hydrogen peroxide. Similarly, the superoxide scavengers 4,5-dihydroxy-1,3-benzene sulfonic acid and 4-hydroxy-2,2,6,6-tetramethylpiperidine-noxyl were without effect on vasodilator responses to BK. Thus, the coronary vasodilator action of BK is independent of superoxide or its derivatives, which can be excluded as hyperpolarizing factors mediating NO-independent vasodilation in the rat.

5,6-Epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450-dependent arachidonate product, is a substrate for cyclooxygenase (COX) and, in some vascular preparations, elicits COX-dependent vasodilation. In the blood perfused rat kidney, 5,6-EET causes COX-dependent renal vasoconstriction, whereas in the rat isolated kidney perfused with a physiological buffer, 5,6-EET produces dose-dependent vasodilation that is unaffected by indomethacin. We examined the possible contribution of platelet COX to the vasoconstrictor action of 5,6-EET. Incubation of labeled 5,6-EET with rat washed platelets yields additional products that elute between 14 to 17 min on high-performance liquid chromatography (HPLC) and cause constriction of the perfused kidney. Indomethacin decreased the formation of these products and reduced the vasoconstrictor capacity of the corresponding HPLC fractions. Thus, platelet COX can metabolize 5,6-EET to vasoconstrictor products that may contribute to the in vivo vasoconstrictor effect of this eicosanoid.

The cytochrome P450-dependent component of the coronary vasodilator action of bradykinin which requires activation of K+ channels was examined in terms of the contribution of phospholipases in the rat Langendorff heart preparation. This component was isolated by inhibition of nitric oxide synthase with nitroarginine and cyclooxygenase with indomethacin, neither of which affects the coronary vasodilator action of bradykinin. However, nitroarginine elevated coronary perfusion pressure from approximately 40 to 130 mm Hg. The phospholipase C inhibitor, U73122 {1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione}, reduced coronary vasodilator responses to bradykinin by greater than 80%. U73122 also diminished the coronary vasodilator action of cromakalim which activates ATP-sensitive K+ channels. The maleimide moiety of U73122 that has the capacity to affect K+ channels inhibited cromakalim-induced coronary vasodilation, but did not affect that to bradykinin. Inhibition of diacylglycerol lipase with RHC 80267 {1,6-bis-(cyclohexyloximinocarbonylamino)-hexane} was without an overall effect on coronary vasodilator responses to bradykinin. The cytosolic phospholipase A2 inhibitor, AACOCF3 {arachidonyl trifluoromethyl ketone¿} decreased responses to bradykinin by up to 90% whereas inhibitors of the secretory form of phospholipase A2 oleyloxyethyl phosphorylcholine and ONO-RS-082 {2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid} were less effective than either AACOCF3 or U73122. The phospholipase inhibitors demonstrated selectivity as they did not affect the coronary vasodilator responses to nitroprusside. We obtained additional evidence for the antiphospholipase activity of the inhibitors by demonstrating their capacity to suppress bradykinin-stimulated increases in the release of prostacyclin, measured as 6-keto prostaglandin F1 alpha. The phospholipase inhibitors did not affect cyclooxygenase activity as the ability of arachidonic acid to stimulate prostaglandin formation was unimpaired. These results indicate that the coronary vasodilator action of bradykinin is linked to the activities of both phospholipase C and A2.

The isolated perfused kidney of the rat was used to address the development of tachyphylaxis to the vasoconstrictor effects of arachidonic acid. Repeated administration of arachidonic acid (3 micrograms at 10-min intervals) to the isolated kidney of the rat, perfused in situ with Krebs-Henseleit solution, led to the development of tachyphylaxis to the renal vasoconstrictor effects of arachidonic acid, using perfusion pressure changes as an index. Vasoconstrictor responses to either angiotensin or the endoperoxide analog, U46619, were unaffected by repeated administration of arachidonic acid. Associated with progressively reduced renal vasoconstrictor responses to arachidonic acid were parallel decrements in the renal venous release of prostanoids, measured by radioimmunoassay. In contrast, the release of prostanoids from the kidney stimulated by angiotensin II was increased after repeated administrations of arachidonic acid. These data suggest that the sequential reduction in renal vasoconstrictor responses to arachidonic acid is due to diminished conversion to prostaglandins, possibly due to inactivation of cyclooxygenase, decreased entry of arachidonic acid into the cell or its increased esterification into phospholipids.

Urinary prostaglandin (PG) and thromboxane (Tx) excretion, measured by radioimmunoassay, were examined in two groups of male Wistar rats made diabetic with streptozotocin, 70 mg/kg, one of which received daily insulin beginning on the 7th day after administration of streptozotocin. In addition, urinary kallikrein excretion and blood pressure were monitored. After the induction of diabetes the profile of urinary eicosanoid excretion was altered. 6-keto-PGF1 alpha and TxB2 excretion increased markedly within 24 to 48 hr and remained elevated for the duration of the study, up to 58 days. PGF2 alpha excretion also increased, the change being apparent after 6 days whereas PGE2 excretion was reduced at this time. Urinary kallikrein excretion was unchanged but blood pressure became elevated above control levels 2 weeks after the induction of diabetes. Insulin treatment, to maintain mean blood glucose levels below 200 mg/dl, resulted in decreased excretion of TxB2, PGF2 alpha and 6-keto-PGF1 alpha. However, excretion of PGE2 and kallikrein were increased after insulin treatment which also prevented the elevation in blood pressure. These studies indicate that insulin treatment of experimental diabetes corrects alterations in renal arachidonic acid metabolism and prevents the associated increase in blood pressure.

Based on the use of inhibitors, cytochrome P450 has been implicated in endothelium-dependent relaxant responses via metabolism of arachidonic acid (AA). However, the contribution of cytochrome P450 and its AA metabolites to the regulation of vascular tone has not been established due, in part, to questions of specificity of cytochrome P450 inhibitors which have not been extensively characterized in terms of their vascular effects. Consequently, we addressed the effects of several inhibitors on vasorelaxant responses of phenylephrine-contracted, rabbit, aortic rings to agents that utilize different transduction mechanisms to determine any actions unrelated to inhibition of cytochrome P450 and/or AA metabolism. Octadecynoic acid (2.5 and 5 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, and eicosatetrayenoic acid (10 and 20 microM), an inhibitor of AA metabolism, were without effect on vasorelaxant responses to acetylcholine, sodium nitroprusside, isoproterenol and diazoxide. 7-Ethoxyresorufin (2-10 microM), a substrate for cytochrome P450, and clotrimazole (2.5-10 microM) which binds to the heme moiety of the enzyme, concentration-dependently reduced responses to acetylcholine but not the other agonists indicating an effect on nitric oxide synthesis although neither affected the conversion of L-arginine to L-citrulline by endothelial cells. SKF 525A (50-200 microM), the prototypical inhibitor of cytochrome P450, which is metabolized to an inhibitory intermediate, also reduced responses to acetylcholine and, in addition, impaired the vasorelaxant activities of isoproterenol and diazoxide.

The inability of chronic treatment with aspirin to cause sustained inhibition of urinary prostaglandin (PG) excretion observed previously prompted us to compare the effects of 9-day treatment of spontaneously hypertensive rats with aspirin, 200 mg/kg/day s.c., flurbiprofen, 2.5 mg/kg/b.i.d. s.c. and indomethacin, 2.5 mg/kg/b.i.d. s.c. on the excretion rate of radioimmunoassayable PGE2 and PGF2 alpha. Conversion of 1-[14C]arachidonic acid and the release of PGs from endogenous substrate by the renal papilla were also examined. In vehicle-treated control rats, PGF2 alpha excretion ranged from 32.2 +/- 6.2 (mean +/- S.E.M.) to 41.6 +/- 7.3 ng/6 h, and was 2- to 4-fold higher than that of PGE2. Within 6 h of administration all three drugs reduced excretion of PGF2 alpha and PGE2 to less than 20% and 35% of control rats, respectively. Thereafter, PGF2 alpha and PGE2 excretion in aspirin-treated rats returned to values similar to the vehicle-treated group, whereas inhibition of PG excretion in indomethacin and flurbiprofen groups was sustained. Urine volume was doubled by aspirin throughout the study. In contrast, urine volume in flurbiprofen- and indomethacin-treated rats was unaffected. Paradoxically, metabolism of 1-[14C]arachidonic acid to PGs by renal papilla dissected on day 10, 2 to 4 h after the last drug dose, was reduced markedly by aspirin as was the release of immunoreactive PGs but was unaffected by flurbiprofen or indomethacin. The failure of long-term aspirin treatment to inhibit urinary PG excretion and the disparity between in vivo and ex vivo indices of PG release emphasize the need to verify their intended action by measuring PGs in biological fluids.

Urinary prostaglandins (PGs) and thromboxane excretion, measured by radioimmunoassay, were examined in male Wistar rats made diabetic with streptozotocin, 70 mg/kg. In addition, immunoreactive (i) 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) production by aortic rings was studied as well as conversion of [14C] arachidonic acid (AA) by aortic rings, polymorphonuclear leukocytes, and macrophages isolated from diabetic and age-matched control rats. The profile of urinary eicosanoid excretion changed after induction of diabetes. iPGF2 alpha excretion transiently increased, reaching a peak at 7 days and declining to control values by 48 days. iPGE2 excretion declined with time after induction of diabetes while marked increases in i thromboxane B2 and i6-keto-PGF1 alpha excretion occurred within 48 h and were maintained for the duration of the study, up to 176 days. However, serum i thromboxane B2 levels were similar in control and diabetic rats. Formation of i6-keto-PGF1 alpha by aortic rings obtained from diabetic rats was approximately one-half that of aortic rings from control rats. Similarly, conversion of [14C]AA revealed a diminished capacity of aortic rings from diabetic rats to synthesize prostacyclin (PGI2) measured as 6-keto-PGF1 alpha. Conversion of [14C]AA by polymorphonuclear leukocytes and macrophages obtained from diabetic rats did not differ from those obtained from control rats. In conclusion, experimental diabetes mellitus is accompanied by temporal alterations in AA metabolism, the functional significance of which is unknown at this time.

Plasma concentration of epoxyeicosatrienoic acids (EETs) derived from cytochrome P450 (CYP)-dependent metabolism of arachidonic acid is increased in women with preeclampsia (PE) as compared to normal pregnancy (N), and is even higher in fetal plasma (Herse et al. Circulation 2012, Jiang et al. Am J Hypertens 2013). We hypothesized that differences in EET synthesis or metabolism in the feto-placental unit underlie the observed differences in circulating EETs.

The activity of large conductance (100-250pS), Ca2+- activated K+ channels (BK) is modified by vasoactive hormones via second messengers, EETs and 20-HETE, generated by cytochrome P450 (CYP). We compared the effects of inhibition of CYP and K+ channels on vasoconstrictor responses to angiotensin II (AII) and the endoperoxide mimetic, U46619, which differ in their requirement for extracellular Ca2+. In the rat isolated perfused kidney, inhibition of CYP with 17-ODYA gradually elevated basal perfusion pressure (PP) without influencing vasoconstrictor responses to AII whereas those to U46619 (30-300ng) were increased several-fold. Inhibition of K+ channels with 1mM TEA enhanced constrictor responses to AII and U46619; viz, PP was increased by 10±5, 81±21 and 155±21 mmHg compared to 2±1, 21±9 and 62±10 mmHg in the control group by 30, 100 and 300 ng U46619, respectively. The inhibitor of BK, charybdotoxin (20nM), but not the inhibitor of small conductance Ca2+-activated K+ channels (15pS), apamin, also enhanced vasoconstrictor responses (U46619>AII). Bolus administration of TEA and charybdotoxin, but not apamin, produced dose-dependent vasoconstriction when PP was elevated to 94±17mmHg from 66±9mmHg by U46619. Thus, 1, 2.5 and 5mg TEA increased PP by 12±4, 29±5 and 49±14 mmHg, respectively, compared to 5±5, 12±5 and 22±7 mmHg, respectively, when PP was elevated to 91±8 mmHg by increasing flow. Similar results were obtained using AII as the constricting agent. These results show that blockade of K+ channels enhances renal vasoconstrictor responses to AII and the thromboxane mimic and that release of CYP-derived eicosanoids moderate constrictor responses.