Abstract-Endothelium-dependent hyperpolarizations and relaxation of vascular smooth muscle induced by acetylcholine and bradykinin are mediated by endothelium-derived hyperpolarizing factors (EDHFs). In bovine coronary arteries, arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), function as EDHFs. The 14,15-EET analog 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI) was synthesized and tested for agonist and antagonist activity. In U46619-preconstricted bovine coronary arterial rings, 14,15-, 11,12-, 8,9-, and 5,6-EET induced maximal concentration-related relaxation averaging 75% to 87% at 10 mol/L, whereas, 14,15-EEZE-mSI induced maximal relaxation averaging only 7%. 14,15-EEZE-mSI (10 mol/L) preincubation inhibited relaxation to 14,15-and 5,6-EET but not 11,12-or 8,9-EET. 14,15-EEZE-mSI also inhibited indomethacin-resistant relaxation to arachidonic acid and indomethacin-resistant and L-nitroarginine-resistant relaxation to bradykinin and methacholine. It did not alter the relaxation to sodium nitroprusside, iloprost, or the K ϩ channel openers bimakalim or NS1619. In cell-attached patches of isolated bovine coronary arterial smooth muscle cells, 14,15-EEZE-mSI (100 nmol/L) blocked the 14,15-EETinduced (100 nmol/L) activation of large-conductance, calcium-activated K ϩ channels. Mass spectrometric analysis of rat renal cortical microsomes incubated with arachidonic acid showed that 14,15-EEZE-mSI (10 mol/L) increased EET concentrations while decreasing the concentrations of the corresponding dihydroxyeicosatrienoic acids. Therefore, 14,15-EEZE-mSI inhibits relaxation to 5,6-and 14,15-EET and the K ϩ channel activation by 14,15-EET. It also inhibits the EDHF component of bradykinin-induced, methacholine-induced, and arachidonic acid-induced relaxation. These results suggest that 14,15-or 5,6 -EET act as an EDHF in bovine coronary arteries. Key Words: vasodilation Ⅲ arachidonic acids Ⅲ endothelium-derived factors Ⅲ acetylcholine Ⅲ bradykinin E poxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid. The vascular endothelium synthesizes and releases EETs in response to vasoactive agonists such as bradykinin and acetylcholine. [1][2][3][4][5] In the coronary circulation, EETs activate smooth muscle membrane large-conductance, calcium-activated K ϩ (BK Ca ) channels to cause hyperpolarization and vascular relaxation. 2 Therefore, they function as endothelium-derived hyperpolarizing factors (EDHFs). Besides EETs, hydrogen peroxide, K ϩ , and endocannabinoids are potential EDHFs. 6 -8 The specific chemical mediator of EDHF activity varies, depending on vascular size, vascular bed, and species. 9 Additionally, in small resistance arteries, electronic spread from the endothelium to the smooth muscle through gap junctions may mediate this activity. 10 The functional characterization of EDHF activity has depended on the use pharmacological inhibitors. To investigate the role of EETs in EDHF-dependent relaxation, inhibitors of cytochrome P450 enzymes are used. In ...

Recently, we reported that 11,12-epoxyeicosatrienoic acid (11,12-EET) potently activates rat mesenteric arterial ATP-sensitive K(+) (K(ATP)) channels and produces significant vasodilation through protein kinase A-dependent mechanisms. In this study, we tried to further delineate the signaling steps involved in the activation of vascular K(ATP) channels by EETs. Whole cell patch-clamp recordings [0.1 mM ATP in the pipette, holding potential (HP) = 0 mV and testing potential (TP) = -100 mV] in freshly isolated rat mesenteric smooth muscle cells showed small glibenclamide-sensitive K(ATP) currents (19.0 +/- 7.9 pA, n = 5) that increased 6.9-fold on exposure to 5 microM 14,15-EET (132.0 +/- 29.0 pA, n = 7, P < 0.05 vs. control). With 1 mM ATP in the pipette solution, K(ATP) currents (HP = 0 mV and TP = -100 mV) were increased 3.5-fold on exposure to 1 microM 14,15-EET (57.5 +/- 14.3 pA, n = 9, P < 0.05 vs. baseline). In the presence of 100 nM iberiotoxin, 1 microM 14,15-EET hyperpolarized the membrane potential from -20.5 +/- 0.9 mV at baseline to -27.1 +/- 3.0 mV (n = 6 for both, P < 0.05 vs. baseline), and the EET effects were significantly reversed by 10 microM glibenclamide (-21.8 +/- 1.4 mV, n = 6, P < 0.05 vs. EET). Incubation with 5 microM 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), a 14,15-EET antagonist, abolished the 14,15-EET effects (31.0 +/- 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET, P = not significant vs. control). The 14,15-EET effects were inhibited by inclusion of anti-G(s)alpha antibody (1:500 dilution) but not by control IgG in the pipette solution. The effects of 14,15-EET were mimicked by cholera toxin (100 ng/ml), an exogenous ADP-ribosyltransferase. Treatment with the ADP-ribosyltransferase inhibitors 3-aminobenzamide (1 mM) or m-iodobenzylguanidine (100 microM) abrogated the effects of 14,15-EET on K(ATP) currents. These results were corroborated by vasodilation studies. 14,15-EET dose-dependently dilated isolated small mesenteric arteries, and this was significantly attenuated by treatment with 14,15-EEZE or 3-aminobenzamide. These results suggest that 14,15-EET activates vascular K(ATP) channels through ADP-ribosylation of G(s)alpha.

Cytochrome P-450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs). EETs relax vascular smooth muscle by membrane hyperpolarization. 14,15-Epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE) antagonizes many vascular actions of EETs. EETs are converted to the corresponding dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). sEH activity in the bovine arterial endothelium and smooth muscle regulates endogenous EETs. This study examined sEH metabolism of 14,15-EE5ZE to 14,15-dihydroxy-eicosa-5(Z)-enoic acid (14,15-DHE5ZE) and the resultant consequences on EET relaxations of bovine coronary arteries (BCAs). BCAs converted 14,15-EE5ZE to 14,15-DHE5ZE. This conversion was blocked by the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). 14,15-EET relaxations (maximal relaxation, 83.4 Ϯ 4.5%) were inhibited by 14,15-DHE5ZE (10 M; maximal relaxation, 36.1 Ϯ 9.0%; p Ͻ 0.001). In sharp contrast with 14,15-EE5ZE, 14,15-DHE5ZE is a 14,15-EET-selective inhibitor and did not inhibit 5,6-, 8,9-, or 11,12-EET relaxations. 14,15-EET and 11,12-EET relaxations were similar in the presence and absence of AUDA (1 M). 14,15-EE5ZE inhibited 14,15-EET relaxations to a similar extent with and without AUDA pretreatment. However, 14,15-EE5ZE inhibited 11,12-EET relaxations to a greater extent with than without AUDA pretreatment. These observations indicate that sEH converts 14,15-EE5ZE to 14,15-DHE5ZE, and this alteration influences antagonist selectivity against EET-regioisomers. 14,15-DHE5ZE inhibited endothelium-dependent relaxations to AA but not endothelium-independent relaxations to sodium nitroprusside. A series of sEH-resistant ether analogs of 14,15-EE5ZE was developed, and analogs with agonist and antagonist properties were identified. The present study indicates that conversion of 14,15-EE5ZE to 14,15-DHE5ZE produces a 14,15-EET-selective antagonist that will be a useful pharmacological tool to identify EET receptor(s) and EET function in the cardiovascular system.

Betulinic acid (I), a pentacyclic triterpene, on derivatization gives six compounds:  3β-hydroxylup-20(29)-en-28 oic acid methyl ester (II), 3β-acetoxylup-20(29)-en-28-oic acid (III), 3β-allyloxylup-20(29)-en-28-oic acid (IV), 3β-p-methylcinnamatoxylup-20(29)-en-28-oic acid (V), 3β-p-methoxycinnamatoxylup-20(29)-en-28-oic acid (VI), and 3β-tri-O-methylgallotoxylup-20(29)-en-28-oic acid (VII). Their antifeedent activity against the agricultural pest tobacco caterpillar larvae (Spodoptera litura F) in a no-choice laboratory study showed the active compounds are V, VI, and VII. Keywords: Antifeedent; betulinic acid derivatives; tobacco caterpillar; Spodoptera litura (F)