Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein-coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2-/-). EP2-/- mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2-/- mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2-/- mice developed profound systolic hypertension, whereas wild-type mice showed no change in systolic blood pressure. Analysis of wild-type and EP2-/- mice on day 5 of pregnancy indicated that the reduced litter size of EP2-/- mice is due to a pre-implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt-sensitive hypertension, and also plays an essential part in female fertility.

Prostaglandins and leukotrienes: advances in eicosanoid biology.

Prostaglandins and leukotrienes are potent eicosanoid lipid mediators derived from phospholipase-released arachidonic acid that are involved in numerous homeostatic biological functions and inflammation. They are generated by cyclooxygenase isozymes and 5-lipoxygenase, respectively, and their biosynthesis and actions are blocked by clinically relevant nonsteroidal anti-inflammatory drugs, the newer generation coxibs (selective inhibitors of cyclooxygenase-2), and leukotriene modifiers. The prime mode of prostaglandin and leukotriene action is through specific G protein-coupled receptors, many of which have been cloned recently, thus enabling specific receptor agonist and antagonist development. Important insights into the mechanisms of inflammatory responses, pain, and fever have been gleaned from our current understanding of eicosanoid biology.

Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice.

Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic beta cells is upregulated by cytotoxic cytokines like IL-1beta. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent beta-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1beta, TNF-alpha, and IFN-gamma for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-gamma + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin-induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.

Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice.

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Metabolism of linoleic acid by prostaglandin endoperoxide synthase from adult and fetal blood vessels.

Linoleic acid (18:2) is converted by prostaglandin endoperoxide synthase in particulate fractions and homogenates of fetal calf aorta to its 9- and 13-hydroperoxy metabolites. These intermediates are then either dehydrated to the corresponding oxo compounds or reduced to monohydroxy products. Alternatively, the hydroperoxyoctadecadienoic acids can be converted to epoxyhydroxyoctadecenoic acids, which are hydrolyzed to trihydroxy metabolites by epoxide hydrolases present in both particulate and cytosolic fractions from aorta. Linoleic acid (Km, 442 microM) is a much poorer substrate for prostaglandin endoperoxide synthase than is arachidonic acid (20:4) (Km, 48 microM). However, the oxygenation of 18:2 by particulate fractions from aorta is linear with time for at least 5 min, whereas the oxygenation of 20:4 is linear for only 15 s. Arachidonic acid strongly inhibits the conversion of 18:2 to monohydroxy (ID50, 10 microM) and trihydroxy (ID50, 140 microM) products. Linoleic acid has a similar, but much weaker effect on the formation of 6-oxoprostaglandin F1 alpha from 20:4. Substantial amounts of both the monohydroxy (9-hydroxy-10, 12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid) and trihydroxy (9,10,11-trihydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid) metabolites of 18:2 were shown by gas chromatography-mass spectrometry to be formed from endogenous substrate during incubation of slices of fetal calf aorta in physiological medium. This raises the possibility that some of these products or their hydroperoxy precursors may have some biological significance.

Effects of selenium-deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortae.

Selenium is an essential component of glutathione peroxidase, which reduces free and esterified hydroperoxides of polyunsaturated fatty acids. Adequate glutathione peroxidase activity could be important for the maintenance of prostacyclin synthesis by blood vessels, since hydroperoxides can inhibit the formation of this substance. We have investigated the effects of dietary selenium deficiency on glutathione peroxidase activity and the synthesis of 6-oxoprostaglandin F1 alpha and monohydroxy and trihydroxy metabolites of polyunsaturated fatty acids by aorta. The latter products can be formed either by the actions of cyclooxygenase or lipoxygenase or by lipid peroxidation. Aortic glutathione peroxidase activity was reduced by over 80% by feeding rats a selenium-deficient diet for 4 weeks, and to undetectable levels after 6 weeks. There were no appreciable differences in the levels of free and esterified oxygenated metabolites of linoleic acid or arachidonic acid between the control and treated groups after 4 weeks. However, after 6 weeks, there were modest, but statistically significant reductions in the formation of 6-oxoprostaglandin F1 alpha and monohydroxy products formed by cyclooxygenase. On the other hand, the amounts of esterified 18:2 metabolites appeared to be higher in aortae from animals on the selenium-deficient diet, although only the increase in esterified 9-hydroxy-10,12-octadecadienoic acid was statistically significant. These results suggest that selenium deficiency can affect the formation of prostacyclin and other oxygenated metabolites of polyunsaturated fatty acids by aorta, possibly by increasing lipid peroxidation. However, the differences between control and selenium-deficient rats after 6 weeks were not very dramatic, in spite of the fact that glutathione peroxidase activity was undetectable. It would therefore appear that additional mechanisms are also involved in controlling the levels of lipid hydroperoxides in aorta.

Binding of prostaglandin E2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion.

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.

Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase.

Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.

Absence of 12/15-lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein e-deficient mice.

BACKGROUND: The enzyme 12/15-lipoxygenase (12/15-LO) has been implicated in the oxidative modification of LDL. In a murine model, we tested the hypothesis that deletion of 12/15-LO decreases atherogenesis by reducing oxidant stress, as measured by 2 indices of lipid peroxidation: isoprostane generation and autoantibody formation to malondialdehyde (MDA)-LDL, an epitope of LDL formed as a result of oxidative modification. METHODS AND RESULTS: 12/15-LO-deficient (12/15-LO(-/-)) mice were crossed with apolipoprotein E-deficient (apoE(-/-)) mice. At 10 weeks of age, atherosclerotic lesion initiation was significantly delayed in the double-knockout mice. The rate of lesion progression was diminished at 8 and 12 months, and even at 15 months, lesion size was reduced 50% (P<0.0005) compared with control apoE(-/-) mice. The urinary and plasma levels of the specific isoprostane 8,12-iso-iPF(2alpha)-VI, as well as IgG autoantibodies against MDA-LDL, were significantly reduced in the double-deficient mice in parallel with decreased atherosclerosis at all time points from 10 weeks to 15 months of age compared with apoE(-/-) controls. CONCLUSIONS: Enzymatic action of 12/15-LO contributes significantly to atherosclerotic lesion initiation and propagation in this murine model. Strong positive correlations exist between lesion size, isoprostane levels, and MDA-LDL autoantibodies, providing in vivo evidence for an enzymatic (12/15-LO) component to lipid peroxidation and atherogenesis.

12/15-Lipoxygenase gene disruption attenuates atherogenesis in LDL receptor-deficient mice.

BACKGROUND: Human 15-lipoxygenase (LO) and its murine analogue 12/15-LO are capable of directly oxidizing esterified fatty acids in lipoproteins and phospholipids. Because these oxidized products possess atherogenic properties, it was suggested that LOs may be involved in enhancing atherogenesis. Previous in vivo tests of the role of LOs in atherogenesis animal models, however, have yielded conflicting results. METHODS AND RESULTS: Aiming to study the role of the 12/15-LO in murine atherogenesis, we crossed LDL-receptor-deficient mice (LDL-R(-/-)) with 12/15-LO-knockout mice and evaluated plaque formation 3 to 18 weeks after initiation of a high-fat diet. Atherosclerotic lesions were considerably reduced in the LDL-R/12/15-LO-double-knockout mice compared with LDL-R(-/-) mice at 3, 9, 12, and 18 weeks, at the aortic root as well as throughout the aorta. The cellular composition of plaques from mice deficient in 12/15-LO did not differ with respect to macrophage and T-lymphocyte content compared with plaques from 12/15-LO littermates. CONCLUSIONS: 12/15-LO plays a dominant role in promoting atherogenesis in LDL-R(-/-) mice.

12/15-lipoxygenase, oxidative modification of LDL and atherogenesis.

Lipoxygenases comprise a family of non-heme iron-containing dioxygenases that stereospecifically insert molecular oxygen into free or esterified polyunsaturated fatty acids. The dual specificity 12/15-lipoxygenases have been implicated in the oxidative modification of low-density lipoproteins and foam cell formation primarily based on in vitro studies. Recent in vivo data obtained with 12/15-lipoxygenase-deficient mice crossbred to apolipoprotein E-deficient mice have established a proatherogenic role for this pathway. In contrast, previous experiments with macrophage expressing 15-lipoxygenase transgenic rabbits have suggested an anti-atherogenic role. Possible explanations are presented that may elucidate these differences.

Basal transepidermal water loss is increased in platelet-type 12-lipoxygenase deficient mice.

The roles of fatty acids in the skin have been under investigation since early reports of the phenotypic abnormalities of mice fed a diet deficient in essential fatty acids. Little is known about the functional significance of fatty acid metabolism by lipoxygenases in epidermis. Here, we have examined the role of platelet-type 12-lipoxygenase which converts arachidonic acid to the oxygenated metabolite 12-hydroperoxyeicosatetraenoic acid, in the skin using platelet-type 12-lipoxygenase-deficient mice generated by gene targeting. Platelet-type 12-lipoxygenase in wild-type mice was localized to the stratum granulosum by immunohistochemical analysis. Platelet-type 12-lipoxygenase-deficient mice lacked immunodetectable platelet-type 12-lipoxygenase in platelets and epidermis, appeared grossly normal, and exhibited an increase in basal transepidermal water loss without alteration in basal mitotic activity. Water loss and mitotic activity in mice with an acetone-disrupted membrane barrier were normal. No defect in ultrastructural properties or content of major fatty acids in dorsal skin or ear inflammation response was apparent in platelet-type 12-lipoxygenase-deficient mice. These results indicate that the platelet-type 12-lipoxygenase pathway in mice is partly responsible for normal permeability barrier function but the mechanism awaits further elucidation.

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.

Role of 'platelet-type' 12-lipoxygenase in skin carcinogenesis.

Murine and human skin express an abundance of lipoxygenase isoforms whose functions are not understood. Substantial data have implicated a role for the 'platelet-type' 12-lipoxygenase (P-12LO) metabolite, 12(S)-hydroxy-eicosatetraenoic acid (12-HETE), in a variety of tumor functions. Using P-12LO deficient mice, we sought to examine the role of the P-12LO pathway in tumor initiation and progression. Two distinct genetic strains of P-12LO deficient and wild-type mice, B6/129 Sv and SENCAR, were evaluated in two-stage carcinogenesis experiments. Carcinoma incidence was significantly reduced in the P-12LO deficient mice of the B6/129 Sv background but not the SENCAR-backcrossed mice. In contrast, papilloma incidence was reduced on the SENCAR background but not in the B6/129 Sv strain mice. A separate experiment employing a complete carcinogenesis protocol failed to find any difference in papilloma or carcinoma incidence. Overall, these data suggest that the P-12LO pathway may contribute to tumor incidence and progression in two-stage, but not complete, carcinogenesis, depending on the genetic background.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT(1)) receptors.

We sought to clone and characterize the murine cysteinyl-leukotriene D(4) receptor (mCysLT(1)R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT(1) receptor (hCysLT(1)R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT(1)R demonstrated high affinity and specific binding for leukotriene D(4) (LTD(4), K(d) = 0.25 +/- 0.04 nM). In competition binding experiments, LTD(4) was the most potent competitor (K(i) = 0.8 +/- 0.2 nM) followed by LTE(4) and LTC(4) (K(i) = 86.6 +/- 24.5 and 100.1 +/- 17.1 nM, respectively) and LTB(4) (K(i) > 1.5 microM). Binding of LTD(4) was competitively inhibited by the specific CysLT(1) receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon), and zafirlukast (Accolate), while the CysLT(1)/CysLT(2) receptor antagonist BAY-u9773 [6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD(4). In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT(1)R, LTD(4) induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD(4) induced the dispersion of pigment granules, consistent with the activation by LTD(4) of a G(alphaq) (calcium) pathway. Functional elucidation of mCysLT(1)R properties as described here will enable further experiments to clarify the selective role of LTD(4) in murine models of inflammation and asthma.

Insight into prostaglandin, leukotriene, and other eicosanoid functions using mice with targeted gene disruptions.

In recent years, there has been an exponential increase in the number of targeted gene disruptions performed in mice. At least 18 different gene knockouts have now been reported that have direct relevance to eicosanoid biology. These include genes that influence substrate availability (phospholipases), metabolism to eicosanoids (e.g., prostaglandin H synthases, lipoxygenases), and eicosanoid action (e.g., receptors for various prostaglandins). This minireview will outline the phenotype of these knockout mice and what has been learned about eicosanoid functions through use of this novel methodology.

Characterization of the murine epidermal 12/15-lipoxygenase.

The murine lipoxygenase (LO) family consists of at least seven members classified according to the HETE (hydroxyeicosatetraenoic acid) metabolite generated during arachidonic acid metabolism and the site of tissue expression. At present there are four 12-lipoxygenases that are functionally distinct, vary in cell and tissue distribution, catalytic activity and each are products of separate, linked genes. They are "platelet-type" 12-LO (P-12LO), "leukocyte-type" 12-LO (L-12LO), "epidermal-type" 12-LO (e-12LO) and the most recently discovered 12(R)-LO. In this report we characterize e-12LO, which was overexpressed in the baculovirus/insect cell expression system. The enzyme functions as a dual specificity 12/15-lipoxygenase with a 12-HETE/15-HETE product ratio of approximately 6:1 with arachidonic acid as substrate. Several other polyunsaturated fatty acids served as substrates for e-12LO such as gamma-linolenic, dihomo-gamma-linolenic and eicosapentaenoic acids. A green fluorescent protein/e-12LO fusion protein was localized to the cytosol of transfected HEK 293 cells. The e-12LO gene was expressed in mouse oocytes and early embryos. Western blot analysis revealed high level expression in postnatal day 3 mouse epidermal lysates. Together these data suggest that e-12LO plays a role in normal epidermal function and as yet an undiscovered role in early development.

Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni.

Periovular granuloma formation during Schistosoma mansoni infection is a complex, multifaceted immunologic response. Products of arachidonic acid metabolism have been shown to contribute to this response through studies in which general inhibitors of lipoxygenase function reduce granulomatous inflammation. To determine which lipoxygenases are important for granuloma development in schistosomiasis, wild type mice or mice deficient for 5-lipoxygenase (5-LO) or "leukocyte-type" 12-lipoxygenase (12-LO) were infected with S. mansoni and studied for responses to schistosome eggs and egg antigens. At the acute stage of infection, when granuloma formation is usually maximal, 5-LO deficient mice developed smaller granulomas around liver-deposited schistosome eggs compared with wild type or 12-LO deficient mice. 5-LO mice also displayed less antibody-mediated (5 h) and cell-mediated, delayed-type (24 h) hypersensitivity to schistosome egg antigens than did the other two infection groups. In an attempt to determine possible mechanisms for the reduced inflammatory responses, we also measured hepatic mRNA levels of cytokines that have been shown to influence granuloma size (IL-4, IL-10, and IFN-gamma). The mRNA levels for IL-10 were significantly lower in 5-LO-deficient mice, but SEA-stimulated spleen cells did not demonstrate a significant difference in IL-10 production between wild type and 5-LO mice. These data suggest that 5-LO plays a role in host responses to schistosomiasis via a mechanism that cannot be explained solely by changes in expression of these cytokines.