Prostaglandins V (1). Synthesis and pharmacology of (±)-11-deoxy-10-hydroxyprostaglandin E1 methyl ester

(±)-11-Deoxy-10-hydroxy-PGE₁ methyl ester (Va) has been synthesised conjugate addition of the cuprate (III) to the 5-tetrahydropyranyloxycyclopentenone (IId).Va was found to be 0.1 times as active as PGE₁ as a uterine stimulant in the anaesthetized pregnant rat, 0.25 times as active as PGE₁ in causing vasodepression in the anaesthetized cat, and approximately equiactive to PGE₁ in inducing bronchoconstriction.     

Effect of a "minimal natriuretic and diuretic" dose of prostaglandin E1 (PGE1) on the intratubular and peritubular pressures in anaesthetized rats

A "minimal natriuretic and diuretic" dose (3.4--4.2 ng/min/kg) of prostaglandin E1 (PGE1) as previously estimated was infused into the aorta in anaesthetized rats. During the PGE1 infusion parameters for renal haemodynamics, Na+ and water excretion, intra- and peritubular pressures were studied ("electronic servo nulling device"). During the solvent period parameters for the infused left kidney did not differ from those on the control side. Diuresis and Na+ excretion were increased significantly due to PGE1 infusion in both series of investigations. Cortical blood flow (radio isotope labelled microsphere technique) and glomerular filtration (inulin clearance) did not change significantly. Oncotic pressure in the afferent arteriole was not affected by PGE1, whereas it was reduced significantly in the efferent arteriole (control, 22.9 +/- 1.7; PGE1, 19.2 +/- 0.9 mmHg). Transit times to the early and late distal tubules were not affected by PGE1. There were no changes in the hydrostatic pressure in the proximal tubule and efferent arteriole, whereas the peritubular capillary hydrostatic pressure was significantly increased (control, 9.6 +/- 0.3; PGE1, 11.2 +/- 0.2 mmHg). The present results indicate that PGE1 is capable of enhancing Na+ and water excretion without affecting RBF and GFR, and peritubular physical factors might play an auxiliary role in this effect.     

The effects of prostaglandins PGE1, PGE2, PGF1a, and PGF2alpha on canine synovial perfusion.

In these experiments we have examined the effects of PGE1, PGE2, PGF1alpha and PGF2alpha on synovial perfusion in the normal canine synovial microcirculation. The effects of the drugs on synovial perfusion were determined indirectly from the changes produced in the rate of clearance of 133Xenon from the joint by their intra-articular injection. Prostaglandins PGE1 and PGE2 were found to be strongly vasodilator with PGE1 being the more active. PGF1alpha appeared to have little or no vasoactive properties in doses up to 1 ugm. (2.8 times 10(-5M)) in our preparation while PGF2alpha was vasodilator at this high dosage only. Neither SC19920 nor diphloretin phosphate antagonished the effects of PGE1 in these experiments.     

Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets.

Prostacyclin (PGX) strikingly increases cyclic AMP concentrations in human platelets. Prostacyclin is approximately 10 times more active than PGD2, 30 times more active than PGE1 and more than 1000 times more active than its stable end product, 6-oxo-PGF1alpha. These results correlate well with the anti-aggregating activity of prostacyclin, compared with PGE1 and PGD2.     

Influence of the position of the side chain hydroxy group on the gastric antisecretory and antiulcer actions of E1 prostaglandin analogs.

The influence of transposing the C-15 hydroxy group of prostaglandin E1 methyl ester (PGE2ME) on gastric antisecretory and antiulcer actions was investigated. The compound (+/-)15-deoxy- 16alpha, beta-hydroxy PGE1ME (SC-28904) was equipotent to the reference standard PGE1ME in suppressing histamine-stimulated gastric secretion in the Heidenhain pouch (HP) dog. In contrast to PGE1ME, SC-28904 was longer acting when administered intravenously and also showed significant oral activity in the histamine-stimulated gastric fistula dog. SC-28904 was also equipotent to PGE1ME (range of active doses of 0.5 to 5.0 mg/kg, s.c.) in inhibiting forced-exertion gastric ulceration in rats. The compound (+/-)15-deocy-17alpha, beta-hydroxy PGE1ME (SC-30963) was an inactive antisecretory agent in the dog at the 1.0 mg/kg i.v. bolus dose. This dose was 100 times greater than the active antisecretory dose of PGE1ME. Likewise, SC-30693, when administered subcutaneously at a 5.0 mg/kg dose, was also totally inactive in preventing gastric ulcers induced by forced exertion in rats. The important implications of this work are that some of the receptor sites for the PGE1 molecule could easily accomodate the side chain hydroxy group either in the C-15 or C-16 position. Moreover, the hydroxy group in the latter position significantly improved the biological activity of PGE1ME.     

Differential sensitivity of osteoclasts and osteoblasts suggests that prostaglandin E1 effects on bone may be mediated primarily through the osteoclasts

Prostaglandin E (PGE) stimulates resorption in bone. Since osteoblast-like osteosarcoma cells secrete PGE2, the possibility that osteoclasts were the major target for PGE was considered. To study this question, it was first established that in isolated bone cells enriched for either osteoclastic (OC) or osteoblastic (OB) characteristics, PGE1 can induce biochemical effects similar to those seen with bovine parathyroid hormone 1-84 (PTH), another potent stimulator of bone resorption. These changes include increased cAMP and hyaluronate synthesis in OC cells, and increased cAMP but decreased citrate decarboxylation in OB cells. By following these markers, it is demonstrated that PGE1 can activate OC cells at doses as low as 1 nM, whereas OB cells require 250 nM. Bone cell responses to various doses of PTH and PGE1 were also compared. In OC cells the lowest effective dose of PGE1 and PTH was similar (1 nM), but increasing response to PGE1 was seen up to 1000 nM in contrast to PTH response which peaked at 20 nM. In addition, the magnitude of PGE1-induced OC cell hyaluronate was two to four times greater than that of PTH at all doses tested. In OB cells, PTH induced significant decreases in citrate decarboxylation at 0.1 nM, compared to 250 nM for PGE1. Half-maximal inhibition of citrate decarboxylation (19% of control) by PTH occurred at 0.5 nM, whereas 500 nM of PGE1 was required for an equivalent effect. Thus, (i) OC cells responded to PGE1 doses that were approximately 200 times lower than the minimum required by OB cells, and (ii) OB cells responded to 100 times lower doses of PTH than PGE1.     

Comparison of coagulation factor Xa and des-(1-44)factor Xa in the assembly of prothrombinase

The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.     

Enhancement of the PGE1 response of macrophages by concanavalin A and colchicine.

The effect of concanavalin A (Con A) and colchicine on the prostaglandin E1 (PGE1)-mediated cyclic AMP generation in rat peritoneal macrophages have been studied. Although Con A and colchicine by themselves did not affect cyclic AMP levels, they greatly enhanced cyclic AMP production induced by PGE1. There was not only augmentation of cyclic AMP levels at maximally active concentrations of PGE1, but also an increased sensitivity to low (inactive) concentrations of PGE1. Except for lentil lectin, none of the other lectins affected PGE1 sensitivity whereas lumicolchicine was as effective as colchicine. In addition, both Con A and colchicine raised the sensitivity to isoproterenol and choleraenterotoxin. Although details of the mechanisms by which Con A or colchicine influenced the membrane-bound adenyl cyclase and PGE1 receptors remain unclear, these observations suggest that certain alterations of the cell membrane may render macrophages more susceptible to the regulating effects of prostaglandins.     

Anti-inflammatory and anti-arthritic effects of new synthetic 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione

We previously reported that 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione (1, HMP) has a strong inhibitory effect on prostaglandin E(2) (PGE(2)) production. In this study, the anti-inflammatory and anti-arthritic effects of HMP were evaluated on LPS-induced RAW 264.7 macrophages and rats with carrageenan-induced paw edema and adjuvant-induced arthritis (AIA). The attenuation of PGE(2) production by HMP was found to be caused by the inhibition of cyclooxygenase-2 (COX-2) activity, but not COX-1 activity. However, HMP did not affect COX-2 at the protein or mRNA levels, whereas it suppressed the releases and expressions of inflammatory cytokines, such as, interleukin-1β (IL-1β) and IL-6 in LPS-induced macrophages. Furthermore, HMP suppressed LPS-induced nitric oxide (NO) production by down regulating the protein and mRNA expressions of inducible nitric oxide synthase (iNOS). In rats with carrageenan-injected acute inflammation, oral administration of HMP (25 or 50mg/kg, po) reduced paw swelling, and PGE(2) release and myeloperoxidase (MPO) activity in tissue. Furthermore, HMP (25 or 50mg/kg, po) significantly reduced paw swelling, arthritic indices and plasma PGE(2) concentrations in rat with AIA. These results show that HMP reduces swelling in a model acute inflammation and inhibits arthritic responses in a model of chronic inflammation via the inhibition of PGE(2) production. These results suggest that HMP is a potential therapeutic agent for the treatment of arthritis and associated disorders.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Synergism between PGE1-metabolites(13,14-dihydro-prostaglandin E1, 15-keto prostaglandin E1, 15-keto-13,14-dihydro-prostaglandin E1) and nitric oxide (NO) on platelet aggregation.

A synergistic antiplatelet effect between prostaglandins (PG), cAMP-stimulators and nitric oxide (NO), a cGMP-stimulator, has already been described. Data on a synergism between NO and the metabolites of PGE1, however, are lacking so far. We therefore tested the antiplatelet activity of the metabolites of PGE1 alone and their synergism with NO on human platelets of 8 healthy volunteers in vitro. 13,14-DH-PGE1 (ID 50 = 10.8 ng/ml platelet rich plasma (PRP)) was the only PGE1 metabolite inhibiting the ADP-induced platelet aggregation, its efficacy being 76.4% of the parent compound PGE1 (ID 50 = 8.25 ng/ml PRP). NO (ID 50 = 0.52 microM) also inhibited platelet aggregation. The combined addition of 13,14-dihydro-prostaglandin E1 (13,14-DH-PGE1) and NO caused an additive effect. The other PGE1-metabolites tested, 15-keto prostaglandin (15-K-PGE1) (ID 50 = 16.2. micrograms/ml PRP) and 15-keto-13,14-dihydro-prostaglandin(15-K-13,14-DH-PGE1) (ID 50 = 14.8 micrograms/ml PRP), neither had any relevant antiaggregatory capacity themselves nor a synergistic effect with NO. These findings could be of clinical relevance as a NO-synergism may occur not only with therapeutically administered PGE1 but also with its biologically active metabolite 13,14-DH-PGE1.     

The effect of prostaglandin E1 on ion currents of NG108-15 cells

The aim of this study was to elucidate the mechanism by which prostaglandin E(1) (PGE(1)) acts on ion currents of whole-cell voltage-clamped NG108-15 neuroblastomaxglioma hybrid cells. Ruptured and perforated patch were used. The holding current at -70 mV, the current-voltage curve produced by ramp pulses from -70 to 0 mV and the T-type and hva (high-voltage-activated) Ca(2+) currents associated with rectangular pulses were recorded. Bath application of PGE(1) (0.2 or 3 microM) reversibly increased the holding current, an effect mimicked by the prostanoid agonist iloprost (5-50 nM). The PGE(1) effect was totally blocked by the cAMP-antagonist Rp-cAMPS whereas H-89, an inhibitor of protein kinase A (PKA), failed to inhibit it, even when applied in the fairly high bath concentration of 30 microM. PGE(1) and iloprost also inhibited the T-type and hva Ca(2+) currents and this effect of PGE(1) was likewise not prevented by H-89. In some of the cells, the PGE(1) effect on holding current could be mimicked by 8-pCPT-2Me-cAMP (100-300 microM), a selective agonist of Epac (exchange protein activated by cAMP), but unlike the PGE(1) effect its action was not abolished by Rp-cAMPS. The effect of PGE(1) on the the holding current and on the T-type Ca(2+) current was diminished when EGTA in the pipette solution was replaced by BAPTA, suggesting that Ca(2+) ions are involved in the PGE(1) effect. It is concluded that the PGE(1) effect is mediated by cAMP and Ca(2+) ions but not by PKA or Epac.     

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.     

Blood pressure and cardiac tissue responses to prostacyclin (PGI2) in various species

The effect of prostacyclin (PGI2) on blood pressure and heart rate (in vivo) and on isolated heart tissue has been investigated in different species. Isolated cardiac tissue had limited responses to PGI2 tested at 10(-13) to 10(-5) M. Cultured neonatal rat heart cells did not respond to PGI2, neither did intact rat hearts or rabbit cardiac tissue. Guinea pig and rat atria showed limited dose-dependent responses to PGI2 at concentrations greater than 10(7) M. In rat atria, 10(-5) M PGI2 produced a limited elevation of tissue cAMP content. When given by intravenous injection or infusion, PGI2 produced hypotension in anaesthetized primates (three species), rat, rabbit, pig, and dog. As a vasodepressor in all species, PGI2 (on a weight basis) was more active than prostaglandins of the B or E type and, in most species tested, it was approximately five times more active than PGE2. Heart responses in intact animals were often paradoxical in that decreases in heart rate often accompanied blood pressure falls.     

Interleukin 1- and tumor necrosis factor-stimulation of prostaglandin E2 synthesis in MDCK cells, and potentiation of this effect by cycloheximide

The effects of interleukin (IL)-1 alpha, IL-1 beta and TNF alpha on prostaglandin-E2 synthesis in Madin-Darby canine kidney (MDCK) cells were investigated. IL-1 beta time- and dose-dependently stimulated prostaglandin-E2 synthesis. While TNF alpha produced a comparatively small but significant stimulation of PGE2 release, coincubation of IL-1 beta with TNF alpha produced a marked synergistic stimulation of PGE2 release. The effect of IL-1 beta and of IL-1 beta and TNF alpha was apparent as early as after 2 h of incubation. The enhanced PGE2 synthesis was inhibited by indomethacin as well as actinomycin D, while cycloheximide surprisingly potentiated PGE2 synthesis in response to both IL-1 beta and TNF alpha. IL-1 alpha alone was ineffective in stimulating a significant release of PGE2 at concentrations as high as 10 nM. However, it also showed a marked synergistic interaction with TNF alpha in stimulating PGE2 release.     

Inflammatory effects of prostacyclin (PGI2) and 6-oxo-PGF1alpha in the rat paw.

In the rat paw prostacyclin was 5--10 times less potent than PGE2 in causing oedema, and 5 times less potent in potentiating carrageenin-induced oedema, which it did in a dose-related manner. Prostacyclin was 5 times more potent than PGE2 in producing hyperalgesia and as potent as PGE2 in restoring carrageenin-induced hyperalgesia. The effects on oedema were longer lasting than those on hyperalgesia. 6-oxo-PGF1alpha was 500 times less potent than PGE2 in causing oedema by itself and in potentiating carrageenin-induced oedema. It had no hyperalgesic activity in this test.     

Prostaglandin E1 binding protein on the surface of primary cultured hepatocytes

Prostaglandin E1 (PGE1) was bound to primary cultured rat hepatocytes in a receptor-dependent manner in serum-free medium at 4 degrees C. When added at a concentration of 2 X 10(-9) M, maximal specific binding occurred within 60-90 min. Trypsin treatment of the cells reduced the binding capacity to about 50% of that of untreated cells. Scatchard-analysis of the binding data showed that the cells had an apparent dissociation constant of 1.2 X 10(-8) M and a binding capacity of 580 fmol (approximately 3.5 X 10(11) PGE1 receptors)/mg of protein. In experiments at 37 degrees C, maximal specific binding occurred within 5 min and was 6-7 times that at 4 degrees C, but the amount of bound PGE1 decreased rapidly after 5 min due to metabolism of PGE1 in the hepatocytes. Thin-layer chromatographic analysis showed that the material bound to the cell surface consisted of intact PGE1 and its metabolites at 37 degrees C, but PGE1 only at 4 degrees C.     

Macrophage biology in the Anx-A1-/- mouse

Historical data suggested that a soluble protein, since identified as annexin-A1 (Anx-A1) was released from macrophages following glucocorticoid stimulation and could modulate eicosanoid production and other functions of these cells. Here, we review some recent findings using a line of Anx-A1(-/-) mice to explore the impact of Anx-A1 gene deletion on macrophage biology. The absence of Anx-A1 selectively alters phagocytic capacity of rodent resident peritoneal macrophages apparently through changes in surface adhesion molecule expression. Anx-A1 is also apparently important in the tonic down-regulation of other macrophage functions such as COX-2 induction, PGE(2) release and the production of reactive oxygen species.     

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.     

PGE1 and prostacyclin suppression of NK-cell mediated cytotoxicity and its relation to cyclic AMP

The capacity of three prostanoids (PGE1, 6-beta-PGI1, PGI2 or prostacyclin) and a phosphodiesterase inhibitor (rolipram) to inhibit NK ("natural killer") cell cytotoxicity and to raise cyclic AMP levels in purified NK cells was compared. PGE1 was about 200 times more potent than prostacyclin both in its ability to raise cyclic AMP and to inhibit NK cell cytotoxicity. The stable prostacyclin analogue, 6-beta-PGI1, had an intermediate potency. A 50% inhibition of cytotoxicity was obtained at approximately 10(-8) M for PGE1, 10(-7) M for 6-beta-PGI1, and 10(-6) M for both prostacyclin and rolipram. These doses raised the level of cyclic AMP by approximately 100%. These results suggest that PGE1 is likely to be more important as an endogenous regulator of lymphocyte cytotoxicity than prostacyclin. The results also provide further evidence that cyclic AMP is the mediator of prostanoid-induced reduction in NK cell activity.