Mike VanRollins

Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids Are Potent Vasodilators in the Canine Coronary Microcirculation

Cytochrome P450 epoxygenases convert arachidonic acid into 4 epoxyeicosatrienoic acid (EET) regioisomers, which were recently identified as endothelium-derived hyperpolarizing factors in coronary blood vessels. Both EETs and their dihydroxyeicosatrienoic acid (DHET) metabolites have been shown to relax conduit coronary arteries at micromolar concentrations, whereas the plasma concentrations of EETs are in the nanomolar range. However, the effects of EETs and DHETs on coronary resistance arterioles have not been examined. We administered EETs and DHETs to isolated canine coronary arterioles (diameter, 90.0+/-3.4 microm; distending pressure, 20 mm Hg) preconstricted by 30% to 60% of the resting diameter with endothelin. All 4 EET regioisomers produced potent, concentration-dependent vasodilation (EC50 values ranging from -12.7 to -10.1 log [M]) and were approximately 1000 times more potent than reported in conduit coronary arteries. The vasodilation produced by 14,15-EET was not attenuated by removal of the endothelium and indicated a direct action of 14,15-EET on microvascular smooth muscle. Likewise, 14,15-DHET, 11,12-DHET, 8,9-DHET, and the delta-lactone of 5,6-EET produced extremely potent vasodilation (EC50 values ranging from -15.8 to -13.1 log [M]). The vasodilation produced by these eicosanoids was highly potent in comparison to that produced by other vasodilators, including arachidonic acid (EC50=-7.5 log [M]). The epoxide hydrolase inhibitor, 4-phenylchalone oxide, which blocked the conversion of [3H]14,15-EET to [3H]14,15-DHET by canine coronary arteries, did not alter arteriolar dilation to 11,12-EET; thus, the potent vasodilation induced by EETs does not require formation of DHETs. In contrast, charybdotoxin (a KCa channel inhibitor) and KCl (a depolarizing agent) blocked vasodilation by 11,12-EET and 11,12-DHET. We conclude that EETs and DHETs potently dilate canine coronary arterioles via activation of KCa channels. The preferential ability of these compounds to dilate resistance blood vessels suggests that they may be important regulators of coronary circulation.

Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.

Functional Implications of a Newly Characterized Pathway of 11,12-Epoxyeicosatrienoic Acid Metabolism in Arterial Smooth Muscle

Epoxyeicosatrienoic acids (EETs) are potent vasodilators derived from cytochrome P-450 metabolism of arachidonic acid. The rapid conversion of EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs) has been proposed as a process whereby EETs are rendered biologically inactive. However, the vascular metabolism of EETs and the vasoactivities of EET metabolites have not been extensively studied. Accordingly, 11,12-EET metabolism was characterized in porcine aortic smooth muscle cells. The cells converted [3H]11,12-EET to 11,12-DHET and to a newly identified metabolite, 7,8-dihydroxy-hexadecadienoic acid (DHHD). 11,12-DHET accumulation in the medium reached a maximum in 2 to 4 hours and then declined, whereas 7,8-DHHD accumulation increased continuously and exceeded the amount of 11,12-DHET by 8 hours. [3H]11,12-EET conversion to radiolabeled 7,8-DHHD was reduced in the presence of unlabeled 11,12-DHET, indicating that 11,12-DHET is an intermediate in the conversion of 11,12-EET to 7,8-DHHD. This is consistent with a pathway whereby 11,12-EET is converted by an epoxide hydrolase to 11,12-DHET, which then undergoes two beta-oxidations to form 7,8-DHHD. In porcine coronary artery rings contracted with a thromboxane mimetic, 11,12-DHET produced relaxation similar in magnitude to that produced by 11,12-EET (77% versus 64% relaxation at 5 mumol/L, respectively). 7,8-DHHD also produced vasorelaxation. Thus, the vasoactivity of 11,12-EET is not eliminated by conversion to 11,12-DHET and 7,8-DHHD. These results suggest that 11,12-DHET and its metabolite, 7,8-DHHD, may contribute to the regulation of vascular tone in the porcine coronary artery and possibly other vascular tissues.

Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes.

1 Whole‐cell Na+ currents (holding potential, −80 mV; test potential, −30 mV) in rat myocytes were inhibited by 8,9‐epoxyeicosatrienoic acid (8,9‐EET) in a dose‐dependent manner with 22 ± 4 % inhibition at 0.5 μM, 48 ± 5 % at 1 μM, and 73 ± 5 % at 5 μM (mean ± s.e.m., n= 10, P < 0.05 for each dose vs. control). Similar results were obtained with 5,6‐, 11,12‐, and 14,15‐EETs, while 8,9‐dihydroxyeicosatrienoic acid (DHET) was 3‐fold less potent and arachidonic acid was 10‐ to 20‐fold less potent. 2 8,9‐EET produced a dose‐dependent, hyperpolarized shift in the steady‐state membrane potential at half‐maximum inactivation (V½), without changing the slope factor. 8,9‐EET had no effect on the steady‐state activation of Na+ currents. 3 Inhibition of Na+ currents by 8,9‐EET was use dependent, and channel recovery was slowed. The effects of 8,9‐EET were greater at depolarized potentials. 4 Single channel recordings showed 8,9‐EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5 Incubation of cultured cardiac myocytes with 1 μM [3H]8,9‐EET showed that 25 % of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86 % of the radioactivity remained as [3H]8,9‐EET while 13 % was converted into [3H]8,9‐DHET. After a 30 min incubation, 1–2 % of the [3H]8,9‐EET uptake by cells remained as unesterified EET. 6 These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9‐EET. 8,9‐EET is a potent use‐ and voltage‐dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

Dihydroxyeicosatrienoic acids are potent activators of Ca2+‐activated K+ channels in isolated rat coronary arterial myocytes

1 Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside‐out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca2+‐activated K+ (BK) channels in smooth muscle cells from rat small coronary arteries (150–300 μm diameter). 2 11,12‐DHET potently activated BK channels with an EC50 of 1.87 ± 0.57 nm (n= 5). Moreover, the three other regioisomers 5,6‐, 8,9‐ and 14,15‐DHET were equipotent with 11,12‐DHET in activating BK channels. The efficacy of 11,12‐DHET in opening BK channels was much greater than that of its immediate precursor 11,12‐EET. In contrast, AA did not significantly affect BK channel activity. 3 The voltage dependence of BK channels was dramatically modulated by 11,12‐DHET. With physiological concentrations of cytoplasmic Ca2+ (200 nm), the voltage at which the channel open probability was half‐maximal (V1/2) was shifted from a baseline of 115.6 ± 6.5 mV to 95.0 ± 10.1 mV with 5 nm 11,12‐DHET, and to 60.0 ± 8.4 mV with 50 nm 11,12‐DHET. 4 11,12‐DHET also enhanced the sensitivity of BK channels to Ca2+ but did not activate the channels in the absence of Ca2+. 11,12‐DHET (50 nm) reduced the Ca2+ EC50 of BK channels from a baseline of 1.02 ± 0.07 μm to 0.42 ± 0.11 μm. 5 Single channel kinetic analysis indicated that 11,12‐DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12‐DHET dose‐dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. 6 We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca2+ and voltage. Hence, like EETs, DHETs may function as endothelium‐derived hyperpolarizing factors.

Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids

Cytochrome P-450-derived epoxyeicosatrienoic acids (EETs) are avidly incorporated into and released from endothelial phospholipids, a process that results in potentiation of endothelium-dependent relaxation. EETs are also rapidly converted by epoxide hydrolases to dihydroxyeicosatrienoic acid (DHETs), which are incorporated into phospholipids to a lesser extent than EETs. We hypothesized that epoxide hydrolases functionally regulate EET incorporation into endothelial phospholipids. Porcine coronary artery endothelial cells were treated with an epoxide hydrolase inhibitor, 4-phenylchalcone oxide (4-PCO, 20 μmol/l), before being incubated with 3H-labeled 14,15-EET (14,15-[3H]EET). 4-PCO blocked conversion of 14,15-[3H]EET to 14,15-[3H]DHET and doubled the amount of radiolabeled products incorporated into cell lipids, with >80% contained in phospholipids. Moreover, pretreatment with 4-PCO before incubation with 14,15-[3H]EET enhanced A-23187-induced release of radiolabeled products into the medium. In contrast, 4-PCO did not alter uptake, distribution, or release of [3H]arachidonic acid. In porcine coronary arteries, 4-PCO augmented 14,15-EET-induced potentiation of endothelium-dependent relaxation to bradykinin. These data suggest that epoxide hydrolases may play a role in regulating EET incorporation into phospholipids, thereby modulating endothelial function in the coronary vasculature.Cytochrome P-450-derived epoxyeicosatrienoic acids (EETs) are avidly incorporated into and released from endothelial phospholipids, a process that results in potentiation of endothelium-dependent relaxation. EETs are also rapidly converted by epoxide hydrolases to dihydroxyeicosatrienoic acid (DHETs), which are incorporated into phospholipids to a lesser extent than EETs. We hypothesized that epoxide hydrolases functionally regulate EET incorporation into endothelial phospholipids. Porcine coronary artery endothelial cells were treated with an epoxide hydrolase inhibitor, 4-phenylchalcone oxide (4-PCO, 20 micromol/l), before being incubated with (3)H-labeled 14,15-EET (14,15-[(3)H]EET). 4-PCO blocked conversion of 14,15-[(3)H]EET to 14,15-[(3)H]DHET and doubled the amount of radiolabeled products incorporated into cell lipids, with >80% contained in phospholipids. Moreover, pretreatment with 4-PCO before incubation with 14,15-[(3)H]EET enhanced A-23187-induced release of radiolabeled products into the medium. In contrast, 4-PCO did not alter uptake, distribution, or release of [(3)H]arachidonic acid. In porcine coronary arteries, 4-PCO augmented 14,15-EET-induced potentiation of endothelium-dependent relaxation to bradykinin. These data suggest that epoxide hydrolases may play a role in regulating EET incorporation into phospholipids, thereby modulating endothelial function in the coronary vasculature.

Optimization of epoxyeicosatrienoic acid syntheses to test their effects on cerebral blood flow in vivo

Epoxyeicosatrienoic acids (EETs), normally present in brain and blood, appear to be released from atherosclerotic vessels in large amounts. Once intravascular, EETs can constrict renal arteries in vivo and dilate cerebral and coronary arteries in vitro. Whether EETs in blood will alter cerebral blood flow (CBF) in vivo is unknown. In the present study, the chemical synthesis of four EET regioisomers was optimized, and their identity and structural integrity established by chromatographic and mass spectral methods. The chemically labile EETs were converted to a sodium salt, complexed with albumin, and infused into anesthetized rats via the common carotid. The objective was to test whether sustained, high levels of intravascular EETs alter CBF. The CBF (cortical H2 clearance) was measured before and 30 min after the continuous infusion of 14,15- (n = 5), 11,12- (n = 5), 8,9- (n = 7) and 5,6-EET (unesterified or as the methyl ester, n = 5 for each). Neither the CBF nor the systemic blood pressure was affected by EETs. Because the infusions elevated the plasma concentrations of EETs about 700-fold above normal levels (1.0 nM), it is unlikely that EETs released from atherosclerotic vessels will alter CBF.

Simultaneous resolution of underivatized regioisomers and stereoisomers of arachidonate epoxides by capillary electrophoresis.

cis-Epoxyeicosatrienoic acids (EETs) and their hydrolysis products (threo-DHETs) have been proposed to be endothelial-dependent hyperpolarizing factors (EDHFs) which upregulate blood flow when tissue perfusion is impaired. Various EET regioisomers and enantiomers are formed from arachidonate by inducible cytochrome P450 epoxygenase isoforms, and tissue EET profiles may vary with diet, time, and disease. Because EET actions and metabolism may be regio- and stereospecific, convenient methods to measure profiles of EET isomers in tissues are needed. In the current studies, we describe two simple capillary electrophoretic methods for resolving EETs. The first method involves capillary electrophoresis with a mixture of neutral and anionic beta-cyclodextrins, which in one step baseline-resolves underivatized EET regioisomers and their enantiomers. Low picogram amounts of EET enantiomers were identified based on migration times and UV spectra. The method was also used to assess the antipode purity of EET standards, and to determine murine hepatic levels of EET enantiomers. The second method involves capillary electrochromatography, which also baseline-resolves underivatized EET and DHET regioisomers in one step. We conclude that in EET assays the major advantages of capillary electrophoresis over reversed-phase HPLC are improved peak efficiency, sensitivity, and resolution, plus precise coelution of deuterated and nondeuterated EETs.