Hepoxilins: a review on their cellular actions

Biochimicab et Biophysics Acta ELSEVIER

Biochimica

et Biophysics

Acta 1215 (1994) 1-8

Review

Hepoxilins: a review on their cellular actions Cecil R. Pace-Asciak

*

Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S IA8, Canada Received 30 June 1994 Keywords:

Hepoxilin

A,; Hepoxilin

B,; Calcium ion; Potassium

ion; Second messenger

system

Contents 1.Summary

1

.....................................................

3. Formation

...........................................

2

hepoxilins...........................................

3

of the hepoxilins

4. Metabolism

2

...................................................

2. Introduction.

of the

5. Biological actions of the hepoxilins

.......................................

........................................ Effects on insulin secretion Effects on calcium ............................................. Effects on potassium channels: regulation of cell volume ........................ ..................................... Effects on vascular permeability. ...................................... Effects on vascular contraction Effects on the CNS. ............................................ Effects on second messengers ....................................... Effects on adenyl cyclase ......................................... ..................................... Inhibition of platelet aggregation Hepoxilin-binding proteins. ........................................ trihydroxy Chemical synthesis of hepoxilins A, and B, and the corresponding trioxilins .................................................. 5.12. Hepoxilin analogs .............................................

5.1. 5.2. 5.3. 5.4. 5.5. 5.6. 5.7. 5.8. 5.9. 5.10. 5.11.

3 3 3 4 4 4 5 5 5 5 6 compounds,

the

6. Future studies ................................................... 6.1. Receptor isolation and expression ..................................... Acknowledgements. References

.................................................

This review is intended to summarize the biological actions of the hepoxilins reported to date. These actions

* Corresponding

author. Fax: + 1 (416) 813 5086.

0005-2760/94/$07.00 6 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2760(94)00124-3

6 6 7

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Summary

6 6

7

appear to have, as their basis, changes in intracellular concentrations of ions including calcium and potassium ions as well as changes in second messenger systems. Recent evidence suggests that the biological actions of the hepoxilins may be receptor-mediated as indicated from data showing the existence of hepoxilin-specific binding proteins in the human neutrophil. Such evidence also

CR. Pace-Asciak/ Biochimica et Biophysics Acta 1215 (I 994) l-8

2

implicates the association of G-proteins both in hepoxilinbinding as well as in hepoxilin action. The potential use of stable analogs of the hepoxilins is discussed as well as the directions in which this area is heading.

2. Introduction The hepoxilins [47] are hydroxy epoxide metabolites formed through the rearrangement of 12S-HPETB, the initial product resulting from the action of 1Zlipoxygenase on arachidonic acid [35,39]. Two hepoxilins are formed, hepoxilin A, and hepoxilin B, (Scheme 1). Hepoxilins contain a trans-epoxide whose configuration is llS, 12X In contrast, the epoxide derived from cytochrome P-450 is a cis-epoxide whose configuration is llS, 12R and 11 R, 12s [5,6].

3. Formation of the hepoxilins A structure similar to hepoxilin B, was initially isolated from human platelets by Jones et al. in 1978 [18]. The lack of isolation in this system of hepoxilin A, is probably due to the latter’s instability in the acidic workup in the reported study. Hepoxilin A, was actually isolated from rat lung and characterised in 1983 [39]. Studies on the mechanism of formation of hepoxilins indicated that both hepoxilins A, and B, were formed through the rearrangement of 1ZHPETE [35]. This rearrangement was carried out by lung homogenates but it could also be carried out

4

by hemin, the ferric iron being the important catalyst [36]. This latter finding was suggestive of a nonenzymatic reaction, but recent evidence indicates that this transformation is carried out in intact tissues through a heat-sensitive process suggestive of an enzymatic process [50]. Heat-sensitive hepoxilin-forming activity has been demonstrated in brain slices, pineal gland and skin of the rat. These tissues transform 12S-HPETE into both hepoxilins A, and B, and this transformation is abolished by tissue boiling. Recently, additional information on this enzymic process was obtained through stereochemical studies on the products of this pathway, i.e., lZHETE, hepoxilin A,, hepoxilin B, and the hepoxilin metabolites, the trioxilins. An equal mixture of 12S- and 12R-HPETE was used as hepoxilin precursor to investigate whether hepoxilins are formed from both substrates as would be expected of a nonenzymic reaction or whether one substrate is exclusively selected, as would be expected of an enzymic reaction. Chiral analysis by HPLC of the products derived in these experiments showed that the hepoxilins are exclusively derived from 12S-HPETE, with 12R-HPETE remaining unreacted when the enzyme system from rat pineal gland is used; in contrast when the 12S/R-HPETE substrates are incubated with hemin, hepoxilins from both 12S- and 12R-HPETE are formed. Analysis of the 12-HETE remaining also indicated both epimers (S and R) were present in the hemin (nonenzymic) reaction (unpublished observations). This stereoselective utilization of 12SHPETE is an important finding to suggest the presence of a ‘hepoxilin synthase’. Eicosapentaenoic acid is also transformed into hepox-

lz-Lipoxygenase COOB

Scheme 1. Scheme depicting the structures of the hepoxilins and their metabolites

and the various enzymes involved in their biosynthesis.

C.R. Pace-Asciak/ Biochimica et Biophysics Acta 1215 (1994) I-8

ilins [37], these products having four double bonds and are therefore termed hepoxilins A, and B,. Hepoxilin B, has also been isolated from tropical marine red algae [31].

4. Metabolism of the hepoxilins Hepoxilin A, is unstable biologically and is metabolized into the trihydroxy product, termed trioxilin A,, through the action of an epoxide hydrolase [40,46]. Hepoxilin A, is also unstable chemically as it is rapidly hydrolyzed in acid media into trioxilin A,. It is because of this intrinsic instability that the products of epoxide ring opening, i.e., the trioxilins, are normally detected [3,4,18]. Hepoxilin A, is much more stable as the methyl ester and methods for the rapid extractive methylation of hepoxilin A, have been successful in the analysis of hepoxilin A, by HPLC (unpublished observations). In contrast, hepoxilin B, is resistant to both enzymatic and nonenzymatic hydrolysis. Hepoxilin epoxide hydrolase is inhibited by trichloropropene oxide, TCPO [46]. When hepoxilin epoxide hydrolase is blocked in this way, hepoxilin A, is further metabolized through a glutathione transferase pathway to form a glutathione conjugate of hepoxilin termed I&4,-C. This metabolite is formed through an enzymatic addition of glutathione to the epoxide moiety to form 11-glutathionyl-12-hydroxy metabolite [41-441.

5. Biological actions of the hepoxilins 5.1. Effects on insulin secretion The first biological actions of the hepoxilins that were described were on the secretion of insulin from rat pancreatic islets of Langerhans [47]. Hence the name ‘hepoxilin’ was coined to relate structure (HydroxyEPOXide) with function (InsuLZiV). Using perifused islets, it was shown that hepoxilin A, stimulated the glucose-evoked secretion of insulin. The initial studies were carried out at high (10 mM) glucose concentrations, but subsequent studies with pure epimers of the hepoxilins showed that both hepoxilins A, and B, were more active in releasing glucose at low glucose concentration (5 mM) (unpublished observations). These studies suggest that the hepoxilins may participate in the basal control of insulin secreted from these cells. In support of this sugggestion, it was shown that the hepoxilins are formed by islets from both endogenous precursors [49] as well as exogenous 1ZHPETE [48] and arachidonic acid [45]. Recent observations using single p-cells have shown that hepoxilin A, at lop9 M concentrations causes an increase in the intracellular concentration of calcium (M. Salter and C.R. Pace-As&k, unpublished results). These findings lend support to the concept that hepoxilins may cause the release of insulin through the release of intracellular calcium (see also below).

3

5.2. Effects on calcium Studies with human neutrophils, pancreatic p-cells and vascular tissue show that hepoxilins act on calcium stores resulting in a rise in intracellular calcium. In neutrophils, hepoxilins were shown to also cause the influx of calcium from the extracellular medium but this appears secondary to the initial action to release calcium from stores. The following sections describe in more detail the effects of these hepoxilins. Calcium transport Early on, it was shown that hepoxilin A, added to Ussing chambers partitioned with the visceral yolk sac membrane from a pregnant guinea pig caused an accumulation of calcium on the side of the membrane to which hepoxilin had been added [14]. Biologically-derived hepoxilin was used in those early studies as chemically s nthetic 4Y compound was not available yet. The transport of Ca was independent of whether the hepoxilin was added to the maternal or fetal side. The radiolabel accumulated at the side to which the hepoxilin was added suggesting that hepoxilin A, may have activated a calcium channel. The activation of calcium channels has recently been observed with hippocampal neurons using the whole cell patch clamp technique [63]. Calcium mobilization Studies with fluorescent dye-loaded human neutrophils indicated that hepoxilin A, at Z.LMconcentrations causes a rapid increase in the intracellular concentration of calcium [15]. This rise in intracellular calcium occurs in calciumfree medium indicating that hepoxilin A, releases calcium from intracellular stores. The rapid rise in intracellular calcium is followed by a slow influx of extracellular calcium which is abolished when calcium-free extracellular medium is used. Several observations have been made regarding these effects of the hepoxilins. First, both epimers of hepoxilin A, are active although the 8R epimer appears more active, suggesting some degree of specificity [15]. Second, the hepoxilin must be in the methyl ester form as the free acid of hepoxilin A, applied to neutrophil suspensions does not cause changes in intracellular calcium (unpublished observations). This suggests that the site of action of the hepoxilin is intracellular, the methyl ester being required to permit the compound to penetrate into the cell where it may be hydrolysed into the free acid by cytosolic esterases. The use of the methyl or the acetoxymethyl ester derivative is employed routinely with fluorescent dyes to effect their uptake into cells. Third, the polar glutathione conjugate of hepoxilin A,, i.e., HxA,-C, is inactive in altering the concentraton of intracellular calcium, suggesting that the epoxide moiety of the molecule is important for the release of calcium from stores (unpublished observations). Fourth, the actions of the hepoxilins are inhibited by the pretreatment of the cells with pertussis

4

C.R. Pace-Asciak/Biochimica

toxin suggesting the involvement of G-proteins in hepoxilin action [15]. Additional studies (see below) suggest that hepoxilin-specific-binding sites occur in the neutrophil which are saturable and competed for by hepoxilin stable analogs. Hepoxilin A, releases calcium from intracellular stores [15,21]. In so doing hepoxilin A, inhibits the receptormediated release of calcium by several chemotactic agents. These include the tetrapeptide, fMLP, platelet-activating factor (PAF) and leukotriene B, [21]. The site of action of hepoxilin A, in inhibiting the actions of these unrelated agents is likely at some common site downstream from their receptors. It would be interesting to determine whether the hepoxilins interfere with the IP, receptor on the ER. Stable analogs of hepoxilin A, having a cyclopropyl group instead of an epoxide have been prepared chemically [12,13] and found to mimic the actions of the parent compound, except that they appear to be weaker than the parent compounds on their own (unpublished observations). The analogs are also weaker than the parent compound in antagonizing the binding of the parent compound to intact human neutrophils. However the analogs are 3-5 times more potent than the parent compound in blocking the calcium releasing actions of the chemotactic agents, suggesting that their action may not only be related to mimicking the action of the parent hepoxilin but they could be acting at a common site downstream from the receptor on which the chemotactic agents act (unpublished observations). Further studies with these potentially important analogs may prove useful in identifying stores of calcium that may be activated during the inflammatory response. 5.3. Effects on potassium

channels: regulation of cell

volume When platelets are subjected to stress such as during exposure to hypotonic medium or to shear forces, the cells swell temporarily due to influx of potassium ions and ensuing water [23]. Cell volume quickly returns to normal as K+ is pumped out of the cell. The process has been termed, regulatory volume decrease (RVD). RVD has been shown to be due to a lipoxygenase product as inhibitors of lipoxygenase render the platelet insensitive to its return to normal volume and the cell stays bloated [27,28]. The responsible lipoxygenase product has been identified as hepoxilin A, [30]. Chemically pure or biologically prepared hepoxilin A, when added to platelets that have been volume expanded and retained in this state by the presence of a lipoxygenase blocker, cause the platelet volume to return towards its normal state [30]. These observations suggest that hepoxilins may activate the opening of K+ channels resulting in the lowering of the intracellular concentration of this ion and accompanied water. Although K+ concentrations have not been measured in these experiments, these observations find precedence from another system, the Aplysia neurons. In these studies, using the

et Biophysics Acta 1215 (1994) 1-8

inside-out patch clamp technique, it was shown that hepoxilins increase the probability of the S-type K+ channel to stay in the open state [2]. Both the 8S and the 8R epimers of hepoxilin A, are capable of opposing volume expansion in the platelet [30]. Recent evidence has indicated that the hepoxilin action in reversing volume expansion is mediated through G-proteins because pertussis toxin blocks the hepoxilin effect [29]. GDPsS has also been used in intact platelets and found to block the hepoxilin effect, although it is not clear how this compound penetrated into the platelet as intact membranes are impermeable to these analogs. Whether the volume regulatory property of hepoxilins is common to other cells as well is not yet known. 5.4. Effects on vascular permeability The involvement of the prostaglandins in the inflammatory response has been known for some time, with PGE, being the most potent causing leakage of plasmatic proteins from microvessels (for reviews see [7,57,61]. Recently hepoxilins were compared with PGE, in their ability to cause plasma leakage from the rat skin using the method involving the administration to the rat of Evans blue dye. Subcutaneous administration of hepoxilin A, did not result in much effect (doses in the pg range were needed to cause an effect) while PGE, was active when ng amounts were administered. However, it was observed that when hepoxilin A, was coinjected with subtreshold concentrations of bradykinin, a remarkable potentiation by hepoxilin A, was observed making hepoxilin A, equipotent to PGE, within a 5 min time course. Within a longer time course (up to 30 min) the hepoxilin effect became less potent probably because the compounds are rapidly metabolised [20]. Interestingly the leakage of proteins caused by the hepoxilins was more localised than that observed with PGE, which tended to become diffuse with time. The hepoxilin effect was highly stereospecific, only the 8R epimer showing activity. The mechanism whereby the hepoxilin potentiates the actions of bradykinin is not known. 5.5. Effects on vascular contraction Whereas hepoxilin A, (8R) is active in potentiating the vascular permeability actions of bradykinin, the 8s epimer only is capable of potentiating the contractile actions of norepinephrine in the de-endothelialized aorta and portal vein [19]. In these experiments, spiral strips of the aorta were set up in organ baths and the hepoxilins were tested after appropriate dose-response curves had been constructed for norepinephrine. Hepoxilin A, was seen to potentiate subthreshold doses of norepinephrine through a time-dependent increase in the tone of the vessel. This rise in tone was gradual but kept increasing up to 90 min. Using this information, dose-response curves to nor-

C.R. Pace-Asciak/Biochitnica et Biophysics Acta 1215 (1994) 1-8

epinephrine were constructed before and after the prolonged (45 min was selected) exposure of the strip to hepoxilin A, showing significant leftward shift by hepoxof hepoxilin initiating this ilin A,. The concentration leftward shift was lo- 8 M, and the hepoxilin response appeared to have a bell-shaped relationship with higher concentrations of hepoxilin A 3. The glutathione conjugate of hepoxilin A, was also tested, and similar potentiation of norepinephrine was observed in the same concentration range by the 8R epimer of HxA,-C, the 8s epimer being mostly inactive [19]. These findings contrast with the calcium results observed in the neutrophils as the glutathione conjugates in those experiments were inactive in mobilizing intracellular calcium. Hence the actions of the hepoxilins on the blood vessels may not be entirely calcium related unless specific receptors for the hepoxilins exist on the blood vessels. Nifedipine was shown to block the actions of hepoxilins on the blood vessels [19] suggesting that while neutrophils do not appear to have surface hepoxilin receptors and that hepoxilins may not affect calcium channels in those cells, the vascular effects of hepoxilins may be mediated through the activation of calcium channels. 5.6. Effects on the CNS Hepoxilins have been tested on their ability to affect neuronal function. Two types of studies have been reported involving Aplysia neurons [52,53] and mammalian CA1 neurons [42,62,63]. The molluscan tetrapeptide, FMRFamide, causes presynaptic inhibition of evoked potential in the Aplysia neurons. It was reported at first that lipoxygenase products, specifically 12-HPETE, mimicked the actions of FMRFamide in the Aplysia. It was subsequently shown that the actions of 12-HPETE were mimicked by hepoxilin A 3, which is endogenously formed by the Aplysia brain [52]. Using pure epimers of hepoxilin A, it was shown that these compounds were also active in the mammalian brain. In hippocampal CA1 neurons, hepoxilin A, hyperpolarized the membrane potential and caused an increase in amplitude and duration of the post-spike train after hyperpolarization. Hepoxilins also increased the amplitude and duration of the inhibitory postsynaptic potential. Recent studies have shown a threshold concentration for these actions in the 3-10 nM range [63]. Biochemical studies with tritiated norepinephrine-loaded hippocampal slices demonstrated that hepoxilin A, at10-6 M concentration inhibited the 4-aminopyridine-induced release of norepinephrine, agreeing with electrophysiological observations of a possible synaptic inhibitory action [51]. Hepoxilin A, was shown to occur in mammalian brain [38], and its metabolism via the epoxide hydrolase pathway was also shown to exist [38]. Upon blockade of the epoxide hydrolase with TCPO, hepoxilin metabolism was diverted to glutathione conjugation with formation of the glutathione conjugate, HxA,-C [44]. HxA,-C was also active

5

on hippocampal neurons, with a threshold within the 3-30 nM concentration range [42,63]. 5.7. Effects on second messengers Hepoxilin A, causes the release of arachidonic acid and diacylglycerol from human neutrophils [33]. Both epimers at C8 (8s and 8R) appear effective to a similar extent. They appear to potentiate the actions of fMLP in the release of these second messengers except that the release of these second messengers appears to be carried out through the stimulation of a phospholipase D mechanism and not phospholipase C as expected of fMLP activation [32,33]. Hence hepoxilins appear to act by stimulating an IP,-independent pathway. Evidence for the hepoxilinevoked activation of the phospholipase D pathway was obtained through the detection of enhanced formation of phosphatidic acid. Additional evidence was obtained through the observation of the accumulation of phosphatidic acid by hepoxilin A, when the phosphatidic acid phosphohydrolase was blocked with propranolol [32]. Hence, hepoxilin A, stimulates phospholipase D resulting in the formation of phosphatidic acid, which in turn is hydrolysed into diacylglycerol and arachidonic acid. The effects of hepoxilins are also blocked by treatment of the neutrophils with pertussis toxin providing additional evidence (see above) in support of a G-protein mediated mode of action of the hepoxilins [33]. 5.8. Effects on adenyl cyclase Direct measurements on the effects of hepoxilins on formation of CAMP were obtained in the pineal gland [54]. In this tissue, stimulation of adenyl cyclase by NECA was inhibited by hepoxilin A,. Again both 8-epimers were active. Biosynthetic studies with the pineal gland have shown that both epimers are formed from 12-HPETE or arachidonic acid substrates. Hence confirmation of the formation and action of hepoxilin A, has been provided to implicate the hepoxilins in pineal function. Previous studies had demonstrated that 12-HPETE was capable of inhibiting the norepinephrine-induced activation of adenyl cyclase in the pineal gland [55]. It appears that in those experiments the actions of 12-HPETE may have been mediated through its conversion into the hepoxilins. CAMP is important for the formation of melatonin in the pineal whose principal function is in the regulation of the reproductive cycle [l] and in the sleep-wake cycle [34]. 5.9. Inhibition

of platelet aggregation

Recently, hepoxilin A, formed through the hypotonicinduced or shear stress-induced activation of human platelets has been shown to inhibit the aggregation of normal human platelets. Only thrombin-induced aggregation was inhibited, ADP-evoked aggregation was not re-

C.R. Pace-Asciak/Biochimica et Biophysics Acta 1215 (1994) l-8

6

sponsive

to biologically

formed or commercial

hepoxilin

A, [26]. 5. IO. Hepoxilin-binding

proteins

Through use of hepoxilin A, of high specific molar activity synthesised in our laboratory [12], we could show that hepoxilin-specific-binding proteins exist in the human neutrophil (unpublished observations). The binding of tritiated ligand was saturable, displaced and antagonised by unlabeled compound, and binding was dependent on temperature and the number of cells used. A single population of binding sites was observed with a K, = 133 nM, and calculated 11.26 . lo6 binding sites/cell. These values differ greatly from those reported for 12-HETE in cultured tumor cells (HL60 carcinoma cell line, K, = 0.44 nM, 66000 binding sites/cell [17] or for LTB, in human neutrophils (K, = 0.46 nM, 19600 binding sites/cell [22]). Since hepoxilin-binding proteins are located intracellularly, recognised through the use of the methyl ester derivative of hepoxilin A, (the free acid is not active in intact human neutrophils either in binding or in calcium release from intracellular stores, unpublished observations), while the sites for both LTB, as well as 12-HETE appear on the cell surface, the difference in the values for K, and the number of binding sites may be related to the different location of the respective receptors. In fact, LTB, and 12-HETE (both as the free acid or methyl ester) are largely inactive in displacing hepoxilin A, bound to neutrophils indicating that different binding proteins exist for these compounds. 5.11. Chemical synthesis of hepoxilins A, and B, and the corresponding trihydroxy compounds, the trio&ins Hepoxilin B, was first chemically synthesised in 1983 [9]. Subsequently, the synthesis of hepoxilin A, was reported from the same group [lo]. The synthetic approach used by this group involved the use of a standard intermediate epoxy aldehyde used for leukotriene B, synthesis. The synthesis of hepoxilin A, utilizing arsonium ylides has been reported [8]. The configuration of the C8 hydroxyl group has been assigned [ 111. A different stereoselective synthetic approach was reported by Lumin et al. making use of the Mitsunobu displacement reaction with palladium catalysis [24]. This route afforded both hepoxilin A, and the corresponding trioxilin A, of known streochemistry at the C8 position. An alternate synthesis of hepoxilin B, epimers has been carried out using a new triynoic acid intermediate which lends itself to the preparation of both the A, and B, compounds in optical purity as well as tritiated and deuterium-labelled compounds [56]. The preparation of trioxilins A, and B, has been reported by several other investigators [25,60]. The stereoselective synthesis of 10(R)-hepoxilin B, and 10(R)-trioxilin B, from (-)-o-tartaric acid has been described [58]. These

investigators also reported on the stereoselective synthesis of the 10(S)-epimers [59]. The pure C8 epimers of the hepoxilin biological metabolite, i.e., the glutathione conjugate of hepoxilin A, (HxA,-C), have been prepared by coupling the pure epimer of hepoxilin A, with N-trifluoroacetylglutathione dimethyl ester [ 111. 5.12. Hepoxilin

analogs

Analogs of hepoxilin A, in which a cyclopropyl group has replaced the epoxide moiety at Cll(S), C12(S) of the native hepoxilin have been chemically synthesised [12] and shown to antagonise the actions of hepoxilin A, (unpublished observations). These cyclopropane analogs also mimic the actions of hepoxilin A, in blocking the calcium mobilizing effects of the chemotactic agents, fMLP, PAF and LTB, (unpublished observations). Hepoxilin A, also blocks the effects of these chemotactic agents but the analogs appear more potent, possibly because their stucture may afford them greater chemical and biological stability. The actions of the analogs on such diverse compounds as these chemotactic agents which act through specific cell surface receptors suggests that the hepoxilins and these analogs may act downstream from the receptors for these chemotactic agents. The analogs may act directly on a calcium store common to these agents or may interfere with the signal transduced by these agents. Further studies with permeabilised cells have indicated that hepoxilin-binding and actions are coupled to a receptor whose activation appears to be coupled to G-proteins. It is not yet known whether hepoxilins interfere with IP, actions. If positive, hepoxilins may indeed turn out to represent an important family of naturally occurring compounds useful in studies on the mechanisms involved in neutrophil activation during inflammation and may possibly find use as a new generation of antiinflammatory drugs.

6. Future studies 6.1. Receptor

isolation and expression

Indeed hepoxilin receptors appear to exist in the human neutrophil. It is important to investigate whether other cells have these receptors as well, and if so to determine which cell affords the highest abundance of the appropriate binding protein for isolation and purification. The stage is set to determine its protein structure. Expression studies would naturally follow to provide more information about the factors which regulate the abundance of this new receptor. Of importance in determining the relative contribution of the hepoxilin system would be animal studies in which the 12-lipoxygenase gene has been disrupted. Such gene knockout studies have recently been reported with the 5-lipoxygenase gene [16], and animals lacking the 12lipoxygenase gene cannot be far behind.

C.R. Pace-Asciak/ Biochimica et Biophysics Acta 1215 (1994) l-8

Acknowledgements I wish to thank all my collaborators during the past decade who have helped in moving our initial observations on the hepoxilins so far ahead into the chemical, biochemical and pharmacological areas. This review summarises these findings. The financial support of the MRC of Canada and of ZymoGenetics Inc. is especially acknowledged.

[16] Funk, C.D. (1993) Targeted disruption of the murine 5-lipoxygenase gene, in International Symposium on Molecular Biology of the Arachidonic Acid Cascade, Kyoto, Japan, December 5-7, Abstract, p. 51. [17] Herbertsson, H. and Hammarstrom, S. (1992) High-affinity binding for 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) in carcinoma cells. FEBS Lett. 298, 249-252. [181 Jones, R.L., Kerry, P.J., Poyser, N.L., Walker, I.C. and Wilson, N.H. (1978) The identification of trihydroxy eicosatrienoic acids as products from the incubation of arachidonic acid with washed blood platelets. Prostaglandins 16, 583-590. O., Couture, R. and Pace-Asciak, C.R. (1992) Hepoxilins sensitize blood vessels to noradrenaline - stereospecificity of action. Br. J. Pharmacol. 105, 297-304. [201 Laneuville, 0. and Pace-Asciak, C.R. (1991) Hepoxilin A, induces vascular permeability in rat skin, in Prostaglandins, Leukotrienes, Lipoxins and PAF (Bailey, J.M., ed.), pp. 335-338, Plenum Press, New York. Dll Laneuville, O., Reynaud, D., Grinstein, S., Nigam, S. and PaceAsciak, C.R. (1993) Hepoxilin A, inhibits the rise in free intracellular calcium evoked by formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B,. Biochem. J. 295, 393397. La Lin, A.H., Ruppel, P.L. and German, R.R. (1984) Leukotriene B, binding to human neutrophils. Prostaglandins 28, 837-849. b31 Livne, A., Grinstein, S. and Rothstein, A. (1987) Volume-regulating behaviour of human platelets. J. Cell Physiol. 131, 354-363. [241 Lumin, S., Falck, J.R., Capdevila, J. and Karara, A. (1992) Palladium mediated allylic Mitsunobu displacement: Stereocontrolled synthesis of hepoxilin A, and trioxilin A, methyl esters. Tetrahedron Lctt. 33, 2091-2094. [251 Lumin, S., Yadagiri, P. and Falck, J.R. (1988) Synthesis of Trioxilin B,. Tetrahedron Lett. 29, 4237-4240. 1261Margalit, A. and Granot, Y. (1994) Endogenous hepoxilin A, produced under short duration of high shear-stress, inhibits thrombin-induced aggregation in human platelets. Biochim. Biophys. Acta 1190, 173-176. 1271Margalit, A. and Livne, A.A. (1991) Lipoxygenase product controls the regulatory volume decrease of human platelets. Platelets 2, 207-214. 1281Margalit, A. and Livne, A.A. (1992) Human platelets exposed to mechanical stresses express a potent lipoxygenase product. Thromb. Haemostas. 2, 207-214. [291 Margalit, A., Livne, A.A., Funder, J. and Granot, Y. (1993a) Initiation of RVD response in human platelets: Mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A,. J. Membrane Biol. 136, 1-9. 1301Margalit, A., Sofer, Y., Grossman, S., Reynaud, D., Pace-Asciak, C.R. and Livne, A. (1993b) Hepoxilin A, is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets. Proc. Natl. Acad. Sci. USA 90, 2589-2592. [311 Moghaddam, M.F., Gerwick, W.H. and Ballantine, D.L. (1990) Discovery of the mammalian insulin release modulator, hepoxilin B,, from the tropical red algae Platysiphonia miniata and Cottoniella filamentosa. J. Biol. Chem. 265, 6126-6130. 1321Nigam, S., Muller, S. and Pace-Asciak, C.R. (1993) Hepoxilins activate phospholipase D in the human neutrophil, in Eicosanoids and other bioactive lipids in cancer, inflammation and radiation injury (Nigam, S., Mamett, L.J., Honn, K.V. and Walden T.L.Jr., eds.), pp. 249-252, Kluwer Academic Publishers (London). 1331Nigam, S., Nodes, S., Cichon, G., Corey, E.J. and Pace-Asciak, C.R. (1990) Receptor-mediated action of hepoxilin A, releases diacylglycerol and arachidonic acid from human neutrophils. Biochem. Biophys. Res. Commun. 171, 944-948. [341 Niles, L.P., Brown, G.M. and Grotta, L.J. (1979) Role of the pineal in diurnal endocrine secretion and rythm regulation. Neuroendocrinology 29, 14-21.

1191 Laneuville,

References HI Alonso, R., Prieto, L., Hernandez,

C. and Mas, M. (1978) Antiandrogenic effects of the pineal gland and metabolism in castrated and intact prepubertal male rats. J. Endocr. 79, 77-83. Dl Belardetti, F., Campbell, W.B., Falck, J.R., Demontis, G. and Rosolowsky, M. (1989) Products of heme-catalyzed transformation of the arachidonate derivative 12-HPETE open S-type Kf channels in Aplysia. Neuron 3, 497-505. 131Bryant, R.W. and Bailey, J.M. (1979) Isolation of a new lipoxygenase metabolite of arachidonic acid, 8,11,12-trihydroxy-5,9,14eicosatrienoic acid from human platelets. Prostaglandins 17, 9-18. 141 Bryant, R.W. and Bailey, J.M. (1980) Isolation of glucose sensitive platelet lipoxygenase products from arachidonic acid. Adv. Prost. Thromb. Res. 6, 95-100. 151 Capdevila, J., Pramanik, B., Napoli, J.L., Manna, S. and Falck, J.R. (1984) Arachidonic acid epoxidation epoxy eicosatrienoic acids are endogenous constituents of rat liver. Arch. Biochem. Biophys. 231, 511-517. b51Capdevila, J.H., Karara, A., Waxman, D.J., Martin, M.V., Falck, J.R. and Guengerich, F.P. (1990) Cytochrome P-450 enzyme-specific control of the regio- and enantiofacial selectivity of the microsomal arachidonic acid epoxygenase. J. Biol. Chem. 265, 10865-10871. [71 Casley-Smith, J.R. (1980) The response of the microcirculation to inflammation, in The cell biology of inflammation (Weissman, G., ed.), pp. 53-82, Elsevier, New York. [81 Chabert, P., Mioskowski, C. and Falck, J.R. (1989) Synthesis of (+ )-hepoxilin A, utilizing arsonium ylides. Tetrahedron L&t. 30, 2545-2548. 191 Corey, E.J., Kang, J., Laguzza, B.C. and Jones, R.L. (1983) Total synthesis of 12(S), lo-hydroxy-trans-11,12-epoxyeicosa-5,9,14Z-trienoic acids, metabolites of arachidonic acid in mammalian platelets. Tetrahedron Lett. 24, 4913-4916. m Corey, E.J. and Su, W.-G. (1984) Total synthesis of biologically active metabolites of arachidonic acid. The two b-hydroxy11,12(S,S)-epoxyeicosa-5,14(Z),9(E)-trienoic acids. Tetrahedron L&t. 25, 5119-5122. Ml Corey, E.J. and Su, W.-G. (1990) (8R)- and (8S)-Hepoxilin A,. Assignment of configuration and conversion to biologically active conjugates with glutathione. Tetrahedron L&t. 31, 2113-2116. CR. (1993) Synthesis of racemic WI Demin, P.M. and Pace-As&k, ll,l%-cyclopropyl analogs of hepoxilins A, and B,. Tetrahedron L&t. 34, 4305-4308. [131 Demin, P.M., Pivnitsky, K.K., Vasiljeva, L.L. and Pace-Asciak, C.R. (1994) Synthesis of methyl [5,6,8,9,14,15-3H,]hepoxilin B, and its conversion into methyl [5,6,8,9,14,15-3H,]hepoxilin A,. J. Labelled Compounds and Radiopharmaceuticals 34, 221-230. [141 Derewlany, L.O., Pace-Asciak, C.R. and Radde, I.C. (1984) Hepoxilin A, hydroxyepoxide metabolite of arachidonic acid stimulates transport of calcium-45 across the guinea-pig visceral yolk sac. Can. J. Physiol. Pharmacol. 62, 1466-1469. D51 Dho, S., Grinstein, S., Corey, E.J., Su, W.G. and Pace-Asciak, C.R. (1990) Hepoxilin A, induces changes in cytosolic calcium, intracellular pH and membrane potential in human neutrophils. Biochem. J. 266, 63-68.

7

8

C.R. Pace-Asciak/Biochimica et Biophysics Acta 1215 (1994) 1-8

[35] Pace-As&&,

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46] [47]

C.R. (1984a) Arachidonic acid epoxides. Demonstration through oxygen-18 labeled oxygen gas studies of an intramolecular transfer of the terminal hydroxyl group of l2.S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid to form hydroxy epoxides. J. Biol. Chem. 259, 8332-8337. Pace-As&k, C.R. (1984b) Hemoglobin- and hemin-catalyzed transformation of 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid. Biochim. Biophys. Acta 793, 485-488. Pace-Asciak, CR. (1986) Formation of hepoxilin A,, B, and the corresponding trioxilins from 12(S)-hydroperoxy-5,8,10,14,17icosapentaenoic acid. Prost. Leukot. Med. 22, l-9. Pace-As&k, C.R. (1988) Formation and metabolism of hepoxilin A, by the rat brain. Biochem. Biophys. Res. Commun. 151, 493498. Pace-Asciak, C.R., Granstrom, E. and Samuelsson, B. (1983) Arachidonic acid epoxides: Isolation and structure of 2 hydroxy epoxide intermediates in the formation of 8,11,12_trihydroxy eicosatrienoic acid and 10,11,12-trihydroxy eicosatrienoic acid. J. Biol. Chem. 258, 6835-6840. Pace-Asciak, C.R., Klein, J. and Spielberg, S.P. (1986a) Epoxide hydratase assay in human platelets using hepoxilin A, as a lipid substrate. Biochim. Biophys. Acta 875, 406-409. Pace-Asciak, C.R., Laneuville, O., Chang, M., Reddy, C.C., Su, W.-G. and Corey, E.J. (1989) New products in the hepoxilin pathway: isolation of 11-glutathionyl hepoxilin A, through reaction of hepoxilin A, with glutathione S-transferase. Biochem. Biophys. Res. Commun. 163, 1230-1234. Pace-As&k, C.R., Laneuville, O., Gurevich, N., Wu, P., Carlen, P.L. and Corey, E.J. (199Oa) Formation of a glutathionyl-conjugate of hepoxilin A, and its action in rat brain. Adv. Prost. Thromb. Leukot. Res. 21, 731-734. Pace-Asciak, C.R., Laneuville, O., Gurevich, N., Wu, P., Carlen, P.L., Su, W.-G., Corey, E.J., Chang, M. and Reddy, CC. (199Ob) Novel hepoxilin ghrtathione conjugates with biological activity, in Biological Oxidation Systems 2 (Reddy, C.C., Hamilton, G.A. and Kadyastha, K.M., eds.), pp. 725-735, Academic Press, New York. Pace-As&k, C.R., Laneuville, O., Su, W.-G., Corey, E.J., Gurevich, N., Wu, P. and Carlen, P.L. (1990~) A glutathione conjugate of hepoxilin A,: Formation and action in the rat central nervous system. Proc. Natl. Acad. Sci. USA 87, 3037-3041. Pace-As&k, C.R., Lee, S.P. and Martin, J.M. (1987) In vivo formation of hepoxilin A, in the rat. Biochem. Biophys. Res. Commun. 147, 881-884. Pace-Asciak, C.R. and Lee, W.-S. (1989) Purification of hepoxilin epoxide hydrolase from rat liver. J. Biol. Chem. 264, 9310-9313. Pace-Asciak, CR. and Martin, J.M. (1984) Hepoxilin, a new family of insulin secretagogues formed by intact rat pancreatic islets. Prost.

Leukot. Med. 16, 173-180. [48] Pace-As&k, C.R., Martin, J.M. and Corey, E.J. (1986b) Hepoxilins, potential endogenous mediators of insulin release. Prog. Lipid Res. 25, 625-628.

[49] Pace-Asciak, C.R., Martin, J.M., Corey, E.J. and Su, W.-G. (1985) Endogenous release of hepoxilin A, from isolated perifused pancreatic islets of Langerhans. Biochem. Biophys. Res. Commun. 128, 942-946. [50] Pace-Asciak, C.R., Reynaud, D. and Demin, P.M. (1993) Enzymatic formation of hepoxilin A, and B,. Biochem. Biophys. Res. Commun. 197, 869-873. [51] Pace-Asciak, CR., Wong, L. and Corey, E.J. (1990d) Hepoxilin A, blocks the release of norepinephrine from rat hippocampal slices. Biochem. Biophys. Res. Commun. 173, 949-953. [52] Piomelli, D., Feinmark, S.J. and Schwartz, J.H. (1987) 12-Hydroperoxyeicosatetraenoic acid (1ZHPETE) a lipoxygenase metabolite of arachidonic acid that produces presynaptic inhibition in Aplysia is metabolized to the hepoxilins A and B in neural tissue. 17th Annual Meeting Of The Society For Neuroscience, New Orleans, Louisiana, USA, November 16-21, Abstract #598. [53] Piomelli, D., Shapiro, E., Zipkin, R., Schwartz, J.H. and Feinmark, S.J. (1989) Formation and action of 8-hydroxy-11,12-epoxy-5,9,14icosatetraenoic acid in Aplysia: A possible second messenger in neurons. Proc. Natl. Acad. Sci. USA 86, 1721-1725. [54] Reynaud, D., Delton, l., Gharib, A., Sarda, N., Lagarde, M. and Pace-Asciak, CR. (1994) Formation, metabolism, and action of hepoxilin A, in the rat pineal gland. J. Neurochem. 62, 126-133. [55] Sakai, K., Fafeur, V., Vulliez-Le-Normand, B. and Dray, F. (1988) 12-Hydroperoxy-eicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineal. Prostaglandins 35, 969976. [56] Vasiljeva, L.L., Manukina, T.A., Demin, P.M., Lapitskaja, M.A. and Pivnitsky, K.K. (1993) Synthesis, properties, and identification of epimeric hepoxilins (-)-(lOR)-B, and (+)-(105)-B,. Tetrahedron 49, 4099-4106. [57] Willis, A.L. (1978) Platelet aggregation mechanisms and their implications in haemostasis and inflammatory disease, in Inflammation, handbook of experimental pharmacology (Ferreira, S.H. and Vane, J.R., eds.), pp. 138-205, Springer-Verlag, Berlin. [58] Wu, W.L. and Wu, Y.L. (1992) Synthesis of (lOR)-hepoxilin B, methyl ester and (lOR)-trioxilin B, methyl ester. J. Chem. Sot. Perkin Trans, 2705-2707. [59] Wu, W.L. and Wu, Y.L. (1993) A concise synthesis of the methyl esters of (lOS)-hepoxilin B, and (lOS)-trioxilin B,. J. Org. Chem. 58, 2760-2762. [60] Yadav, J.S., Chander, M.C. and Reddy, K.K. (1992) Stereoselective synthesis of lo(S), 11(R), 12(R)-trihydroxyeicosa-5Z,8Z,14Z-trienoic acid from D-mannose. Tetrahedron Lett. 33, 135-138. [61] Zurier, R.B. (1988) Prostaglandins and Inflammation, in Biology and Chemistry of Prostaglandins and Related Eicosanoids (CurtisPryor, P.B., ed.), pp. 595-607, Churchill Livingstone, New York. [62] Carlen, P.L., Gurevich, N., Wu, P.H., Su, W.-G., Corey, E.J. and Pace-Asciak, CR. (1989) Brain Res. 497, 171-176. [63] Carlen, P.L., Gurevich, N., Zhang, L., Wu, P.H., Reynaud, D. and Pace-Asciak, C.R. (1994) Neuroscience 58, 493-502.