Prostaglandin D2 receptor 2 (DP2) is an important anti-inflammatory and antiallergic drug target. While inactive DP2 structures are known, its activation mechanisms and biased signaling remain unclear. Here, we report cryo-EM structures of an apo DP2-Gi complex, a DP2-Gi complex bound to the endogenous ligand Prostaglandin D2 (PGD2), and a DP2-Gi complex bound to indomethacin, an arrestin-biased ligand, at resolutions of 2.5 Å, 2.8Å, and 2.3 Å, respectively. These structures reveal a distinct binding pose of PGD2 and indomethacin and provide key insights into receptor activation and transducer coupling. Combining the structural data with functional studies, we uncover the molecular basis for biased signaling of indomethacin toward β-arrestin over G proteins. Notably, a phospholipid binding site was identified at the DP2-G protein interface that modulates DP2-G protein interactions. Together, our functional and structural findings provide insights into DP2 activation, biased signaling, drug interactions, and lipid regulation, enabling rational design of safer antiallergy therapeutics targeting this key immune receptor.

The physical characteristics and behavior of the ATP-binding cassette (ABC) A1, A7, and apolipoprotein (apo) E knockout (KO) mice with lipid transport dysfunction were investigated. These KO mice exhibited adequate growth, and their body masses increased steadily. No remarkable changes were observed in their blood pressure and heart rate. However, there was a slight increase in the heart rate of the ABCA7 KO mice compared with that of the wild-type (WT) mice. ABCA1 and apoE KO mice showed hypo- and hyper-cholesterol concentrations in the plasma, respectively. With regard to the cerebrum, however, the weight of the ABCA1 KO mice was lighter than those of the other genotypes. Furthermore, the cholesterol, triglyceride and phospholipid concentrations, and fatty acid composition were generally similar. Compared with the WT mice, ABCA1 KO mice stayed for a shorter time in the closed arm of the elevated plus maze, and performed worse in the initial stage of the Morris water maze. To thermal stimuli, the ABCA1 and apoE KO mice showed hyper- and hypo-sensitivities, respectively. Only the response of the ABCA1 KO mice was significantly inhibited by pretreatment with indomethacin. A low concentration of the prostaglandin E metabolites was detected in the plasma of the ABCA1 KO mice. Thus, ABCA1 is thought to play a specific role in the neural function.

Crocetin (apo carotenoid dicarboxylic acid) is a common constituent of saffron. Its importance is well documented in Chinese medicine. Some studies have reported the inhibitory effect on inflammation in rats. The aim of the current experimental investigation to scrutinize the anti-inflammatory effect of Crocetin using the lipo polysaccharide (LPS) induced mouse macrophages (RAW 264.7) in vitro and complete Freund's adjuvant-induced arthritis model and to explore in vivo possible mechanism of action. RAW 264.7 macrophages were used for estimation of the effect of crocetin on the cyclooxygenase (COX-2), nitric oxide (NO)production, anti-inflammatory and along with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). Single intraperitoneal injection of complete freund's adjuvant (CFA) was used to induce arthritis. The rats were divided into different group and received the oral administration of crocetin in a dose-dependent manner with indomethacin till 28 days. The paw edema and body weight was estimated at regular interval of time. The biochemical parameters, hematological and pro-inflammatory cytokines such as tumor necrosis factor receptor 1 (TNF-R1), IL-6, and IL-1β, Vascular endothelial growth factor (VEGF); heme oxygenase-1/nuclear factor erythroid 2-related factor 2 (HO-1/Nrf-2) expression were estimated at end of the experimental study. Crocetin inhibited the COX-2 catalyzed prostaglandin (PGE2) and inducible nitric oxide synthase catalyzed NO production on RAW 264.7. The paw edema and body weight was significantly (P < 0.001) modulated by the Crocetin in a dose-dependent manner. Crocetin treatment increased the level of red blood cells (RBC), hemoglobin (Hb) and decreased level of white blood cells (WBC), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase (SGPT), and serum glutamic-oxaloacetic transaminase (SGOT) parameters, with reduction of TNF-α, IL-6, and IL-1β.The protective effect of crocetin was substantiated with a reduction in expression of IL-6, IL-1β, VEGF, and TNF-R1, respectively. Crocetin also increased the HO-1/Nrf-2 and decreased the nuclear factor kappa-B (NF-κB) mRNA, protein expression. On the basis of the result, we can conclude that the reduction of HO-1/Nrf-2 expression, as well as inflammatory mediators, may be involved in the protective effect of Crocetin in the CFA model.

Cogent evidence points to the involvement of neurosteroids in the regulation of dopamine (DA) neurotransmission and signaling, yet the neurobiological bases of this link remain poorly understood. We previously showed that inhibition of 5α-reductase (5αR), a key neurosteroidogenic enzyme, attenuates the sensorimotor gating deficits induced by DA receptor activation, as measured by the prepulse inhibition (PPI) of the acoustic startle reflex. To extend these findings, the present study was aimed at the assessment of the role of other key neurosteroidogenic enzymes in PPI, such as 17α-hydroxylase/C17,20 lyase (CYP17A1), 3α- and 3β-hydroxysteroid dehydrogenase (HSD), in Sprague-Dawley rats. The PPI deficits induced by the DAergic non-selective agonist apomorphine (APO, 0.25mg/kg, SC) were dose-dependently attenuated by the selective CYP17A1 inhibitor abiraterone (ABI, 10-50mg/kg, IP) in a fashion akin to that of the 5αR inhibitor finasteride (FIN, 100mg/kg, IP). These systemic effects were reproduced by intracerebroventricular injection of ABI (1 μg/1 μl), suggesting the involvement of brain CYP17A1 in PPI regulation. Conversely, the PPI disruption induced by APO was not significantly affected by the 3α- and 3β-HSD inhibitors indomethacin and trilostane. Given that CYP17A1 catalyzes androgen synthesis, we also tested the impact on PPI of the androgen receptor (AR) antagonist flutamide (10mg/kg, IP). However, this agent failed to reverse APO-induced PPI deficits; furthermore, AR endogenous ligands testosterone and dihydrotestosterone failed to disrupt PPI. Collectively, these data highlight CYP17A1 as a novel target for antipsychotic-like action, and suggest that the DAergic regulation of PPI is modulated by androgenic neurosteroids, through AR-unrelated mechanisms.

Particulate matter (PM) induces oxidative stress and cardiovascular adverse health effects, but the mechanistic link between the two is unclear. We hypothesized that PM enhanced oxidative stress in vascular endothelial cells and investigated the enzymatic sources of reactive oxygen species and their effects on mitogen-activated protein kinase (MAPK) activation and vasoconstriction. We measured the production of extracellular H2O2, activation of extracellular signal-regulated kinases1/2 (ERK1/2) and p38 MAPKs in human pulmonary artery endothelial cells (HPAEC) treated with urban particles (UP; SRM1648), and assessed the effects of H2O2 on vasoconstriction in pulmonary artery ring and isolated perfused lung. Within minutes after UP treatment, HPAEC increased H2O2 production that could be inhibited by diphenyleneiodonium (DPI), apocynin (APO), and sodium azide (NaN3). The water-soluble fraction of UP as well as its two transition metal components, Cu and V, also stimulated H2O2 production. NaN3 inhibited H2O2 production stimulated by Cu and V, whereas DPI and APO inhibited only Cu-stimulated H2O2 production. Inhibitors of other H2O2-producing enzymes, including Nomega-methyl-L-argnine, indomethacin, allopurinol, cimetidine, rotenone, and antimycin, had no effects. DPI but not NaN3 attenuated UP-induced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38 MAPKs. Knockdown of p47phox gene expression by small interfering RNA attenuated UP-induced H2O2 production and phosphorylation of ERK1/2 and p38 MAPKs. Intravascular administration of H2O2 generated by glucose oxidase increased pulmonary artery pressure. We conclude that UP induce oxidative stress in vascular endothelial cells by activating NAD(P)H oxidase and the mitochondria. The endothelial oxidative stress may be an important mechanism for PM-induced acute cardiovascular health effects.

The detrimental effects of ultraviolet B (UVB) irradiation have been connected with the enhanced generation of reactive oxygen species (ROS) by UVB. However, the exact source of ROS produced by UVB has not been clearly revealed yet. In this study, we determined the source of ROS production and its role in the UVB-induced activation of nuclear factor (NF)-kappaB in HaCaT human keratinocytes. UVB irradiation generated ROS in a dose-dependent manner, and this was significantly inhibited by diphenylene iodonium (DPI), apocynin (Apo) and neopterine (Neo), inhibitors of the NADPH oxidase, and indomethacin (Indo), a cyclooxygenase (COX) inhibitor, but not by the mitochondrial electron transport inhibitors and other cytosolic enzyme inhibitors. In addition, these inhibitors of the NADPH oxidase and COX significantly blocked the UVB irradiation-induced nuclear translocation of NF-kappaB. These results suggest that the NADPH oxidase and COX may be major sources for the UVB-induced ROS generation, and play an essential role in the activation of NF-kappaB which is involved in the expression of a variety of genes induced by UVB in HaCaT cells. These results further suggest that these enzymes may be good targets for the preventive strategy of UVB-induced skin injury.

Prostanoids have been implicated in the transcriptional control of several genes. Since prostanoid synthesis inhibitors are commonly used in subjects with coronary heart disease we studied the effect of cyclooxygenase (COX) inhibition on apolipoprotein AI (apoAI) expression in a human hepatoma cell line (HepG2) transfected with full-length apoAI promoter attached to the chloramphenicol acetyl transferase (CAT) reporter gene. To control for transfection efficiency, the cells were cotransfected with the plasmid pCMV.SPORT-beta-gal containing the beta-galactosidase gene driven by the cytomegalovirus promoter. Treatment of these cells with varying concentrations of indomethacin (INDO, 0, 50, 100, and 300 micromol/L) resulted in a dose-dependent decrease in apoAI promoter activity (% acetylation corrected for beta-galactosidase activity: were 46.1 +/- 2.6, 29.9 +/- 1.2, 25.2 +/- 2.9, and 17.2 +/- 2.8, respectively, P <.001). INDO treatment did not cause significant changes in beta-galactosidase activity. A similar reduction in apoAI promoter activity was found after treating the cells with 50 micromol/L acetylsalicylic acid (ASA) (31.8 +/- 1.8%, P <.001), suggesting that the effect of INDO is related to COX inhibition rather than a peculiar effect of INDO. Nuclear run-off assays indicated that treatment of cells with 50 micromol/L INDO resulted in 31.4% reduction in apo A1 transcription rate (P <.0002). Northern blot analysis of RNA from HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed that the apoAI mRNA concentration relative to G3PDH mRNA was 4,043.0 +/- 84.6 and 3,064.0 +/- 49.8 in control and INDO-treated cells, respectively (P <.0006). Kinetic studies of apoAI mRNA in HepG2 cells indicated that the half-life of apoAI mRNA was not significantly altered with 50 micromol/L INDO treatment. Apo AI mRNA half-life was 25.3 hours in control cells and 26.9 hours in INDO-treated cells. Western blot analysis of culture media of HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed a significant reduction in apoAI protein (6,760.0 +/- 318.1 v 4,773.0 +/- 112.0 arbitrary units, P <.004). Treatment of cells with either arachidonic acid (COX substrate) or various prostanoids including prostaglandin I(2), thromboxane B(2), (+/-)5-HETE, or (+/-)12-HETE did not significantly alter apoAI promoter activity. However, prostaglandin E(1) and E(2) at the highest concentration tested (50 nmol/L) significantly repressed apoAI promoter activity. COX activity measurements in HepG2 cells verified the efficacy of COX inhibition by INDO. It is concluded that COX inhibition with INDO or ASA downregulates apoAI expression at the transcriptional level. This effect could not be attributed to either arachidonic acid excess or to a deficiency in various prostanoids tested.

In order to investigate the consequences of stress susceptibility on vascular function, the authors assessed the respective contributions of nitric oxide (NO), prostanoids, and endothelium-derived hyperpolarizing factor to the vascular tone in rats with a constitutionally determined high and low susceptibility to behavioral stressors. In mesenteric resistance arteries mounted in a small vessel myograph and precontracted with l-phenylephrine hydrochloride (phenylephrine), the NO-synthase inhibitor N omega-nitro-l-arginine (l-NOARG, 100 microM) elicited a smaller increase of vascular tone in apomorphine-susceptible (APO-SUS) rats (P < 0.01). Addition of indomethacin (10 microM), in the presence of l-NOARG, resulted in a smaller decrease of vascular tone in APO-SUS rats (P < 0.01). Although acetylcholine-induced relaxation in phenylephrine-precontracted arteries was not different (P > 0.1), the individual components contributing to this relaxation were. In arteries precontracted with 125 mM K+, and incubated with indomethacin, acetylcholine-induced relaxation was not significantly different (pEC(50) and E(max): P > 0.1). Sensitivity (pEC(50): P < 0.05) and maximum relaxation (E(max): P < 0.001) to sodium nitroprusside, in the presence of 125 mM K+, was more pronounced in APO-SUS rats. In phenylephrine-precontracted arteries, in the presence of l-NOARG and indomethacin, maximum relaxation to ACh was reduced in APO-SUS rats (E(max): P < 0.05). This study showed that in rats with a high susceptibility to stressors, the contribution of NO to vascular tone was decreased as was the ratio of vasoconstrictor and vasodilator cyclooxygenase products in alpha-adrenergic precontracted arteries. End-organ sensitivity to NO was greater in APO-SUS rats, possibly due to up-regulation. Moreover, the contribution of endothelium-derived hyperpolarizing factor to acetylcholine-induced vasodilation was reduced in APO-SUS rat arteries.

High serum lipoprotein(a) (Lp(a)) is a risk factor for vascular disorders. Our preliminary observations suggest that, in some patients with coronary heart disease with high serum Lp(a) levels, administration of aspirin reduced Lp(a) levels. Therefore, we aimed to analyze the effects of aspirin on the production of apo(a), the expression of apolipoprotein(a) (apo(a)) mRNA and the transcriptional activity of apo(a) gene promoter. Aspirin (5 mM) reduced the apo(a) levels in culture medium of human hepatocytes and suppressed apo(a) mRNA expression to 73% and 85% of the controls, respectively. Aspirin also reduced the transcriptional activity of apo(a) gene transfected into HepG2 hepatoma cells in a dose-dependent manner, with a maximal effect at 5 mM (44.3 +/- 1.5% of the control). Sodium salicylate (5 mM) also reduced apo(a) gene transcription, whereas indomethacin (10 microM) had no effect. Deletion analysis of apo(a) gene promoter showed that promoter region extending from -30 to +138 is critical for the effect of aspirin. Furthermore, enhanced production, mRNA expression, and gene transcription of apo(a) by interleukin-6 were also inhibited by aspirin. These results demonstrate that aspirin reduces apo(a) production from hepatocytes via reduction of the transcriptional activity of apo(a) gene with suppression of apo(a) mRNA expression. The suppression of apo(a) production by aspirin may at least in part play a role in the anti-atherogenic effect of aspirin in vascular disorders.

The mechanism of the hypolipidemic effect of n-3 fatty acids was studied using isolated rat hepatocytes maintained in culture. EPA and DHA caused a significant reduction in the incorporation of 3[H]-leucine into apoB associated with the VLDL produced by hepatocytes in culture when compared to that in presence of palmitic acid. Presence of indomethacin, an inhibitor of cyclo-oxygenase reversed the effect of EPA on VLDL synthesis while diethyl carbamazine an inhibitor of lipoxygenase did not show any effect suggesting that the effect of EPA may be mediated through prostaglandins. This was further tested by invivo experiments where animals were fed fish oil containing diet with and without aspirin, which inhibits formation of prostaglandins. The incorporation of 3[H]-leucine into apo B and 14[C]-acetate into cholesterol of VLDL produced by hepatocytes from aspirin treated animals were significantly high. The reversal of the effect of n-3 fatty acids by agents which inhibit the formation of prostaglandin suggests that the n-3 fatty acids may exert their effect on VLDL production by liver cells through prostaglandins.

Apoptosis induced by an antibody to CD95/APO-1/FAS in the colon carcinoma cells COLO 205 and HT-29 is suppressed by the phorbol ester TPA. Inhibition is much more effective in COLO 205 than in HT-29 cells. The TPA effect is abrogated by the protein kinase C (PKC)-specific inhibitor Go6983 indicating a role of PKC in this process. Bryostatin 1, unlike TPA, is unable to suppress apoptosis, but inhibits the TPA-induced suppression of apoptosis. TPA also inhibits indomethacin-induced apoptosis in COLO 205 cells. COLO 205 and HT-29 cells contain the PKC isoenzymes alpha, beta(II) delta, epsilon, eta, mu and zeta. Expression and activity of PKC alpha are at least 5 times higher in COLO 205 than in HT-29 cells. This correlates with the fact that inhibition of CD95-mediated apoptosis by TPA is more prominent in COLO 205 than in HT-29 cells. Thus, these findings suggest that PKC alpha has an important role in the TPA-induced inhibition of apoptosis.

The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.

Hydralazine (HDZ), a p.o. effective antihypertensive drug, was evaluated for its genotoxic effects in both rodent and human cultured cells and in the intact rat. Dose-dependent amounts of DNA fragmentation, as measured by the alkaline elution technique, and of DNA repair synthesis, as revealed by autoradiography, were produced in primary cultures of metabolically competent rat hepatocytes by subtoxic HDZ concentrations ranging from 0.32 to 1.0 mM. A similar potency in inducing DNA repair synthesis was displayed by HDZ in primary cultures of hepatocytes from four human donors. A modest reduction of both DNA fragmentation (-13%) and DNA repair (approximately -50%) was observed in hepatocytes obtained from rats pretreated with indomethacin in order to reduce prostaglandin synthetase activity. In contrast, neither in rat nor in human hepatocytes, differences in N-acetyltransferase activity resulted in meaningful changes of the same end points. V79 cells, which are essentially deficient of monooxygenases catalyzing the biotransformation of xenobiotics, were as sensitive as hepatocytes to the DNA-damaging activity of HDZ. Moreover, after exposure to 0.1 to 0.3 mM HDZ, a modest (2.1- to 2.8-fold), but significant, increase in the frequency of mutation to 6-thioguanine resistance was observed in V79 cells in the absence of a metabolic activation system. In rats, a single p.o. dose of 80 mg/kg produced a clastogenic effect in the liver, but not in the bone marrow, and the p.o. administration for 14 successive days of approximately 46 mg/kg/day increased the average diameter of liver basophilic foci initiated by diethylnitrosamine.(ABSTRACT TRUNCATED AT 250 WORDS)