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.     

Bacterial-induced hepoxilin A3 secretion as a pro-inflammatory mediator

Bacterial infections at epithelial surfaces, such as those that line the gut and the lung, stimulate the migration of neutrophils through the co-ordinated actions of chemoattractants secreted from pathogen-stimulated epithelial cells. One such factor involved in attracting polymorphonuclear leukocytes across the epithelium and into the lumen has until recently remained elusive. In 2004, we identified the eicosanoid, hepoxilin A(3), to be selectively secreted from the apical surface of human intestinal or lung epithelial cells stimulated with Salmonella enterica serotype Typhimurium or Pseudomonas aeruginosa, respectively. In this role, the function of hepoxilin A(3) is to guide neutrophils, via the establishment of a gradient, across the epithelial tight junction complex. Interestingly, interruption of the synthetic pathway of hepoxilin A(3) blocks the apical release of hepoxilin A(3)in vitro and the transmigration of neutrophils induced by S. typhimurium both in in vitro and in vivo models of inflammation. Such results have led to the discovery of a completely novel pathway that is not only critical for responses to bacterial pathogens but also has broad implications for inflammatory responses affecting mucosal surfaces in general. Thus, the objective of this review was to highlight the recent findings that implicate hepoxilin A(3) as a key regulator of mucosal inflammation.     

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.     

The mechanism of water transport in the epidermis

Possible mechanisms of water transport through Stratum Corneum (SC) in the process of insensible perspiration are investigated. Electrometric methods developed for measuring water flow density through SC are used. Data showing the reverse osmotic mechanism of water transport through the SC selective membrane are obtained. It is shown that the water flow density through SC controlling the evaporation rate from the skin surface in the process of insensible perspiration depends upon the skin capillary pressure.     

The role of lipoxygenases in epidermis

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The permeability barrier in mammalian epidermis

The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.     

The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity

Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3). 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.     

Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V_max of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N’-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH^−/− mice; and iv) liver homogenates of sEH^−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.     

Formation and metabolism of hepoxilin A3 by the rat brain

Incubation of homogenates of the rat cerebral cortex with arachidonic acid led to the appearance of hepoxilin A3, analysed as its stable trihydroxy derivative, trioxilin A3, by high resolution gas chromatography/electron impact mass spectrometry. Using the stable deuterium isotope dilution technique, it is estimated that the cerebral cortex generates 5.0 +/- 0.2 ng/mg protein of hepoxilin A3. The formation of this product was stimulated by the addition of exogenous arachidonic acid (12.9 +/- 1.5 ng/mg protein) and blocked by boiling of the tissue. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C at a concentration of 75 microM did not result in a blockade of hepoxilin formation. Three other regions were also tested for their ability to form hepoxilin A3 upon stimulation with exogenous arachidonic acid, i.e. median eminence, 11.7 +/- 1.6 ng/mg protein, pituitary, 12.3 +/- 0.7 ng/mg protein; pons, 26.6 +/- 0.2 ng/mg protein. In a separate study, 14C-labelled hepoxilin A3 was transformed into 14C-labelled trioxilin A3 by homogenates of the rat whole brain, demonstrating the presence of epoxide hydrolases in the CNS which utilise the hepoxilins as substrates. This is the first demonstration of the occurrence of the hepoxilin pathway in the central nervous system.     

The epidermis as a graft

Laboratory epidermal autotransplantation was performed on the surface of a full-thickness skin defect using mongrel female rats. Epidermal graft represented suction blister roofs, formed as the result of the donor skin site treatment with lowered up to -0.6 kg/cm2 pressure. It contained all epidermal cell layers. Following 1, 7 and 28 days after the transplantation recipient bed sites containing grafted epidermis were excised and histological study war performed. It was demonstrated that epidermal graft received by the method described was able to grow as well as to differentiate on the surface of a full-thickness skin defect.     

The choice of cell fate in the epidermis of Drosophila

In Drosophila, neural precursors are formed in a spaced pattern separated by intervening epidermal cells. Segregation of neural and epidermal lineages relies on cellular interactions. Failure of this cell communication, as in the mutants Notch (N), Delta, and shaggy, results in most or all of the cells becoming neural. Cells mutant for N and shaggy, but not Delta, autonomously adopt the neural fate when adjacent to wild-type cells in mosaics. Furthermore, wild-type cells adopt the epidermal fate if adjacent cells express a lower level of N activity than themselves, but produce neural precursors if adjacent cells express a higher level of N activity. This shows that there is competition between the cells and that the N protein is required for the mechanism whereby the cells choose between alternative fates. It also suggests that N acts as a receptor for an inhibitory signal emanating from the neural precursors.     

The fibrillin-marfan syndrome connection

A few years ago no one would have suspected that the well-known disorder of connective tissue, Marfan syndrome, could be caused by mutations in a recently discovered extracellular component, fibrillin. Likewise, nobody would have predicted that fibrillin represents a small family of proteins that are associated with several pheno-typically overlapping disorders. The fibrillins are integral constituents of the non-collagenous microfibrils, with an average diameter of 10 nm. These aggregates are distributed in the extracellular matrix of virtually every tissue. Microfibrillar bundles provide the external coating to elastin in elastic fibers, and serve an anchoring function in non-elastic tissues. At higher resolution, individual microfibrils have a “beads-on-a-string” appearance resulting from the head-to-tail polymerization of multiple fibrillin aggregates. Structurally, fibrillin contains a series of repeated sequences homologous to the epidermal growth factor calcium-binding motif. Characterization of fibrillin mutations in Marfan syndrome patients, together with the elucidation of the structure of the fibrillin proteins, have provided new insights, and raised new questions, about the function of the 10 nm microfibrils. For example, it is possible that the fibrillins, in addition to serving a structural function, might also be involved in regulating cellular activities and morphogenetic programs. It is fitting that the long search for the Marfan syndrome gene has brought a novel group of proteins to the forefront of extracellular matrix biology.