12-Hydroxyheptadecatrienoic acid
12-Hydroxyheptadecatrenoic acid -hydroxyheptadeca-5Z,8E,10E-trienoic acid, or 12 is a 17 carbon metabolite of the 20 carbon polyunsaturated fatty acid, arachidonic acid. It was first detected and structurally defined by P. Wlodawer, Bengt I. Samuelsson, and M. Hamberg as a product of arachidonic acid metabolism made by microsomes isolated from sheep seminal vesicle glands and by intact human platelets. 12-HHT is less ambiguously termed 12--hydroxy-5Z,8E,10E-heptadecatrienoic acid to indicate the S stereoisomerism of its 12-hydroxyl residue and the Z, E, and E cis-trans isomerism of its three double bonds. The metabolite was for many years thought to be merely a biologically inactive byproduct of prostaglandin synthesis. More recent studies, however, have attached potentially important activity to it.
Production
Primary source
and cyclooxygenase-2 metabolize arachidonic acid to the 15-hydroperoxy, 20 carbon prostaglandin intermediate, PGG2, and then to the 15-hydroxy, 20 carbon intermediate, prostaglandin H2. Thromboxane synthase further metabolizes PGH2 to the 20 carbon product, Thromboxane A2, the 17 carbon product, 12-HHT, and the 3 carbon product Malonyldialdehyde. Platelets express cycloxygenase and thromboxane synthase enzymes, producing PGG2, PGH2, and TXA2 in response to platelet aggregating agents such as thrombin; these metabolites act as autocrines by feeding back to promote further aggregation of their cells of origin and as paracrines by recruiting nearby platlets into the response as well as exerting effects on other nearby tissues such as contracting blood vessels. These effects combine to trigger blood clotting and limiting blood loss. 12-HHT is a particularly abundant product of these pro-clotting responses, accounting for about one third of the total amount of arachidonic acid metabolites formed by physiologically stimulated human platelets. Its abundant production during blood clotting, the presence of cyclooxygenases and to a lesser extent thromboxane synthase in a wide range of cell types and tissue, and its production by other pathways imply that 12-HHT has one or more important bioactivities relevant to clotting and, perhaps, other responses.Further metabolism
12-HHT is further metabolized by 15-hydroxyprostaglandin dehydrogenase in a wide variety of human and other vertebrate cells to its 12-oxo derivative, 12-oxo-5Z,8E,10E-heptadecatrienoic acid. Pig kidney tissue also converted 12-HHT to 12-keto-5Z,8E-heptadecadienoic acid and 12-hydroxy-heptadecadienoic acid.Acidic conditions cause 12-HHT to rearrange in a time- and temperature-dependent process to its 5-cis isomer, 12-hydroxy-5E,8E,10E-heptadecatrienoic acid.
Activities and clinical significance
Early studies
Fourteen years after the first publication on its detection in 1973, 12-HHT was reported to stimulate fetal bovine aortic and human umbilical vein endothelial cells to metabolize arachidonic acid to Prostacyclin I2, a powerful inhibitor of platelet activation and stimulator of Vasodilation ; it did not, however, alter arachidonic acid metabolism in human platelets. Shortly thereafter, 12-HHT was reported to inhibit the chemotaxis-blocking effect of a human monocyte-derived factor on human moncytes while the immediate metabolite of 12-HHT, 12-oxo-HT, was reported to stimulate the chemotasis of human neutrophils. and to inhibit platelet aggregation responses to various agents by stimulating platelets to raise their levels of Cyclic adenosine monophosphate, an intracellular signal that serves broadly to inhibit platelet activation. These studies were largely overlooked; in 1998 and 2007 publications, for example, 12-HHT was regarded as either inactive or without significant biological activity. Nonetheless, this early work suggested that 12-HHT may serve as a contributor to monocyte- and neutrophil-based inflammatory responses and 12-oxo-HT may serve as a counterpoise to platelet aggregation responses elicited or promoted by TXA2. Relevant to the latter activity, a later study showed that this inhibitory effect was due to the ability of 12-oxo-HT to act as a partial antagonist of the Thromboxane receptor: it blocks TXA2 binding to its receptor and thereby the responses of platelets and possibly other tissues to TXA2 as well as agents that depend on stimulating TXA2 production for their activity. Thus, 12-HHT forms simultaneously with, and by stimulating PGI2 production, inhibits TXA2-mediated platelet activation responses while 12-oxo-HT blocks TXA2 receptor binding to reduce not only TXA2-induced thrombosis and blood clotting but possibly also vasospasm and other actions of TXA2. In this view, thromboxane synthase leads to the production of a broadly active arachidonic acid metabolite, TXA2, plus two other arachidonic acid metabolites, 12-HHT and 12-oxo-HT, that serve indirectly to stimulate PGI2 production or directly as a receptor antagonist to moderate TXA2's action, respectively. This strategy may be essential for limiting the deleterious thrombotic and vasospastic activities of TXA2.12-HHT is a BLT2 receptor agonist
is an arachidonic acid metabolite made by the 5-lipoxygenase enzyme pathway. It activates cells through both its high affinity Leukotriene B4 receptor 1 and its low affinity BLT2 receptor ; both receptors are G protein coupled receptors that, when ligand-bound, activate cells by releasing the Gq alpha subunit and pertussis toxin-sensitive Gi alpha subunit from Heterotrimeric G proteins. BLT1 receptor has a high degree of ligand-binding specificity: among a series of hydroxylated eicosanoid metabolites of arachidonic acid, it binds only LTB4, 20-hydroxy-LTB4, and 12-epi-LTB4; among this same series, BLT2 receptor has far less specificity in that it binds not only LTB4, 20-hydroxy-LTB4, and 12-epi-LTB4 but also 12-HETE and 12-HETE and 15-HpETE and 15-HETE. The BLT2 receptor's relative affinities for finding LTB4, 12-HETE, 12-HpETE, 12-HETE, 15-HETE, and 20-hydroxy-LTB4 are ~100, 10, 10, 3, 3, and 1, respectively. All of these binding affinities are considered to be low and therefore indicating that some unknown ligand might bind BLT2 with high affinity. In 2009, 12-HHT was found to bind to the BLT2 receptor with ~10-fold higher affinity than LTB4; 12-HHT did not bind to the BLT1 receptor. Thus, the BLT1 receptor exhibits exquisite specificity, binding 5,12-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid but not LTB4's 12 or 6Z isomers while the BLT2 receptor exhibits a promiscuous finding pattern. Formyl peptide receptor 2 is a relevant and well-studied example of promiscuous receptors. Initially thought to be a second and low affinity receptor for the neutrophil tripeptide chemotactic factor, N-formyl-met-leu-phe, subsequent studies showed that it was a high affinity receptor for the arachidonic acid metabolite, lipoxin A4, but also bound and was activated by a wide range of peptides, proteins, and other agents. BLT2 may ultimately prove to have binding specificity for a similarly broad range of agents.The production of LTB4 and expression of BLT1 by human tissues are largely limited to bone marrow-derived cells such as the neutrophil, eosinophil, mast cell, and various types of lymphocytes and accordingly are regarded primarily as contributing to the many human defensive and pathological inflammatory responses which are mediated by these cell types. Drugs that inhibit LTB4 production or binding to BLT1 are in use or development for the latter diseases. In contrast, the production of 12-HH2 and expression of BLT2 receptors by human tissues is far wider and more robust than that of the LTB4/BLT2 receptor axis. Recent studies indicate that the role of the 12-HHT/BLT2 receptor axis in human physiology and pathology may be very different than those of the LTB4/BLT1 axis.
Recent studies on 12-HHT/BLT2 receptor activities
Inflammation and allergy
12-HHT stimulates chemotactic responses in mouse bone marrow mast cells, which naturally express BLT2 receptors, as well as in Chinese hamster ovary cells made to express these receptors by transfection. These findings suggest that the 12-HHT/BLT2 receptor pathway may support the pro-inflammatory actions of the LTB4/BLT1 pathway.On the other hand, the immortalized human skin cell line HaCaT expresses BLT2 receptors and responds to ultraviolet B radiation by generating toxic reactive oxygen species which in turn cause the HaCaT cells to die by activating apoptotic pathways in a BLT2 receptor-dependent reaction. Topical treatment of mouse skin with a BLT2 receptor antagonist, LY255283, protects against UVB radiation-induced apoptosis and BLT2-overexpressing transgenic mice exhibited significantly more extensive skin apoptosis in response to UVB irradiation. Furthermore, 12-HHT inhibits HaCaT cells from synthesizing interleukin-6, a pro-inflammatory cytokine associated with cutaneous inflammation, in response to UVB radiation. These results suggest that the 12-HHT/BLT2 axis can act to suppress inflammation by promoting the orderly death of damaged cells and blocking IL-6 production. Opposition between the pro-inflammatory LTB4/BLT1 and anti-inflammatory actions of the 12-HHT/BLT2 axes occurs in another setting. In a mice model of ovalbumin-induced allergic airway disease, 12-HHT and its companion cyclooxygenase metabolites, Prostaglandin E2 and Prostaglandin D2, but not 12 other lipoxygenase or cycloxygenase metabolites, showed a statistically significantly increase in bronchoalveolar lavage fluid levels after intratracheal ovalbumin challenge; after this challenge, only 12-HHT, among the monitored BLT2 receptor-activating ligands stereoisomer of 12-HETE, and 15 attained levels capable of activating BLT2 receptors. Also, BLT2 knockout mice exhibited a greatly enhanced response to ovalabumin challenge. Finally, BLT2 receptor expression was significantly reduced in allergy-regulating CD4+ T cells from patients with asthma compared to healthy control subjects. Unlike LTB4 and its BLT1 receptor, which are implicated in contributing to allergen-based airway disease in mice and humans, 12-HHT and its BLT2 receptor appear to suppress this disease in mice and may do so in humans. While further studies to probe the role of the 12-HHT/BLT2 axis in human inflammatory and allergic diseases, the current studies indicate that 12-HHT, acting through BLT2, may serve to promote or limit, inflammatory and to promote allergic responses.