8S-lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes.

To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor alpha (PPARalpha) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPARalpha, PPARgamma, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPARalpha.     

Biological role of hepoxilins: upregulation of phospholipid hydroperoxide glutathione peroxidase as a cellular response to oxidative stress?

The 12S-lipoxygenase (12S-LOX) pathway of arachidonic acid (AA) metabolism is bifurcated at 12(S)-hydroperoxy-5Z,8Z,10E (12S-HpETE) in the reduction route to form 12S-hydroxy-eicosatetraenoic acid (12S-HETE) and in 8(S/R)-hydroxy-11(S),12S-trans-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA3) synthase pathway, previously known as isomerization route, to form hepoxilins. Earlier we showed that the HXA3 formation is restricted to cellular systems devoid of hydroperoxide reducing enzymes, e.g. GPxs, thus causing a persistent oxidative stress situation. Here, we show that HXA3 at as low as 100 nM concentration upregulates phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA and protein expressions, whereas other metabolites of AA metabolism 12S-HpETE and 12S-HETE failed to stimulate the PHGPx. Moreover, the decrease in 12S-HpETE below a threshold value of the hydroperoxide tone causes both suppression of the overall 12S-LOX activity and a shift from HXA3 formation towards 12S-HETE formation. We therefore propose that under persistent oxidative stress the formation of HXA3 and the HXA3-induced upregulation of PHGPx constitute a compensatory defense response to protect the vitality and functionality of the cell.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, delta 6-trans-leukotriene B4, 12-epi-delta 6-trans-leukotriene B4, omega-hydroxy-leukotriene B4, omega-carboxy-leukotriene B4, 15S-hydroxyeicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to LTB4 on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The omega-hydroxy-LTB4 appeared more potent than LTB4 both for inducing a contraction and for releasing prostanoids whereas the omega-carboxy-LTB4 was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by FPL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB4 (and other hydroxy acids) actions on the guinea-pig lungs.     

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.     

Investigation of the mechanism of biosynthesis of 8-hydroxyeicosatetraenoic acid in mouse skin

One of the many changes induced by topical application of phorbol ester or calcium ionophore A23187 to mouse skin is the appearance of an enzymic activity which will convert arachidonic acid to its 8-hydroxyeicosatetraenoic acid metabolite (8-HETE) (Gschwendt, M., et al (1986) Carcinogenesis 7, 449-455). Induction of this activity is lower in strains of mice with a weak inflammatory response to TPA, and the 8-HETE may be involved in the inflammation or hyperplasia. To further characterize the activity, we first measured the chirality of the product; it is almost exclusively the 8DS)-hydroxy enantiomer (8S-HETE). The 8(S)-HETE is formed from octadeuterated arachidonic acid with complete retention of deuterium labels, indicating that a keto intermediate is not involved in the biosynthesis. Using arachidonic acids labeled with a prochiral tritium in either the 10DR or 10LS positions, we found that the biosynthesis of 8S-HETE is associated with the stereoselective abstraction of the 10DR hydrogen from the 10-carbon of the substrate. This stereoselective hydrogen removal conforms to the properties of an 8S-lipoxygenase. This is the only lipoxygenase known to catalyze solely 8S-oxygenation of arachidonic acid. The recent characterization of stereoselective biological effects for other HETEs serve as strong precedents to suggest that 8S-HETE has a specific role in the cellular tissue response to TPA.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: Myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, Δ⁶-trans-leukotriene B₄, 12-epi-Δ⁶-leukotriene B₄, ω-hydroxy-leukotriene B₄, ω-carboxy-leukotriene B₄, 15S-hydroxy-eicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to TLB₄ on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The ω-hydroxy-LTB₄ appeared more potent than LTB₄ both for inducing a contraction and for releasing prostanoids whereas the ω-carboxy-LTB₄ was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by PFL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB₄ (and other hydroxy acids) actions on the guinea-pig lungs.     

Cytochrome P450-type hydroxylation and epoxidation in a tyrosine-liganded hemoprotein, catalase-related allene oxide synthase

The ability of hemoproteins to catalyze epoxidation or hydroxylation reactions is usually associated with a cysteine as the proximal ligand to the heme, as in cytochrome P450 or nitric oxide synthase. Catalase-related allene oxide synthase (cAOS) from the coral Plexaura homomalla, like catalase itself, has tyrosine as the proximal heme ligand. Its natural reaction is to convert 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE) to an allene epoxide, a reaction activated by the ferric heme, forming product via the Fe(IV)-OH intermediate, Compound II. Here we oxidized cAOS to Compound I (Fe(V)=O) using the oxygen donor iodosylbenzene and investigated the catalytic competence of the enzyme. 8R-hydroxyeicosatetraenoic acid (8R-HETE), the hydroxy analog of the natural substrate, normally unreactive with cAOS, was thereby epoxidized stereospecifically on the 9,10 double bond to form 8R-hydroxy-9R,10R-trans-epoxy-eicosa-5Z,11Z,14Z-trienoic acid as the predominant product; the turnover was 1/s using 100 μm iodosylbenzene. The enantiomer, 8S-HETE, was epoxidized stereospecifically, although with less regiospecificity, and was hydroxylated on the 13- and 16-carbons. Arachidonic acid was converted to two major products, 8R-HETE and 8R,9S-eicosatrienoic acid (8R,9S-EET), plus other chiral monoepoxides and bis-allylic 10S-HETE. Linoleic acid was epoxidized, whereas stearic acid was not metabolized. We conclude that when cAOS is charged with an oxygen donor, it can act as a stereospecific monooxygenase. Our results indicate that in the tyrosine-liganded cAOS, a catalase-related hemoprotein in which a polyunsaturated fatty acid can enter the active site, the enzyme has the potential to mimic the activities of typical P450 epoxygenases and some capabilities of P450 hydroxylases.     

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.     

Regional biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids from arachidonic acid in the rat stomach tissue

The present study was conducted to determine regional differences in the biosynthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the rat stomach tissue (fundus, corpus and pyloric antrum) from radioactive arachidonic acid (AA). The radioactive metabolites were validated by RP-HPLC using non-radioactive AA as substrate. PGE(2) was the major prostanoid in the tissue(.) The relative ratio of PGE(2):PGF(2)alpha:PGD(2) in the whole stomach was 1:0.5:0.1. Regionally, the fundus biosynthesized the largest amount of all three cyclo-oxygenase products. Among the lipoxygenase metabolites, 15S-HETE was the predominant product, while 12S-HETE was found to be the lowest. The relative ratio of 15S-HETE:5S-HETE:12S-HETE in the whole stomach was 1:0.6:0.4. Interestingly, the generation of lipoxygenase products was the highest in the pyloric antrum when compared to fundus or corpus. Thus, the regional differences in the biosyntheses of gastric PGs and monohydroxy fatty acids may be relevant to our understanding of corresponding differences in mucosal resistance or susceptibility to gastric disease.     

Double dioxygenation by mouse 8S-lipoxygenase: specific formation of a potent peroxisome proliferator-activated receptor alpha agonist

Mouse 8S-lipoxygenase (8-LOX) metabolizes arachidonic acid (AA) specifically to 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE), which will be readily reduced under physiological circumstances to 8S-hydroxyeicosatetraenoic acid (8S-HETE), a natural agonist of peroxisome proliferator-activated receptor alpha (PPAR alpha). Here, we investigated whether 8-LOX could further oxygenate AA and whether the products could activate PPARs. The purified recombinant 8-LOX converted AA exclusively to 8S-HPETE and then to (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8S,15S-diHPETE). The kcat/Km values for 8S-HPETE and AA were 3.3x10(3) and 2.7x10(4) M(-1) s(-1), respectively. 8-LOX also dioxygenated 8S-HETE and 15S-H(P)ETE specifically to the corresponding 8S,15S-disubstituted derivatives. By contrast, 15-LOX-2, a human homologue of 8-LOX, produced 8S,15S-diH(P)ETE from 8S-H(P)ETE but not from AA nor 15S-H(P)ETE. 8S,15S-diHETE activated PPAR alpha more strongly than 8S-HETE did. The present results suggest that 8S,15S-diH(P)ETE as well as 8S-H(P)ETE would contribute to the physiological function of 8-LOX and also that 8-LOX can function as a potential 15-LOX.     

Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression.

The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.     

Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid.

Prostaglandin A2 is a major constituent of the gorgonian Plexaura homomalla, and there is evidence that its biosynthesis involves a noncyclooxygenase pathway. The coral contains an 8(R)-lipoxygenase and an allene oxide synthase; from arachidonic acid, the sequential action of these enzymes gives an allene epoxide, the cyclization of which forms an analogue of prostaglandin A2 (PGA2) with no 15-hydroxyl group. In this study we examined the metabolic fate of 15-hydroxyeicosatetraenoic acid (15-HETE), which via analogous reactions could lead to PGA2. The 8(R)-lipoxygenase metabolized preferentially the 15(R) enantiomer of 15-HETE, and this reaction was stimulated fivefold by including 1 M NaCl in the incubation. Further enzymic steps were detected by comparing the metabolic profiles of the 8(R)-hydroperoxy-15(R)-hydroxy intermediate with that of its 8(S),15(S) enantiomer. Two main products were formed exclusively from the 8(R),15(R) enantiomer: an allene epoxide and the comparatively stable epoxide, 8,9-epoxy-10,15-dihydroxyeicosa-5,11,14-trienoic acid. Formation of the allene oxide was inferred from detection of its hydrolysis and cyclization products. It cyclized to give two isomers of PGA2 which have a "cis" arrangement of the side chains. The main hydrolysis product (8,15-dihydroxy-9-ketoeicosa-5,11,13-trienoic acid) was unstable and prone to oxygenation, giving 8,14,15-trihydroxy-9-ketoeicosa-5,10,12-trienoic acids after reduction of the 14-hydroperoxide. We conclude that metabolism of a 15-hydroxy eicosanoid is a potential route to the A series prostaglandins, although the low yield and lack of stereochemical control suggest that this is not the natural pathway of biosynthesis in P. homomalla. Unexpectedly, the major end products of the pathway are trihydroxy ketols and the single diastereomer of a stable epoxyalcohol.     

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.     

Alterations of MCF-7 human breast cancer cell after prostaglandins PGA1 and PGF2 alpha treatment

Treatment of monolayer cultures of MCF-7 cells with prostaglandins PGA1 and PGF2 alpha inhibited cell proliferation, reduced the rate of labeled precursor incorporation into DNA, RNA, and protein, and induced morphological changes in a dose-dependent manner. The rate of [3H]thymidine incorporation was increased by PGA1 at 10(-10)-10(-8) M, while a sharp decrease was observed at 10(-6)-10(-4) M (p less than 0.05 and p less than 0.005). PGF2 alpha inhibited [3H]thymidine incorporation at all concentrations tested. Similar results were obtained for [3H]uridine incorporation with both PGs. PGA1 inhibited [3H]leucine incorporation at 10(-4) M, but increased incorporation at 10(-10)-10(-6) M. At the ultrastructural level, neither PG induced morphological alterations at 10(-12)-10(-8) M. However, at 10(-6)-10(-4) M both PGA1 and PGF2 alpha diminished the number and size of cell surface projections; some cells appeared to completely lack microvilli. Disorganization of mitochondrial cristae and increased electron density of the matrix were also evident.     

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B₂ production; ASA selectively affected TXB₂ production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB₂ while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y₁₂ blocker) affected platelet aggregation associated with profound inhibition of TXB₂ generation. P2Y₁₂ receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.     

Platelet-neutrophil interactions. 12S,20- and 5S,12S-dihydroxyeicosapentaenoic acids: two novel neutrophil metabolites from platelet-derived 12S-hydroxyeicosapentaenoic acid

Dietary marine n-3 polyunsaturated fatty acids have demonstrated an antiinflammatory potential in epidemiologic and intervention studies in humans. Proposed mechanisms, involving only leukocytes, fall short of explaining this potential completely. Enriched by dietary means with eicosapentaenoic acid (EPA), stimulated human platelets release substantial amounts of eicosapentaenoic acid and 12S-hydroxyeicosapentaenoic acid (12S-HEPE) in addition to 12S-hydroxyeicosatetraenoic acid (12S-HETE) derived from arachidonic acid. Human neutrophils metabolize 12S-HETE to 5S,12S-DiHETE when stimulated, whereas unstimulated neutrophils produce 12S,20-DiHETE. This study was undertaken to characterize metabolism of 12S-HEPE in human neutrophils. We demonstrate herein for the first time that 12S-HEPE is metabolized by human neutrophils. In unstimulated neutrophils 20-hydroxylation to 12S,20-DiHEPE occurs, whereas in stimulated neurtrophils 5-lipoxygenation to 5S,12S-DiHEPE takes place. The structures of these metabolites were characterized by their relative retention times on reversed-phase high pressure liquid chromatography, by their UV absorbance spectra, and by gas-liquid chromatography-mass spectrometry. With increasing amounts of 12S-HEPE, stimulated neutrophils produced increasing amounts of 5S,12S-DiHEPE, which is virtually inactive biologically. Concomitantly, production of the potent chemokinetic and chemoattractant arachidonic acid derivative leukotriene B4 decreased. Thus, 12S-HEPE can compete with endogenous arachidonic acid for 5-lipoxygenation in stimulated human neutrophils. 12,20-DiHEPE, LTB5, and 5S,12S-DiHEPE were detectable after coincubating EPA-enriched platelets with unenriched neutrophils, and arachidonic acid-derived 5-lipoxygenase products were decreased. We conclude that 12S-HEPE can participate in platelet-neutrophil interactions in a manner similar to 12S-HETE. By providing competing substrates for neutrophil 5-lipoxygenase, platelets might contribute to the antiinflammatory potential of dietary n-3 fatty acids through platelet-neutrophil interaction.     

Structure, biochemistry and biology of hepoxilins: an update

Hepoxilins are biologically relevant epoxy-hydroxy eicosanoids synthesized through the 12S-lipoxygenase (12S-LOX) pathway of the arachidonic acid (AA) metabolism. The pathway is bifurcated at the level of 12S-hydroperoxy-eicosatetraenoic acid (12S-HpETE), which can either be reduced to 12S-hydro-eicosatetraenoic acid (12S-HETE) or converted to hepoxilins. The present review gives an update on the biochemistry, biology and clinical aspects of hepoxilin-based drug development. The isolation, cloning and characterization of a rat leukocyte-type 12S-LOX from rat insulinoma RINm5F cells revealed a 12S-LOX possessing an intrinsic 8S/R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA(3)) synthase activity. Site-directed mutagenesis studies on rat 12S-LOX showed that the HXA(3) synthase activity was impaired when the positional specificity of AA was altered. Interestingly, amino acid Leu353, and not conventional sequence determinants Met419 and Ile418, was found to be a crucial sequence determinant for AA oxygenation. The regulation of HXA(3) formation is dependent on the cellular overall peroxide tone. Cellular glutathione peroxidases (cGPxs) compete with HXA(3) synthase for 12S-HpETE as substrate either to reduce to 12S-HETE or to convert to HXA(3), respectively. Therefore, RINm5F cells, which are devoid of GPxs, are capable of converting AA or 12S-HpETE to HXA(3) under basal conditions, whereas cells overexpressing cGPx are unable to do so. HXA(3) exhibits a myriad of biological effects, most of which are associated with the stimulation of intracellular calcium or the transport of calcium across the membrane. The activation of HXA(3)-G-protein-coupled receptors explains many of the extracellular effects of HXA(3), including AA- and diacylglycerol (DAG) release in human neutrophils, insulin secretion in rat pancreatic beta-cells or islets, and synaptic actions in the brain. The availability of stable analogs of HXA(3), termed 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid derivatives (PBTs), recently made several animal studies possible and explored the role of HXA(3) as a therapeutic in treatment of diseases. Thus, PBT-3 induced apoptosis in K562 tumour cells and inhibited growth of K562 CML solid tumours in nude mice. HXA(3) inhibited bleomycin-evoked lung fibrosis and inflammation in mice and the raised insulin level in the circulation of rats. At low glucose concentrations (0-3 mm), HXA(3) also stimulated insulin secretion in RINm5F cells through the activation of IRE1alpha, an endoplasmic reticulum-resident kinase. The latter regulates the protein folding for insulin biosynthesis. In conclusion, HXA(3)-mediated signaling may be involved in normal physiological functions, and hepoxilin-based drugs may serve as therapeutics in diseases such as type II diabetes and idiopathic lung fibrosis.     

Cartilage growth inhibition and necrosis in vitro caused by prostaglandin A

This paper details experiments using the Fell technique of organ culture of 8-day chick embryo femoral and tibial rudiments to test the effects of prostaglandin A1 (PGA1) on cartilage growth. Growth was studied during 8 days in vitro by measurement of rudiment length and wet and dry weight, and by histology. PGA1 inhibited explant growth in a dose-related manner. Linear growth was significantly decreased by 20 and 25 microgram/ml PGA1 at 2, 4, 6 and 8 days, and by 15 microgram/ml at 6 and 8 days. Linear growth was unaffected by 1 and 10 microgram/ml doses. Weight measurements were significantly reduced by 25 microgram/ml PGA1 (2, 4 and 8 days) and by 20 microgram/ml (8 days). Chondroblast degeneration was caused by doses of 15, 20 and 25 microgram/ml PGA1. Progressive degeneration was seen at the 25 microgram/ml concentration after 2 days in vitro. Cellular changes as early as 27 h in vitro were seen using electron microscopy. Tritiated thymidine autoradiography confirmed reduced chondroblast proliferation in the presence of PGA1 (25 microgram/ml). The mechanisms of prostaglandin-induced changes in embryonic cartilage remain uncertain. The possible role of intracellular cyclic nucleotides in the reaction is discussed.