Structure requirements for antiproliferative and cytotoxic activities of marine coral prostanoids from the Japanese stolonifer Clavularia viridis against human myeloid leukemia cells in culture

The structural requirements for antiproliferative and cytotoxic activities of marine coral prostanoids from Japanese stolonifer Clavularia viridis and related compounds were examined in HL-60 cells in culture. From our data on the structure-activity relationship of these compounds, we elucidated that 1) the alkylidenecyclopentenone structure in these prostanoids was required for the antiproliferative and cytotoxic activities against HL-60 cells, but the epoxy prostanoids without cross-conjugated cyclopentenone system had the activities; 2) the presence of hydroxyl group at C-12 position in the prostanoids enhanced the activities, but the stereospecificity of the 12-hydroxyl group was not required for the activities; 3) the introduction of halogen atom at C-10 position of the prostanoids potentiated the activities (Cl greater than Br = I greater than H); 4) the introduction of blocking groups for blocking beta-oxidation to the alpha-side chain of the prostanoids did not cause the marked increase of the activities; 5) the presence of dienone (C5-6 and C7-8) in the structure potentiated the activities. These results provide the basis for drug-design of a new class of antitumor agent from marine coral prostanoids.     

Prostanoids and their receptors that modulate dendritic cell-mediated immunity

Dendritic cells (DC) are essential for the initiation of immune responses by capturing, processing and presenting antigens to T cells. In addition to their important role as professional APC, they are able to produce immunosuppressive and pro-inflammatory prostanoids from arachidonic acid (AA) by the action of cyclooxygenase (COX) enzymes. In an autocrine and paracrine fashion, the secreted lipid mediators subsequently modulate the maturation, cytokine production, Th-cell polarizing ability, chemokine receptor expression, migration, and apoptosis of these extremely versatile APC. The biological actions of prostanoids, including their effects on APC-mediated immunity and acute inflammatory responses, are exerted by G protein-coupled receptors on plasma membrane. Some COX metabolites act as anti-inflammatory lipid mediators by binding to nuclear receptors and modulating DC functions. Although the role of cytokines in DC function has been studied extensively, the effects of prostanoids on DC biology have only recently become the focus of investigation. This review summarizes the current knowledge about the role of prostanoids and their receptors in modulating DC function and the subsequent immune responses.     

Role of prostaglandins in the urinary bladder: an update

Our knowledge of prostanoids is rapidly increasing. In this review we survey the factors governing the synthesis of prostanoids by the urinary bladder, their role in the maintenance of normal bladder function, the pattern of their secretion in bladder disease and the possible use of prostanoids in the treatment of bladder pathology.     

Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry

Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E(2) (PGE(2)), PGD(2), PGF(2α), 6-keto PGF(1α), and TXB(2), in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C(18) column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL (r(2)>0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng (r(2)>0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.     

Effects of ischemia/reperfusion on brain tissue prostanoids and leukotrienes in newborn pigs.

We investigated the hypothesis that cerebral prostanoid and peptidoleukotriene (LTs) (LTC4/D4/E4/F4) synthesis are increased during postischemic reperfusion of newborn pig brains. Prostanoids and LTs extracted from brain tissue were determined by RIA in sham-control piglets and at 1h, 3h, or 12h after a 20-min period of total cerebral ischemia. During reperfusion following ischemia, all regional brain tissue (cerebrum, brain stem and cerebellum) prostanoids (6-keto-PGF1 alpha, TXB2, PGE2 and PGF2 alpha) were increased at 1h compared with those in sham-control piglets. Only cerebral and brain stem 6-keto-PGF1 alpha and cerebral TXB2 remained elevated at 3h postischemia and all prostanoids returned to control levels by 12h postischemia. Brain tissue LTs were lower than prostanoids and were not altered 1, 3, or 12h following ischemia. These data indicate that 1) newborn pig brain tissue prostanoids are increased initially, and then returned to control levels at later stages of reperfusion following ischemia; 2) LTs are present in newborn pig brain tissue, but are not increased by ischemia/reperfusion injury and therefore probably do not play a significant role in cerebral ischemia-reperfusion injury.     

Vascular and neural mechanisms of ACh-mediated vasodilation in the forearm cutaneous microcirculation.

The relative contribution of endothelial vasodilating factors to acetylcholine (ACh)-mediated vasodilation in the forearm cutaneous microcirculation is unclear. The aims of this study were to investigate the contributions of prostanoids and cutaneous C fibers to basal cutaneous blood flow (CuBF) and ACh-mediated vasodilation. ACh was iontophoresed into the forearm, and cutaneous perfusion was measured by laser-Doppler flowmetry. To inhibit the production of prostanoids, four doses of acetylsalicylic acid (ASA; 81, 648, 972, and 1,944 mg) were administered orally. Cutaneous nerve fibers were blocked with topical anesthesia. Cyclooxygenase inhibition did not change basal CuBF or endothelium-mediated vasodilation to ACh. In contrast, ASA (972 and 1,944 mg) significantly reduced the C-fiber-mediated axon reflex in a dose-dependent fashion. Blockade of C-fiber function significantly reduced axon reflex-mediated vasodilation but did not affect basal CuBF or endothelium-dependent vasodilation. The findings suggest that prostanoids do not contribute significantly to basal CuBF or endothelium-dependent vasodilation in the forearm microcirculation. In contrast, prostanoids are mediators of the ACh-provoked axon reflex.     

Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.     

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

NO and prostanoids blunt endothelin-mediated coronary vasoconstrictor influence in exercising swine

Withdrawal of the endothelin (ET)-mediated vasoconstrictor influence contributes to metabolic coronary vasodilation during exercise. Because production of nitric oxide (NO) and prostanoids increases with increasing shear stress and because NO and prostanoids are able to modify the release of ET, we hypothesized that the withdrawal of ET-mediated coronary vasoconstriction during exercise is mediated through NO and/or prostanoids. To test this hypothesis, 19 chronically instrumented swine were studied at rest and while running on a treadmill up to 85-90% of maximal heart rate. Blockade of ET(A)/ET(B) receptors with tezosentan resulted in an increase in coronary venous O(2) levels (i.e., in coronary vasodilation) at rest, which waned at increasing levels of exercise intensity. Inhibition of either NO synthase [N(omega)-nitro-l-arginine (l-NNA)] or cyclooxygenase (indomethacin) did not affect the response to tezosentan under resting conditions but unmasked a vasodilator response to tezosentan during exercise. The vasodilator response to tezosentan during exercise increased progressively after combined administration of l-NNA and indomethacin. These findings suggest that NO and prostanoids act synergistically to inhibit the vasoconstrictor influence of ET on the coronary circulation during exercise, thereby facilitating the exercise-induced vasodilation of coronary resistance vessels.     

Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells

In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.     

Lipidomic analysis reveals prostanoid profiles in human term pregnant myometrium

Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD₂ and PGF_2α being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE₂ and 13,14-dihydro-15-keto-PGE₂ were the only prostanoids to increase significantly at early and late labour (p≤0.001). Our data suggest that PGF_2α plays an important role in parturition, whilst the increase in PGE₂ could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA₂ was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.     

Tissue prostanoids as biomarkers for chemoprevention of colorectal neoplasia: correlation between prostanoid synthesis and clinical response in familial adenomatous polyposis

Recent studies indicate that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), lowers mucosal prostanoid levels and regresses colorectal adenomas in patients with familial adenomatous polyposis (FAP). To determine whether they are biomarkers for sulindac-mediated chemoprevention of colorectal adenomas, levels of 5 prostanoids [prostaglandin (PG) D2, PGE2, PGF2alpha, thromboxane B2, and 6-keto-PGF1alpha] in the normal-appearing rectal mucosa from 7 FAP patients with a history of subtotal colectomy and ileorectal anastomosis and 4 FAP patients without surgery, were measured in the absence or presence of exogenously added arachidonic acid before the initiation and at the end of 3 months of sulindac treatment. The addition of arachidonic acid resulted in a uniform increase in the levels of all 5 prostanoids although this increase was selectively attenuated in patients with ileorectal anastomosis who took sulindac. In the latter patients, arachidonic acid also augmented the inhibition of prostanoid synthesis by sulindac. In contrast, sulindac failed to attenuate the increase in prostanoid levels resulting from arachidonic acid in patients without previous surgery. Importantly, when measured in the presence of arachidonic acid, the reduction in the levels of all 5 prostanoids due to sulindac was statistically correlated with a reduction in the size and number of adenomas in the two groups of patients combined. These results suggest that tissue prostanoids measured in the presence of arachidonic acid may serve as sensitive and reliable biomarkers in monitoring the clinical responsiveness of FAP patients undergoing chemoprevention for colorectal neoplasia with NSAIDs.     

Vasoactive prostanoids are generated from arachidonic acid by COX-1 and COX-2 in the mouse

Generation of vasoactive prostanoids from arachidonic acid by cyclooxygenase (COX)-1 and COX-2 was investigated in anesthetized mice. Intravenous injections of the prostanoid precursor arachidonic acid increased pulmonary arterial pressure and decreased systemic arterial pressure. Pulmonary pressor and systemic depressor responses were attenuated by SC-560 and nimesulide, inhibitors of COX-1 and COX-2, in doses that did not alter responses to injected prostanoids. Pulmonary pressor responses to arachidonic acid were blocked and a depressor response was unmasked, whereas systemic depressor responses were not altered, by a thromboxane receptor antagonist. Pulmonary and systemic pressor responses to angiotensin II injections and systemic pressor responses to angiotensin II infusion were not modified by COX-1 or COX-2 inhibitors but were attenuated by losartan. Systemic depressor responses to arachidonic acid were smaller in COX-1 and COX-2 knockout mice, whereas responses to angiotensin II, norepinephrine, U-46619, endothelin-1, and PGE(1) were not different in COX-1 and COX-2 knockout and wild-type control mice. These results suggest that vasoactive prostanoids with pulmonary pressor and systemic vasodepressor activity are formed by COX-1 and COX-2 and are consistent with Western blot analysis and immunostaining showing the presence of COX-1 and COX-2. These data suggest that thromboxane A(2) (TxA(2)) is formed from the precursor by COX-1 and COX-2 in the lung and are in agreement with immunofluorescence studies showing thromboxane synthase. The present data suggest that COX-1- or COX-2-derived prostanoids do not modulate responses to angiotensin II or other vasoactive agents and that prostanoid responses are similar in CD-1 and C57BL/6 and in male and female mice.     

Formation of prostanoids in human umbilical vessels perfused in vitro

Four major prostanoids (6-keto-PGF1 alpha, PGE2, PGF2 alpha and TXB2) were measured by specific radioimmunoassays in the outputs from human umbilical vessels perfused in vitro. As evaluated by scanning electron microscopy (SEM) only few blood platelets were attached to the vessel wall. After an initial flush with decreasing concentrations of all four prostanoids, a stable stage was reached, lasting for 4-5 hours. During this stage the production could be inhibited by indomethacin and only slightly stimulated with arachidonic acid. The TXA2 synthetase inhibitor UK 38485 depressed the TXB2 production, while only slightly affecting the other three prostanoids at very high concentrations. The arteries produced relatively more 6-keto-PGF1 alpha than did the vein.     

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.     

The contribution of prostaglandins versus prostacyclin in ventricular remodeling during heart failure

Although the role of Cox-2 in the heart's response to physiologic stress remains controversial (i.e. expression in myocytes versus other resident myocardial cells) the ever expanding role of prostanoids in multiple models of heart failure cannot be denied. Due to the fact that prostanoids are metabolized rather quickly (half life of seconds to minutes) it is believed these signaling mediators act in a paracrine fashion at the site of production. Evidence to date is quite convincing that these bioactive lipid derivatives are involved in physiologic homeostatic regulation as well as beneficial and maladaptive ventricular remodeling in heart failure. Thus, this review will assess the direct contribution of each PG on remodeling in the left ventricle (e.g. hypertrophy, functional effects, and fibrosis).     

Prostaglandins and oxylipins of corals

Literature data for the period from 1969 to 2010 on the distribution and chemical structures of prostaglandins, prostanoids, eicosanoids, and other cyclic and acyclic oxylipins from corals are summarized. The chemical structures of 97 compounds are given.     

Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.