Hepoxilin analogs, potential new therapeutics in disease.

We have chemically synthesized several stable analogs of the naturally occurring hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

Effect of 8-isoprostaglandin F2alpha on the newborn rat pulmonary arterial muscle and endothelium.

8-Isoprostaglandin F2alpha (8-iso-PGF2alpha) is a bioactive lipid peroxidation product that is a vasoconstrictor at high concentrations. Paradoxically, at lower, and possibly physiological, concentrations, it is a pulmonary vascular muscle's relaxant. Its effects on newborn pulmonary vasculature are unknown. We hypothesized that the pulmonary arterial 8-iso-PGF2alpha responses may be developmentally regulated. Therefore, the purpose of this study was to evaluate and compare 8-iso-PGF2alpha effects between 1- and 2-wk-old newborn and adult rat isolated intrapulmonary arteries (100 microm) mounted on a myograph. Force after 8-iso-PGF2alpha stimulation was greatest in the adult (P < 0.01). In newborns, force was significantly increased by the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) (P < 0.01) and was suppressed by blockade of the thromboxane (Tx) A2 receptor. Whereas 8-iso-PGF2alpha induced a significant dose-dependent relaxation of adult precontracted vessels in the presence of a TxA2 mimetic (U-46619; 1 microM), contraction was observed in the 1-wk-old rat. This 8-iso-PGF2alpha-induced contraction was abolished by endothelium removal and l-NAME and was attenuated by the cyclooxygenase inhibitor ibuprofen. In the presence of a TxA2/prostaglandin H2 receptor blocker, 8-iso-PGF2alpha induced NO-mediated relaxation, the magnitude of which was greater in the newborn, compared with the adult (P < 0.01). When exposed to 8-iso-PGF2alpha in vitro, only the newborn lung secreted TxB2. We conclude that, in contrast to its relaxant effect in the adult, 8-iso-PGF2alpha induces contraction of the pulmonary arteries in the early postnatal period, which is likely to be mediated by endothelium-derived TxA2. This phenomenon may contribute to the maintenance of a higher pulmonary vascular resistance in the early postnatal period.

A lipoxygenase product, hepoxilin A(3), enhances nerve growth factor-dependent neurite regeneration post-axotomy in rat superior cervical ganglion neurons in vitro.

Hepoxilins are 12-lipoxygenase metabolites of arachidonic acid found in the CNS. They can modulate neuronal signaling but their functions are not known. We examined the effects of hepoxilin A(3) on neurite outgrowth post-axotomy in an in vitro model of spinal cord transection using superior cervical ganglion neurons. In the absence of nerve growth factor, hepoxilin A(3) did not support neuronal survival, or regeneration post-axotomy but did significantly enhance neurite regeneration in the presence of nerve growth factor. As early as 1 h post-injury hepoxilin A(3)-treated cultures (+nerve growth factor) had significantly more neurites than controls (nerve growth factor alone). Average hourly rates of outgrowth in hepoxilin A(3)-treated cultures were significantly higher than in controls for at least 12 h post-injury, suggesting that the effect of hepoxilin A(3) is maintained in vitro for several hours post-injury. In uninjured neurons hepoxilin A(3) caused a rapid but transient increase in intracellular calcium in the somata; by 2 min post-addition, calcium levels decreased to a new stable plateau significantly higher than pre treatment levels. In injured neurons, hepoxilin A(3) addition immediately post-transection caused a rapid transient increase in intracellular calcium in cell bodies; however, peak calcium levels were significantly lower than in uninjured neurons and the new baseline lower than in uninjured cells. In uninjured cells hepoxilin A(3) addition in zero calcium produced the same pattern, a transient elevation and subsequent decline to a new stable baseline significantly above rest but in injured cells levels fell rapidly to pretreatment values. Taken overall, these findings demonstrate a novel role for hepoxilins as a potentiator of neurite regeneration. They also provide the first evidence that this lipoxygenase metabolite can alter intracellular calcium in neurons by causing release of calcium from intracellular stores and modulating calcium influx mechanisms.

The hepoxilin analog PBT-3 inhibits heparin-activated platelet aggregation evoked by ADP.

We have previously shown that PBT-3, a stable synthetic analog of hepoxilins, inhibits the aggregation of human platelets in vitro evoked by collagen through inhibition of thromboxane A(2) formation and action on the TP receptor. We now show that PBT-3 is capable of potently inhibiting the second phase of aggregation evoked by ADP in both washed human platelets and platelet-rich plasma (PRP), a phase associated with thromboxane formation. Aspirin blocks this second phase as well; so does the thromboxane receptor antagonist SQ 29,548. When ADP-evoked aggregation in PRP is activated by heparin through an enhancement of thromboxane formation, PBT-3, aspirin as well as SQ 29,548 block this activation through different mechanisms. These data confirm the inhibitory action of PBT-3 on aggregation of human platelets through inhibition of both thromboxane formation and blockade of thromboxane receptor action and suggest that this family of compounds may be useful in the treatment of thrombotic disorders in combination with heparin.

Novel platelet antiaggregating substances.

We describe herein a novel action of four stable analogs of the hepoxilins. These analogs inhibit to different degrees, the aggregation of washed human platelets evoked by collagen. One of the analogs, PBT-3, is particularly effective with an IC(50) = 8 x 10(-7) M. The other analogs are about 5-fold less active, but all analogs are about 500-fold more active than the native hepoxilins. The PBT analogs inhibit the collagen-enhanced formation of thromboxane A(2) and HHT but do not affect the formation of 12-HETE or the release of arachidonic acid except at doses higher than those needed to block platelet aggregation. These results demonstrate that these novel compounds may have potential for development into drugs in the treatment of thromboxane-mediated cardiovascular disease.

Study of binding of 12(S)-hydroxy-5(Z),8(Z),10(E), 14(Z)-eicosatetraenoic acid to bovine serum albumin using dynamic surface tension measurements.

In a recent paper,(1) we demonstrated that molecular interactions between biopolymers and other smaller molecules can be detected by means of dynamic surface tension measurements. In the present paper, we demonstrate that the same methodology can be employed for investigating dose effects and specificity of molecular interactions. Three similar lipids were chosen for this study: 12(S)-hydroxy-5(Z), 8(Z),10(E),14(Z)-eicosatetraenoic acid (12(S)-HETE-free acid), methyl 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoate (12(S)-HETE-methyl ester), and 5(Z),8(Z),11(Z), 14(Z)-eicosatetraenoic acid (arachidonic acid-free acid). These substances were added to a fatty acid free bovine serum albumin (BSA) aqueous solution at different lipid concentrations. The characteristic tension response indicates that molecular interactions between 12(S)-HETE-free acid and BSA exist. The detected interactions are concentration dependent: at a molecular ratio of lipid to protein of 1:1, the binding of 12(S)-HETE-free acid to BSA is hydrophobic in nature; at the molecular ratio of lipid to protein of 10:1, a secondary binding occurs and is hydrophilic in nature. Similar molecular interactions were not detected between 12(S)-HETE-methyl ester or arachidonic acid-free acid and BSA, indicating that the interactions between 12(S)-HETE-free acid and BSA are specific. As an independent means, surface elasticity is used to probe the molecular interactions at the interface. In the case of 12(S)-HETE-free acid but not its methyl ester or arachidonic acid, distinct higher surface elasticities were observed at lipid concentrations in excess of a molecular ratio of lipid to protein of 1:1. This finding reinforces the above stipulations.

Stereoselective actions of hepoxilins A3 and B3 and their cyclopropane analogs (HxdeltaA3 and HxdeltaB3) on bradykinin and PAF-evoked potentiation of vascular leakage in rat skin.

Native hepoxilins (Hx) A3 and B3 as well as their synthetic cyclopropane analogs, HxdeltaA3 and HxdeltaB3 are inactive on their own in causing changes in vascular permeability in rat skin measured by leakage of plasma-bound Evans Blue dye. Several of these compounds, however, were observed to potentiate the leakage of dye evoked by bradykinin (BK) and platelet-activating factor (PAF). The syn epimer of HxA3 was effective in potentiating dye leakage evoked by BK but not by PAF. The syn epimer of HxB3, on the other hand, was capable of potentiating both BK- and PAF-evoked plasma protein leakage. The anti epimer of both hepoxilins was inactive. In contrast, the anti epimer of the cyclopropane analog HxdeltaA3 potentiated only the BK-evoked changes, whereas the anti epimer of HxdeltaB3 potentiated only the PAF-evoked changes in dye leakage. The corresponding other epimer of each compound was inactive. Our findings indicate that the hepoxilin cyclopropane analogs appear to mimic the actions of the native compounds.

Hepoxilins raise circulating insulin levels in vivo.

We have demonstrated over a decade ago that hepoxilins cause the release of insulin from isolated pancreatic islets of Langerhans in vitro. However, no studies are available so far to indicate whether these compounds are active in vivo. The present study is the first to our knowledge which demonstrates that hepoxilins administered intra-arterially in the anaesthetized rat cause the release of insulin in the circulation. This release is dependent on the glucose status of the rat. Hence, animals fasted overnight do not respond to hepoxilin administration, while animals that have had free access to food respond to hepoxilins with a rise in insulin concentrations in blood. The hepoxilin effect is rapid and varies with different hepoxilins, the most potent of which is hepoxilin A(3) (HxA(3)) (both the 8S and the 8R enantiomers). Administration of 100 microg HxA(3) produces a rise in blood insulin equivalent to that caused by the administration of 5 mg glucose. In view of earlier evidence showing that these compounds cause a rise in intracellular calcium levels in vitro at a <1 microg/ml concentration through a receptor-mediated mechanism, we speculate that the actions of hepoxilins in causing the release of insulin from the pancreas may be due to alterations in calcium levels within the beta-cell. We believe that hepoxilins may represent new lead compounds as therapeutics in type II diabetes mellitus.

Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.

Biochemical, hemodynamic, and vascular evidence concerning the free radical hypothesis of nitrate tolerance.

Tolerance to nitroglycerin (NTG) may be due to increased superoxide anion production. Hemodynamic parameters and biochemical markers of free radical production were measured in 20 healthy male subjects at baseline, 3 h after acute transdermal NTG (0.6 mg/h), and after 5 days of continuous therapy. Transdermal NTG therapy was continued, and 2 days later all subjects received 2 g of oral vitamin C, or placebo, in a double-blind, randomized, crossover fashion. In another study of eight male subjects, forearm plethysmography was used to assess the venous responses to sublingual NTG at baseline, after 5 days of sustained transdermal NTG therapy (0.6 mg/h), and after 2 g of oral vitamin C or placebo. Systolic blood pressure decreased in response to acute transdermal NTG therapy but returned to normal after sustained NTG therapy, indicating the development of tolerance. The venous volume responses to sublingual NTG were significantly diminished after sustained therapy with transdermal NTG. Plasma lipid peroxidation products, 8-iso-PGF2 alpha, and vitamin C were unchanged by acute and sustained therapy with transdermal NTG. Vitamin C failed to restore either the hemodynamic or venous effects of NTG. These results do not support the hypothesis that nitrate therapy and tolerance is associated with increased free radical production.

Hepoxilin signaling in intact human neutrophils: biphasic elevation of intracellular calcium by unesterified hepoxilin A3.

We have previously shown that the methyl ester of hepoxilin A3 causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A3 is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188+/-14 S.D., free acid = 135+/-11 S.D. nM, P < 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88+/-8 S.D., free acid = 107+/-15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calcium-free medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189+/-12; free acid, 145+/-6 S.D. nM, P<0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A3 demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.

The hepoxilins. A review.

This manuscript reviews the literature on hepoxilins to date. It affords a review of the structures, nomenclature, biosynthesis, catabolism and biological actions of the hepoxilins and some of their chemical analogs. Some unpublished data are also presented. The primary biological action of the hepoxilins appears to relate to their ability to release calcium from intracellular stores through a receptor-mediated action. The receptor is intracellular, and appears to be G-protein coupled. The conversion of hepoxilin into its omega-hydroxy catabolite has recently been demonstrated through the action of an omega-hydroxylase. This enzyme is different from that which oxidizes leukotriene B4, as the former activity is lost when the cell is disrupted, while leukotriene B4-catabolic activity is recovered in both the intact and disrupted cell. Additionally, hepoxilin catabolism is inhibited by CCCP, a mitochondrial uncoupler, while leukotriene catabolism is unaffected. As hepoxilins cause the translocation of calcium from intracellular stores in the endoplasmic reticulum to the mitochondria, it is speculated that hepoxilin omega-oxidation takes place in the mitochondria, and the omega-oxidation product facilitates accumulation of the elevated cytosolic calcium by the mitochondria. The biological activity of stable analogs of the hepoxilins is also described which inhibit the calcium-releasing actions of neutrophil inflammatory mediators.

In vivo stimulation of 12(S)-lipoxygenase in the rat skin by bradykinin and platelet activating factor: formation of 12(S)-HETE and hepoxilins, and actions on vascular permeability.

In this study we set out to investigate whether the inflammatory agents, bradykinin (BK) and platelet activating factor (PAF), affect the lipoxygenase pathway in rat skin in vivo and whether the main products so formed may be involved in the inflammatory actions of these agents. In vitro preparations of epidermis were also investigated to determine whether lipoxygenases are stimulated by these agents. We also investigated the actions of arachidonic acid and 12(S)-HPETE as substrates for the lipoxygenases. Our results indicated that 12-lipoxygenase is actively and selectively stimulated in a dose-dependent way in both preparations by the administration of BK and PAF; the main product, 12-HETE, was shown by chiral analysis to be exclusively of the S-configuration, indicating that 12(S)-lipoxygenase was present in the rat skin and was stimulated by these inflammatory agents. Hepoxilins were also formed but to a lesser extent in both in vivo and in vitro preparations. In separate experiments, 12(S)-HETE administered intradermally on its own (40 ng/site), increased vascular permeability as also seen with bradykinin (100 ng/site) and PAF (10 ng/site). However, unlike previously observed with hepoxilin A3 administration, 12(S)-HETE did not stimulate the action of BK on vascular permeability, suggesting that the two compounds may have different mechanisms of action to enhance inflammation. These observations suggest that the vascular permeability and plasma extravasation observed with both inflammatory agents (BK and PAF) may be mediated at least in part through the activation of 12(S)-lipoxygenase, resulting in enhanced formation of 12(S)-HETE which causes acute inflammation.

Detection of 8-epi prostaglandin F2alpha in an extract of epidermal secretion of the catfish from the Arabian Gulf.

We provide herein mass spectrometric evidence for the existence of F2 isoprostanes in an epidermal secretion of the catfish from the Arabian Gulf. The compounds were extracted, purified by thin layer chromatography (TLC) and derivatized into the pentafluorobenzyl trimethylsilyl (PFB-TMSi) derivatives and quantified using SIM-negative ion gas chromatography-mass spectrometry based on the addition of tetradeuterated 8-epi PGF2alpha as internal standard prior to sample workup. Freshly collected secretions from the male catfish contained 5.63 and 4.17 ng/ml (two determinations) and from the female catfish 3.60 and 3.81 ng/ml (two determinations) of free 8-epi PGF2alpha. The level of these compounds increases approximately 8-fold upon storage of the extract at -20 degrees. In view of the potent reported actions of F2 isoprostanes on vasoconstriction and as thromboxane receptor antagonists, the presence of isoprostanes in the catfish secretions may contribute to the reported ulcer healing properties of these secretions.

Hepoxilin A3 is oxidized by human neutrophils into its omega-hydroxy metabolite by an activity independent of LTB4 omega-hydroxylase.

Hepoxilin A3-methyl ester is taken up by intact human neutrophils where it is first hydrolyzed into the free acid which is subsequently converted into a single major metabolite. The structure of this metabolite was determined through mass spectral analysis of several derivatives, and through identity with an authentic compound prepared by chemical synthesis. The metabolite was identified as omega-hydroxy-hepoxilin A3 showing that the epoxide functionality of the parent hepoxilin is not opened during incubation with human neutrophils. All attempts to investigate hepoxilin metabolism in broken cells, despite the presence of protease inhibitors (Aproteinin, PMSF, DFP) and supplementation with NADPH were unsuccessful. Metabolism of hepoxilin A3 required the intact cell, while parallel experiments with LTB4 as substrate demonstrated that this eicosanoid was metabolized into its omega-hydroxy metabolite regardless of whether intact or broken cell preparations were used provided that NADPH was present in the latter. Hepoxilin metabolism in intact cells was inhibited dose-dependently by CCCP (0.01-100 microM), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A3 occurs in intact human neutrophils through omega-oxidation, is likely located in the mitochondrial compartment of the cell (inhibition by CCCP) and is carried out by an activity that is independent of the well characterized, relatively stable microsomal LTB4 omega-hydroxylase.

Docosahexaenoic acid causes accumulation of free arachidonic acid in rat pineal gland and hippocampus to form hepoxilins from both substrates.

Hepoxilins (Hx) are biologically active metabolites of arachidonic acid (AA) formed regioselectively from 12(S)-HPETE by 'hepoxilin synthase'. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the course of our studies we investigated whether docosahexaenoic acid (DHA) was a substrate for hepoxilin formation. We used two tissues, the pineal gland and hippocampal slices. Tissues were incubated alone or with AA (20 microg/ml) or DHA (20 microg/ml). After 60 min at 37 degrees C, samples were acid-extracted to convert Hx into their stable trioxilin (TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to determine the structures of the DHA metabolites, and as PFB-TMSi derivatives by GC/MS in the NICI mode using SIM to simultaneously quantify TrX products of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593. Results show good conversion of both substrate fatty acids by the rat pineal gland and hippocampal slices, into the 3-series (21.3 +/- 5.8 and 12.5 +/- 2.2 ng/microg protein, respectively) and 5-series TrX (12.3 +/- 2.7 and 2.9 +/- 0.4 ng/microg protein, respectively). Surprisingly though, experiments with DHA, in both tissues, also showed formation of TrX derived from endogenous AA (3-series) (10.4 +/- 8.3 and 3.1 +/- 2.1 ng/microg protein, respectively). These experiments demonstrate previously unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated during DHA stimulation of the tissue, we carried out separate experiments with hippocampal slices in which the neutral lipids and phospholipids were labeled with [14C]AA. DHA caused a time-dependent appearance of free [14C]AA which was released mostly from the TG pool. Measurement of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results demonstrate that DHA competes with AA for acylation into the metabolically active TG fraction, and both fatty acids are converted into hepoxilins of the corresponding series.

Hepoxilin-evoked intracellular reorganization of calcium in human neutrophils: a confocal microscopy study.

Hepoxilin A3 has previously been shown to cause a rapid dose-dependent rise in intracellular calcium in intact human neutrophils in suspension. Two components have been observed, an initial rapid phase of intracellular calcium rise, followed by a slow decline to plateau levels that remain above the original baseline calcium levels. These changes have been suggested to involve the release of calcium from intracellular stores in the ER (initial rapid phase), while the slower rate of decline (plateau phase) was presumed to be due to calcium influx as it was abolished in zero calcium extracellular medium. The present study used confocal microscopy to examine the response to hepoxilin A3 at the subcellular level. Our results show that calcium dynamics in response to hepoxilin A3 varies in different subcellular compartments within the cell and that hepoxilin A3 evoked a persistent accumulation of calcium in organelles. The hepoxilin-evoked calcium sequestration was eliminated by prior exposure to CCCP, a mitochondrial uncoupler. CCCP also eliminated the plateau phase of the calcium response in cell suspension, suggesting that this phase was due to mitochondrial accumulation of calcium rather than calcium influx. Experiments with DiI-loaded cells, a membrane marker, showed that the nuclear calcium was not elevated by hepoxilin addition to the cells. These results demonstrate that hepoxilins evoke the release of calcium from the ER which is taken up by the mitochondria where it is tightly sequestered. These results offer an explanation of observations previously made with cell suspensions in which hepoxilin A3 was shown to inhibit the calcium mobilizing effects of chemotactic agents.

12-HETE and 12-HPETE potently stimulate intracellular release of calcium in intact human neutrophils.

We describe here the effects of two 12-lipoxygenase products, 12-HETE (12-hydroxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid) and 12-HPETE (12-hydroperoxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid), on the release of intracellular calcium in intact human neutrophils using the INDO-1 AM fluorescent dye technique. Both products dose dependently stimulate intracellular release, with 12-HETE being more powerful than 12-HPETE. The threshold concentration for 12-HETE was 5 ng/ml (1.5 x 10-8 M), while that for 12-HPETE was 10 ng/ml. The (12S) regioisomer was slightly more active than the (12R) isomer. The laser potency of 12-HPETE may be due to its conversion into the less active hepoxilins as incubation of neutrophils with (12S/R)-HPETE in a nonradioactive assay, using fluorescent ADAM esters of the products, generated mostly hepoxilin A3 (8-hydroxy-(11S,12S) epoxyeicosa (5Z,9E,14Z)trienoic acid), indicative of an enzymatic process. In contrast, boiled neutrophil preparations converted 12-HPETE primarily into hepoxilin B3 which previously showed to be derived nonenzymatically. This data demonstrates that 12-HETE, known to be generated in significant amounts by platelets, can act transcellularly to modify intracellular concentrations of calcium in neutrophils. This may in turn affect the responsiveness of these cells to other chemotactic factors.