Odette Laneuville

Hepoxilin A3 (HxA3) is formed by the rat aorta and is metabolized into HxA3-C, a glutathione conjugate

In this paper we describe the release of hepoxilin A3 (HxA3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A3 (TrXA3), and a second pathway involves conjugation of HxA3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A3-C (HxA3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA3 and HxA3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA3 and to a lesser extent HxA3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

Hepoxilins sensitize blood vessels to noradrenaline--stereospecificity of action.

1 . The vascular activity of two stereoisomers of hepoxilin A3 (HxA3) (8R and 8S) and of its glutathione conjugate, hepoxilin A3‐C (HxA3‐C) (8R and 8S), was investigated on rat helicoidal strips of thoracic aorta and longitudinal strips of portal vein. 2 . Neither of the hepoxilins tested had a direct effect on the tone of the aortic strip or on the spontaneous contractions of the portal vein. However, the noradrenaline (NA)‐induced response of these vessels, as expressed by the dose required for half maximal contraction, (EC50) was greater in HxA3 (8S)‐ and HxA3‐C (8R)‐treated aorta. Increased frequency and strength of spontaneous contractions of the portal vein were detected at lower concentrations of NA in the presence of hepoxilins. 3 . The threshold dose for both hepoxilins was 10−8 m and their effect was not dose‐related beyond 10−8 m. The effect of hepoxilin appeared after a 45 min incubation period and could be observed even if the compounds were washed out after 15 min. 4 . Stereochemical specificity was observed. The 8S isomer of HxA3 was active in potentiating the NA‐induced contraction of these vessels while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3‐C was active while the 8S isomer was inactive. In both tissues, HxA3 (8S) was more potent than its glutathione conjugate, HxA3‐C (8R). 5 . In calcium‐free buffer or in the presence of a calcium channel blocker (nifedipine 1 μm), no potentiation of NA‐induced contraction by hepoxilins could be observed, suggesting the involvement of extracellular calcium in the actions of hepoxilins. 6 . These experiments suggest that hepoxilins may be involved in the modulation of vascular tone and contractility.

New products in the hepoxilin pathway: isolation of 11-glutathionyl hepoxilin A3 through reaction of hepoxilin A3 with glutathione S-transferase.

We describe herein the metabolism of hepoxilin A3 (HxA3) by glutathione S-transferase (GST) into a glutathione conjugate. The reaction was carried out with HxA3 (unlabelled and 14C-labelled) and glutathione (unlabelled and tritium labelled). When two isomers of HxA3 were reacted with GST, two products were formed. Only one product was formed when a single isomer of HxA3 was used. The isomeric product HxB3 was marginally active indicating considerable specificity in the reaction with GST. The products were characterized by retention of tritium from glutathione and by comparison of their migration on high performance liquid chromatography with authentic reference compounds. The products bear the structure, 11-glutathionyl HxA3.

Neurokinin A-induced contraction of guinea-pig isolated trachea: potentiation by hepoxilins.

1 Hepoxilin A3 (8R and 8S isomers) (HxA3), hepoxilin A3‐C (8R and 8S isomers) (HxA3‐C) and trioxilin A3 (8S isomer) (TrXA3, the stable derivative of HxA3) were tested for their effects on helicoidal strips of guinea‐pig isolated tracheae. 2 None of the compounds (10−9−10−6 m) tested had a direct effect on resting tension of trachea. 3 HxA3 (8S) and HxA3‐C (8R) (10−8 m) produced a significant leftward shift of the log concentration‐response curves to neurokinin A (NKA) (EC50 (n m), control = 29.0 ± 2.8, HxA3 (8S) = 21.7 ± 3.7, HxA3‐C (8R)= 13.8 ± 3.8, n = 6 for each). Also the maximal response to NKA was significantly increased when the tissues were exposed to these hepoxilins (% of the maximal response to NKA, control = 100, HxA3 (8S) = 114.5 ± 5.3, HxA3‐C (8R) = 139.0 ± 6.2, n = 6 for each). The threshold concentrations for both hepoxilins was 10−8 m and their effects were dose‐related. 4 Stereochemical specificity was observed. The 8S‐isomer of HxA3 was active in potentiating the NKA‐induced contraction of the trachea while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3‐C was active while the 8S isomer was inactive. The trihydroxy metabolite of the active isomer of HxA3 (8S), i.e. TrXA3 (8S) (10−6 m), was inactive in potentiating the NKA‐induced contraction of the trachea. 5 It is concluded that hepoxilins sensitize the guinea‐pig isolated trachea to the potent bronchoconstrictor, NKA.

Hepoxilin A3 induces heat shock protein (HSP72) expression in human neutrophils

In this paper we show that hepoxilin A3 induces the expression of heat shock protein expression in human neutrophils at a concentration of 100 nM using Western blotting techniques employing the use of a commercial monoclonal antibody to HSP72. No regiospecificity was observed as the 8S enantiomer of HxA3 was as active as the 8R enantiomer of HxA3. Comparison of the effects of HxA3 with 12S-HETE and PGA1 indicated that HxA3 was as effective as 12S-HETE although PGA1 was essentially inactive at the same concentration used for these 12-lipoxygenase products.

Hepoxilin A3 Induces Vascular Permeability in the Skin

Hepoxilins are 11S,12S-epoxy-8(R,S)-hydroxy metabolites of arachidonic acid formed through the 12-lipoxygenase pathway from the intermediate, 12S-HPETE (1, 2). Evidence to date shows that the hepoxilins are formed through an Fe+3 catalysed rearrangement of 12-HPETE, which although retaining both oxygen atoms of 12-HPETE through an intramolecular isomerisation, the hydroxyl group at carbon 8 is racemic (3, 4). During the past few years we have been interested in profiling the biological properties of the 8R and 8S isomers of HxA3. The present report demonstrates that the hepoxilins are active in causing vascular permeability in the rat skin, and although they are not powerful on their own, they greatly potentiate the actions of subthreshold concentrations of bradykinin.