Experimental hyperthyroidism in rats suppresses in vitro prostaglandin metabolism in lung and kidney.

Metabolism of prostaglandin (PG) F2alpha and PGE2 was depressed 40--62% in 100,000 g cytoplasmic supernatants of lungs and kidneys prepared from rats made hyperthyroid by 18 daily L(-) thyroxine injections (200microgram, s--c). These hyperthyroid rats had elevated serum thyroxine levels, cardiac hypertrophy and thyroid atrophy. There were no differences in soluble protein concentrations, NAD+ utilisation by endogenous enzymes and substrates, or in the NAD+ dependence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) between the supernatants prepared from hyperthyroid rats and saline-injected controls. Thyroxine did not inhibit PG metabolism in vitro up to 260 micrometer. These results suggest that thyroxine specifically decreases intracellular levels of PG-metabolising enzymes, especially of the rate-limiting 15-PGDH. Metabolism of PGF2alpha and PGE2 by 15-PGDH was faster in smaller rats and declined with increasing animal weight. These studies imply that some of the clinical features of hyperthyroidism in man might be caused by deficiencies in PG metabolism.     

Indomethacin increases 15-PGDH mRNA expression in HL60 cells differentiated by PMA

We previously reported an induction of 15-hydroxyprostaglandin dehydrogenase type I mRNA (15-PGDH) expression accompanied by a decrease in prostaglandin E2(PGE2) levels during cord blood monocytes differentiation into preosteoclastic cells by 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3). These results suggested a role of prostaglandin (PG) enzymes in adhesion and/or differentiation of monocytes.In the present work, we studied modulation of gene expression of PG metabolism enzymes mRNAs in HL60 cells differentiated by phorbol myristate acetate (PMA) into the monocyte/macrophage lineage. We showed that adhesion of HL60 induced by PMA causes an increase of cyclooxygenase 2 (COX 2) and 15-PGDH mRNAs. When adding indomethacin, a non steroidal antiinflammatory drug known to inhibit COX activity, the cells remained attached and expressed large amounts of 15-PGDH mRNA while COX 2 mRNA expression remained unchanged. Indomethacin, in association with PMA can consequently exert a dual control on key enzymes of PGE2 metabolism without modifying adhesion of the cells.     

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.     

PRESENCE OF 15-HYDROXY PROSTAGLANDIN DEHYDROGENASE, PROSTAGLANDIN-Δ13-REDUCTASE AND PROSTAGLANDIN E-9-KETO(α)-REDUCTASE IN THE FROG SPINAL CORD

Metabolism of prostaglandin E₁ (PGE₁) and F_1α (PGF_1α) was studied in the frog spinal cord, using a hemisected preparation in vitro and tissue homogenates (whole honiogenate and tissue fractions). In the intact tissue, PGE, was converted to three Metabolites, 1 to 111, whereas only Metabolites 11 and 111 werc detected in experiments with PGF_1α. Work with tissue homogenatcs confirmed that PG transformation is enzymatic, and endproducts were identified as PGF_1α (Metabolite 1), 15-kcto metabolite (Metabolite 11) and 15-keto-13,14-dihydro metabolite (Metabolite 111). The 15-keto-13,14-dihydro metabolite was formed via the 15-keto metabolite which is consistent with findings elsewhere. These results establish the presence in the frog spinal cord of two pathways for PG metabolism, consisting one of the 15-hydroxy prostaglandin dehydrogenase (15-PGDH) and the prostaglandin-A13- reductase (13-PGR), the other of the prostaglandin E 9-keto(α)-reductase (9K-PGR). 9K-PGR is regarded as an inactivating enzyme because amphibian spinal neurons are less responsive to PGF, than to PGE₁. In the intact or in the homogenized tissue, PGE, is metabolized more efficiently by the 15-PGDH/13-PGR than by the 9K-PGR route. The 15-PGDH metabolizes PGE, more readily than PGF_1α. The present findings, together with the previous demonstration of active PG synthesis in the tissue and the potent actions of exogenous PGs, strongly suggest that the PGs play an important role in the function of neurons in the frog spinal cord.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Prostaglandin catabolism in fetal and maternal tissues--a study of 15-hydroxyprostaglandin dehydrogenase and delta 13 reductase with specific assay methods

Recent studies have demonstrated that extraductal tissues such as lung are important sources of prostaglandin E2 which maintains the patency of ductus arteriosus in fetuses and prematurely-born infants. Also, organs such as lung are known to be active in the catabolism of PGE2. Earlier studies of enzymes involved in the catabolism of PGE2 such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and delta 13 reductase all used non-specific methods. In the present report, we studied 15-PGDH in fetal and maternal rat lung, kidney, and fetal lamb lung, kidney and ductus arteriosus with the use of a specific substrate (15-S)-[15(3)H-PGE2]. In addition, we measured the activity of delta 13 reductase in these tissues by measuring the conversion of [1-14C]-15-keto PGE2 to [1-14C]-15-keto-13,14-dihydro PGE2. The results from these studies demonstrated that in fetal rat lung and kidney, 15-PGDH activities increased rapidly while delta 13 reductase remained unchanged during late gestation. Ductus arteriosus possessed little 15-PGDH activities. These results strongly suggest that extraductal regulation of PGE2 metabolism is important in determining ductal caliber in fetuses and prematurely delivered neonates.     

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF_1α was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF_1α was recovered in the urine. The metabolic pattern very closely resembles that of PGI₂ in rats. Metabolites were found which represented 15-dehydrogenation, β-oxidation, ω and ω-1-hydroxylation and oxidation.Previous work showed that 6-keto PGF_1α is very poorly oxidized by 15-PGDH. We administered 15-[H³]-PGI₂ and 15-[H³]-6-keto PGF_1α to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI₂ and 6-keto PGF_1α were both oxidized to the 15-keto product, although the rate of oxidation of PGI₂ was greater than that of 6-keto PGF_1α. We concluded that the administered PGI₂ was oxidized by 15-PGDH before hydrolysis to 6-keto PGF_1α. A portion of the dose is probably hydrolyzed before 15-dehydrogenation.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrenaline, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 μg/ml (0.83 × 10⁻⁵M) for MIX, 18 μg/ml (1 × 10⁻⁴M) for theophylline and 133 μg/ml (6.8 × 10⁻⁴M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyryl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10⁻²M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogeneous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2α on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonism may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

The role of nitric oxide in the metabolism of labeled glucose in isolated rat uterus. Influence of 17β-estradiol

The influence of nitric oxide (NO) on the production of ¹⁴CO₂ from labeled glucose in uteri isolated from ovariectomized-estrogenized rats was studied. Nitroprusside, an NO donor (NP), 200 μM increased the formation of labeled CO₂ from [U-¹⁴C]glucose. This effect was blunted by hemoglobin (Hb) 20 μg/mL, an NO scavenger. The addition of N-monomethyl arginine (NMMA), an inhibitor of NO synthase decreased the stimulatory action of NP at 400 mM. Incubation of uterine strips in the presence of NP plus acetylsalicylic acid (ASA) 10⁻⁴ M (a cyclooxygenase inhibitor), inhibited the stimulatory action of NP on glucose metabolism. PGE₂ (10⁻⁷ M) added to the incubation medium containing NP and ASA reversed the effect of the inhibitor. Neither NP nor Hb nor NMMA modified the ¹⁴CO₂ production from labeled glucose in uterine strips from ovariectomized rats. The addition of NP to the incubating medium increased PGE accumulation by uterine strips from rats treated with estradiol, but not in ovariectomized animals. These results suggest that NO exerts a positive influence on glucose metabolism and PGE synthesis in isolated rat uteri from estrogenized animals.     

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.     

Inhibition of 15-hydroxy prostaglandin dehydrogenase activity in rabbit gastric antral mucosa by 13-hydroperoxyoctadecadienoic acid

The effect of 13-hydroperoxyoctadecadienoic acid (13-HPODE), a hydroperoxy adduct of linoleic acid (LA), on the activities of prostaglandin (PG) synthesizing and catabolizing enzymes in rabbit gastric antral mucosa was examined. 13-HPODE had no effect on the synthesis of PGE₂, PGF_2α and PGD₂ from exogenous arachidonic acid in the microsomal fraction of the gastric mucosa at concentrations ranging from 5–20 μM. On the other hand, at 1–10 μM, it inhibited the activity of 15-hydroxy PG dehydrogenase (PGDH), which catalyzes the initial step of catabolism of PGs, in a dose-dependent manner. The concentration required for 50% inhibition was approximately 1 μM. Experiments utilizing LA, 13-hydroxyoctadecadienoic acid and Fe²⁺ indicated the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on the PGDH activity. These results suggest that 13-HPODE has the potential to increase the levels of biologically active PGs in gastric mucosa by preventing their inactivation and may have functional effects within the stomach.     

Synthesis of 6-keto-PGF1α by ram seminal vesicle microsomes

At low substrate/enzyme ratios, and in the absence of reduced glutathione (GSH), the major prostaglandin (PG) biosynthesised by the ram seminal vesicle cyclo-oxygenase from arachidonic acid was 6-keto-PGF1α. The addition of nanomolar amounts of reduced GSH suppressed biosynthesis of this product and stimulated the formation of PGE2; 1-epinephrine enhanced the conversion of the substrate but had not effect on the type of product formed. 15-Hydroperoxy arachidonic acid selectively inhibited formation of 6-keto-PGF1α (IC50 100 μM) but blocked synthesis of all cyclooxygenase products at concentations greater than 1 mM. At substrate concentrations of 30 μM or greater, synthesis of 6-keto-PGF1α was inhibited and PGE2 and PGF2α were the main products formed.     

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal andembryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A₂ (PLA₂) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF_2α production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF_2α decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF_2α Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 μM) induced a decrease in PGE and PGF_2α production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.     

Steroid-independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles

The content of prostaglandins of the E-group (PGE) or F-group (PGF) was determined by radioimmunoassay in rat ovaries and in homogenates of cultured Graafian follicles. Intraperitoneal administration of luteinizing hormone (NIH-LH-S18; 10 μg/rat) at 9.00 h on any day of the estrous cycle caused an increase in ovarian PGE content within 5 h. The response was greatest on the day of proestrus (940% rise), i.e. when the ovary contains large follicles, and least at metestrus (80%). Follicles explanted from proestrous rats before the preovulatory gonadotropin surge responded to addition of LH (1–5 μg/ml) to the culture medium with a 10 to 30-fold increase in PGE and a 5-fold increase in PGF accumulation over a 5-h-period. Follicle stimulating hormone (NIH-FSH-S9; 10 μg/ml) caused a similar rise in follicular PGE accumulation, even after treatment of the FSH preparation with excess of an antiserum to the β-subunit of LH. Stimulation of follicular PG accumulation was unimpaired during suppression of progesterone and estrogen synthesis by aminoglutethimide. It is concluded that these steroids play no part in the mediation of the LH-effect on follicular prostaglandin formation.     

Stimulation of rat colonic mucosal prostaglandin synthesis by calcium and carbamylcholine: Relationship to alterations in cyclic nucleotide metabolism

The present study examined the relationships between prostaglandin (PG) synthesis and cyclic nucleotide metabolism in rat colonic mucosal slices. Ca²⁺, Ca²⁺ plus A23187 and carbamylcholine all increased [¹⁴C]-arachidonate release from prelabeled slices and stimulated production of PGE. Actions of A23187 and carbamylcholine required Ca²⁺ and were suppressed by tetracaine or mepacrine, whose known actions include inhibition of acyl hydrolase activity. Exogenous arachidonate or linoleate stimulated PGE synthesis in the absence of Ca²⁺ or in the presence of the inhibitors, suggesting a role for Ca²⁺ dependent acyl hydrolase activity in the mediation of the actions of Ca²⁺, A23187 and carbamylcholine on PGE synthesis. Accumulation of both cAMP and cGMP in colonic mucosal slices was enhanced by carbamylcholine, Ca²⁺, Ca²⁺ plus A23187, arachidonate or linoleate. Stimulatory actions of each of these agents on PGE production and cyclic nucleotide accumulation were inhibited by O₂ exclusion or indomethacin (100 μg/ml). The results support a role for local PG production in the mediation of carbamylcholine and Ca²⁺ actions on cyclic nucleotides. Endogenous ionic, neurohumoral and dietary factors may modulate colonic mucosal PG synthesis and cyclic nucleotide content, and thereby influence the physiologic expression of the actions of these putative local cellular regulators.     

Resveratrol Induces a Mitochondrial Complex I-dependent Increase in NADH Oxidation Responsible for Sirtuin Activation in Liver Cells

Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1–5 μm) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC₅₀ ∼1 μm). In HepG2 cells, this complex I activation increases the mitochondrial NAD⁺/NADH ratio. This higher NAD⁺ level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.     

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD⁺ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD⁺ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD⁺ levels and expression levels of NAD⁺ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD⁺ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD⁺ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD⁺ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD⁺ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD⁺. The agonist-induced rise in NAD⁺ shows striking parallels to well-known second messengers and raises the possibility that NAD⁺ is acting in a similar manner in this model.