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Despite recent advances in our understanding of the biological processes leading to the development and progression of cancer, there is still a need for new and effective agents to treat this disease. Phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are non-enzymatically oxidized products of α-linolenic acid that are present in seeds and vegetable oils. They have been shown to possess anti-inflammatory and apoptosis-promoting activities in macrophages and leukemia cells, respectively. In this work, seven PhytoPs (PP1-PP7) and one PhytoFs (PF1) were evaluated for their cytotoxic, chemosensitization, and anti-migratory activities using the MCF-7 and MDA-MB-231 breast cancer cell lines. Among the tested compounds, only three PhytoPs had a significant effect on cell viability compared to the control group: Ent -9-L 1 -PhytoP (PP6) decreased cell viability in both cell lines, while 16-F 1t -PhytoP (PP1) and 9-L 1 -PhytoP (PP5) decreased viability of MCF-7 and MDA-MB-231 cells, respectively. When combined with a sub-cytotoxic dose of doxorubicin, these three PhytoPs displayed significantly enhanced cytotoxic effects on MCF-7 cells while the chemotherapeutic drug alone had no effect. In cellular motility assays, Ent -9-( RS )-12- epi -ST-Δ 10 -13-PhytoF could significantly inhibit cellular migration of MDA-MB-231 cells. In addition, Ent -9-( RS )-12- epi -ST-Δ 10 -13-PhytoF also enhanced cellular adhesion of MDA-MB-231 cells.

Background: Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting β(2) adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E(2) (PGE(2))-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro., Methods: Using an extensive range of pharmacological tools, the relaxant potential of PGE(2) and selective agonists for the EP(1-4) receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated., Results: In agreement with previous studies, it was found that the EP(2) receptor mediates PGE(2)-induced relaxation of guinea pig, murine and monkey trachea and that the EP(4) receptor mediates PGE(2)-induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP(2) receptor-deficient mice (Ptger2). In contrast to previous publications, a role for the EP(4) receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP(2) agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies., Conclusion: Identification of the receptor mediating PGE(2)-induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP(2) receptor agonists in clinical studies.

Prostanoids and cannabinoids have ocular hypotensive and neuroprotective properties. The effect of the prostanoid AH13205 (EP2), the thromboxane-mimetic U46619, the cannabinoid (CB) agonists WIN55212-2 and CP 55,940, endothelin-1 (ET-1) and 8-bromo-cAMP on the membrane currents of trabecular meshwork (TM) cells were measured using the patch-clamp technique and compared to their effects on TM contractility. Previous studies show relaxation of TM to AH 13205 and other substances that elevate cAMP, while U46619 and endothelin-1 contract TM. This study shows that after contraction (100%) with carbachol (10(-6)m), the CB agonist CP 55,940 dose-dependently reduced contractility to 83+/-4% (n=9) (10(-6)m) and 61+/-10%, (n=7) (10(-5)m). In the presence of both the CB1 antagonist AM251 (10(-6)m) and CP 55,940 (10(-5)m), the contractile response to carbachol reached 84+/-3% (n=6) of the original level. In patch-clamp experiments, membrane permeable 8-bromo-cAMP (10(-4)m) had no effect on currents of TM cells. In contrast, AH 13205 and two cannabinoids reversibly enhanced outward current through high-conductance Ca(2+)-activated K(+) channels (BKCa, BK, maxi-K) to the following values (in % of the initial value at 100 mV): AH 13205 (10(-5)m): 200+/-28% (n=6), CP 55,940 (10(-6)m): 196+/-33% (n=7), CP 55,940 (10(-5)m): 484+/-113% (n=7), WIN55212-2 (10(-5)m): 205+/-41% (n=10). Iberiotoxin (10(-7)m) completely blocked these responses. The current response to CP 55,940 (10(-5)m) could be partially blocked by the CB1 antagonist AM251 (10(-6)m). Conversely, the contractile agents in this study either caused a transient reduction in outward current (ET-1(5x10(-8)m)) or had no effect (U46619 (10(-6)m)). We conclude that stimulation of EP2 and CB1 receptors in TM is coupled to the activation of BKCa channels via a non-diffusible second messenger cascade. This effect may contribute to the relaxant activity of EP2 and CB1 agonists in isolated TM strips, modulating ocular outflow.

Novel 9,11-ethano analogues of prostaglandin endoperoxides with a nitrogen in position 13 were synthesized. (1)H NMR spectra of the obtained compounds were studied. All prostanoids administered perorally at doses of 2.5-10.0 microg x kg(-1) had specific dose-dependent effects on the B-cellular immunity estimated under in vivo conditions on the model of the B-cellular immune response. In terms of the direction of their activities, eight of the studied compounds were found to be immunostimulators, whereas other three compounds displayed immunosuppressing effect. Two of the compounds increased the amount of antibody-forming cells (AFC) per 10(6) spleen cells by 1.9 times in comparison with the respective parameter of control group.

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E(2), an EP(2)receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP(1)and EP(3)receptor agonist (sulprostone), a EP(4)receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D(2)showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D(2)may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP(2)receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D(2)and AGN 192093 need further study., (Copyright 2001 Academic Press.)

Von Willebrand factor (vWF) is a large plasma glycoprotein that mediates platelet adhesion at sites of vascular injury. We have previously reported that the pathological type 2B (formerly named type IIB) variant of vWF promotes platelet activation through phospholipase A(2)-mediated release of arachidonic acid. The present report shows that adrenaline (1 microM) potentiates type 2B vWF-induced platelet aggregation, serotonin secretion, rise in cytosolic Ca(2+) concentration, and pleckstrin phosphorylation, as well as thromboxane B(2) production. The hormone also increases the partially inhibited release of serotonin observed in platelets pretreated with the anti-GPIIb-IIIa antibody LJCP8 but does remove the total inhibition on the secretion caused by the anti-GPIb antibody LJIB1. Adrenaline also increases type 2B vWF-elicited tyrosine phosphorylation of proteins with apparent molecular masses of 60 and 80 kDa. Furthermore, adrenaline potentiates the rise in cytosolic Ca(2+) and the release of thromboxane B(2) in platelets stimulated with arachidonic acid (2 microM) as well as the increase in Ca(2+) induced by the thromboxane mimetic U46619 (0.3 microM). Platelet pretreatment with yohimbine or 13-azaprostanoic acid, which are antagonists of the alpha(2)-adrenergic and thromboxane receptors, respectively, or with acetylsalicylate and indomethacin, both of which act as inhibitors of thromboxane formation, abolishes the potentiating effect of adrenaline. These observations lead to the conclusion that the potentiating action of adrenaline on type 2B vWF-promoted platelet responses is due to an increase in both the formation and activating action of thromboxanes.

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists. 2. In human monocytes that were adherent to plastic, neither prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F(2 alpha) (PGF(2 alpha)) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNF alpha-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells. 3. Exposure of human monocytes to LPS (3 ng ml-1, approximately EC84) resulted in a time-dependent elaboration to TNF alpha which was suppressed in cells pretreated with prostaglandin E1 (PGe1), PGE2 and cicaprost. This effect was concentration-dependent with mean pIC50 values of 7.14, 7.34 and 8.00 for PGE1, PGE2 and cicaprost, respectively. PGD2, PGF(2 alpha) and U-46619 failed to inhibit the generation of TNF alpha at concentrations up to 10 microM. 4. With respect to PGE2, the EP-receptor agonists, 16,16-dimethyl PGE2 (non-selective), misoprostol (EP2/EP3-selective), 11-deoxy PGE1 (EP2-selective) and butaprost (EP2-selective) were essentially full agonists as inhibitors of LPS-induced TNF alpha generation with mean pIC50 values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE1, another EP2-selective agonist, AH 13205, inhibited TNF alpha generation by only 21% at the highest concentration (10 microM) examined. EP-receptor agonists which have selectively for the EP1- (17-phenyl-omega-trinor PGE2) and EP3-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNF alpha generation. 5. Pretreatment of human monocytes with the TP/EP4-receptor antagonist, AH 23848B, at 10, 30 and 100 microM suppressed LPS-induced TNF alpha generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves. 6. Given that AH 13205 was a poor inhibitor of TNF alpha generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 and 30 microM AH 13205 inhibited the generation of TNF alpha by 31% and 53%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves at either concentration examined. 7. Since PGD2 and 17-phenyl-omega-trinor PGE2 (EP1-agonist) did not suppress TNF alpha generation, the EP1/EP2/DP-receptor antagonist, AH 6809, was employed to assess if EP2-receptors mediated the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 microM AH 6809 did not affect LPS-induced TNF alpha generation but produced a parallel 3.5 fold rightwards shift of the PGE2 concentration-response curve. 8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNF alpha generation. One of these probably represents i.p. receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP2- and/or EP-4- and/or i.p.) or novel EP-receptors mediate the inhibitory effect of PGE2 on TNF alpha generation.

1. Prostanoids induce a wide range of biological actions which are mediated by specific membrane-bound receptors. We have recently shown that the E-type prostaglandins, PGE1 and PGE2, effectively inhibit eosinophil aggregation induced by platelet-activating factor (PAF). In an attempt to determine which prostanoid receptor(s) were involved, we investigated the effects of a range of selective prostanoid agonists and antagonists on eosinophil homotypic aggregation induced by PAF. 2. Both PGE1 and PGE2 (10(-10) to 10(-6) M) induced a concentration-related inhibition of the aggregation response induced by PAF. PGE1 was more effective than PGE2 but PGE2 was slightly more potent than PGE1 (approximate IC50 values for PGE1 and PGE2 of 1.5 x 10(-8) M and 5 x 10(-9) M, respectively). 3. The EP2-selective agonists, 11-deoxy-PGE1, butaprost and AH13205, and the EP2/EP3-selective agonist, misoprostol, also inhibited PAF-induced aggregation. The rank order of potency for EP2-selective agonists was 11-deoxy-PGE1 > misoprostol > butaprost = AH13205. The protein kinase A inhibitor, KT5720 (10(-6) M), reversed the inhibitory effects of 11-deoxy-PGE1 (10(-6) M) and AH13205 (10(-5) M). 4. The EP1/EP3-selective agonist, sulprostone, and the EP1-selective agonist, 17-phenyl-omega-trinor PGE2, had no significant inhibitory activity when tested at concentrations up to 10(-6) M. The EP4-receptor antagonist, AH23848B, had no effect on PAF-induced aggregation and did affect the inhibitory activity of PGE1. 5. The IP-selective agonist, cicaprost (up to 10(-6) M), and the IP/EP1-receptor agonist, iloprost (up to 10(-5) M), had no significant effect on PAF-induced eosinophil aggregation. However, iloprost significantly augmented the inhibitory effects of a maximally inhibitory concentration of PGE2. 6. PGD2 (10(-5) M) had no effect on eosinophil aggregation and the inhibitory activity of PGE1 on PAF-induced eosinophil aggregation was not altered by the DP-selective receptor antagonist, BWA868C. 7. The results presented here suggest that the inhibition of PAF-induced eosinophil aggregation by prostanoids is mediated by the occupation of EP2-receptors. It is important to note that the effects of naturally occurring prostanoids, such as PGE2, on eosinophil aggregation occur at low concentrations highlighting a potential role for EP2 receptors in regulating eosinophil function in vivo.

Many nonsteroidal antiinflammatory agents (NSAIDs) bind to prostaglandin endoperoxide synthase (PGHS) and induce a conformational change in the PGHS apoprotein that renders it resistant to cleavage by trypsin at Arg277. In the present study, the trypsin protection assay was modified to permit detection of conformational changes at times as short as 5 s after the addition of inhibitor. The kinetics of the induction and reversal of trypsin resistance in apoPGHS-1 by a series of NSAIDs and isozyme-specific PGHS-1 and PGHS-2 inhibitors were determined. All compounds induced resistance to trypsin cleavage at a rapid rate. The conformational change induced by competitive inhibitors was reversed on prolonged incubation with trypsin (approximately 5 min). In contrast, the resistance induced by irreversible inhibitors was not lost during a 5 min incubation with trypsin. All of the selective PGHS-2 inhibitors protected against tryptic cleavage of apoPGHS-1 but did not inhibit the protein's cyclooxygenase activity. The results suggest that induction of trypsin resistance is a reflection of the initial association of reversible as well as irreversible inhibitors with the apoprotein.

Spontaneous contractile activity of strips of human myometrium obtained from non-pregnant donors at the time of hysterectomy was inhibited by the selective prostanoid DP receptor agonists BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin) and ZK110841 ((5Z,13E)-(9R,11R,15S)-9 beta-chlor-15-cyclohexyl-11,15-dihydroxy-16,17,18,19, 20-pentanor-5,13-prostadienoic acid) with pEC50 values of 8.4 and 7.3 respectively but prostaglandin D2 produced a biphasic effect. In the presence of the TP receptor antagonist L670596 ((-)-6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid), contractile activity induced by the FP receptor agonist, cloprostenol ([1R-[1 alpha(Z),2 beta(1E,3R),3 alpha,5 alpha]]-7-[2-[4-(3- chlorophenoxy)-3-hydroxy-7-butenyl]-3,5-dihydroxycyclopentyl]-5-he ptenoic acid), was inhibited by BW 245C (pEC50 = 7.5), ZK110841 (pEC50 = 6.7) and prostaglandin D2 (pEC50 = 6.3). Under these conditions both prostaglandin J2 and 9 alpha,11 beta-prostaglandin F2 were inhibitory partial agonists. All compounds were antagonized by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)h ydantoin), but the pKB values were both concentration-dependent (pKB versus BW 245C at 10 nM = 9.1, at 50 nM = 8.3) and agonist-dependent (pKB at 10 nM versus BW 245C = 9.1, versus ZK110841 = 7.4). Both agonist and antagonist potencies support the existence of DP receptors in human myometrium. The concentration and agonist dependence of the action of BW A868C suggests that putative DP receptor agonists relax human myometrium by more than one mechanism. These observations may be explained by the existence of subtypes of DP receptor in human myometrium.

The cloning of the genes that encode for prostaglandin (PG) receptors has resolved much of the complexity and controversy in this area by confirming the classification proposed by Coleman, et al. Two issues that remained unresolved were (1) the inability of the EP2 agonist butaprost to interact with the cloned putative EP2 receptor and (2) molecular biological confirmation of a fourth PGE2-sensitive receptor, which was pharmacologically designated EP4. In order to provide clarification, we attempted to clone further PGE2-sensitive receptors. By using a cDNA probe that encodes for the human EP3A receptor, a cDNA clone that encoded for a novel PGE2-sensitive receptor was obtained by screening a human placenta library. This cDNA clone was transfected into COS-7 cells for pharmacological studies. The cDNA clone obtained from human placenta had only about 30% amino acid identity with cDNAs for other PG receptors, including those that encode for the previously proposed murine and human EP2 receptors. Radioligand binding studies on the novel EP receptor expressed in COS-7 cells revealed that selective EP2 agonists such as butaprost, AH 13205, AY 23626 and 19(R)-OH PGE2 all competed with 3H-PGE2 for its binding sites, whereas selective agonists for other PG receptor subtypes had minimal or no effect. This receptor was coupled to adenylate cyclase and EP2 agonists caused dose-related increases in cAMP. It appears that the cDNA described herein encodes for the pharmacologically defined EP2 receptor. Ocular studies revealed that AH 13205 decreased intraocular pressure in normal and ocular hypertensive monkeys by a mechanism that does not appear to involve inhibition of aqueous humor secretion.

A fourth PGE receptor subtype, the EP4 receptor, has recently been described in the pig saphenous vein (PSV). Similar to the EP2 receptor, it mediates relaxation and is linked to stimulation of adenylate cyclase. The aim of this study was to determine whether or not the EP receptor present in the rabbit jugular vein (RJV), currently classified as an atypical EP2 receptor, is of the EP4 subtype. The relaxant activities of four EP2 agonists, 11-deoxy PGE1, 16,16-dimethyl PGE2, butaprost, and AH 13205, on the RJV and PSV have been examined, and the effect of the EP4 receptor antagonist AH 23,848B studied. The EP2 agonists showed a similar order of potency on the two preparations. 11-Deoxy PGE1 and 16,16-dimethyl PGE2 were potent agonists on the EP4 receptors of the PSV and on the RJV giving approximately equi-effective concentration ratios (EECs) of 2.0-6.6 and 2.8-9.9, respectively, compared to PGE2 (EEC = 1), and so do not discriminate between EP2 and EP4 receptors. Butaprost was less active on these preparations (EEC 42-43) than on classical EP2 receptors, and AH 13205 was much less active (EEC 3100-2780). While these results suggest that the EP receptors on the RJV are of the EP4 subtype, this was not confirmed using the EP4 receptors antagonist AH 23,848B.

Objective: Our purposes were to assess the effects of prostaglandin (PG) E2 and PGF2 alpha on myometrial contractility in pregnant sheep and baboons in an in vitro superfusion study, and to characterize further the PGE-sensitive (EP) receptor subtype involved in the myometrial response to PGE2 by using the selective prostanoid EP2 agonist AH 13205., Methods: Strip preparations of uterine muscle from 15 sheep (107-145 days' gestational age) and ten baboons (158-185 days' gestation) were studied. Cumulative concentration-response curves (CRC) were constructed to oxytocin (4.2 pmol/L to 0.42 mumol/L, PGE2 (0.1 nmol/L to 1 mumol/L), and PGF2 alpha (1 nmol/L to 100 mumol/L), and 50% effective concentration (EC50) values (mean and 95% confidence interval) were calculated. We also tested the hypothesis that PGE2-induced myometrial relaxation in pregnant baboons could be mediated by EP2-prostanoid receptors. Myometrial strips were stimulated by oxytocin (0.42 nmol/L), and CRCs to the EP2-agonist AH 13205 (0.1 nmol/L to 10 mumol/L) were constructed., Results: Prostaglandin F2 alpha stimulated myometrial activity in a concentration-related fashion in all preparations from both sheep and baboons. The EC50 in the sheep myometrium for PGF2 alpha (52 nmol/L, 95% confidence interval [CI] 25-110) was significantly (P < .05) lower than that in baboon myometrium (183 nmol/L, 95% CI 93-355). Oxytocin stimulated myometrial activity in preparations of both sheep (EC50 = 0.29 nmol/L, 95% CI 0.11-0.71) and baboon (EC50 = 0.31 nmol/L, 95% CI 0.18-0.52). In contrast, responses to PGE2 were species-related: PGE2 caused concentration-related stimulation of myometrial activity in sheep tissue (EC50 = 3.2 nmol/L, 95% CI 2.0-5.0), but induced concentration-related inhibition of activity in baboon myometrium (50% inhibitory concentration [IC50] = 21 nmol/L, 95% CI 2.2-203). A concentration-related inhibitory response to AH 13205 (IC50 = 3.56 nmol/L, 95% CI 1.28-5.99) was obtained in the baboon. In contrast, AH 13205 failed to inhibit comparable myometrial strip preparations from pregnant sheep., Conclusions: The present studies suggest that both sheep and baboon myometrium contain prostanoid receptors that mediate stimulation. In addition, baboon myometrium, like that from the human, contains AH 13205-sensitive EP receptors (EP2 receptors), which mediate inhibition. The pregnant baboon may therefore represent a suitable animal model for investigations into the use of EP2 agonists for the prevention of premature labor in humans.

The purpose of this study was to determine which receptors mediate the dilator effects of prostaglandins (PGs) on the ductus arteriosus of the fetal rabbit. Isolated rings of the vessel from fetal New Zealand White rabbits were precontracted with indomethacin (1 microM) and potassium (25 mM) in 100 to 110 mmHg oxygen, and the dilator effects of a range of synthetic prostanoids were quantified by cumulative relaxant concentration-effect curves. The potencies of agonists were quantified with reference to PGE2 by the equieffective molar ratio (EMR): EC50 test agonist/EC50 PGE2. The effects on these responses of available antagonists were also studied. None of a range of synthetic prostanoids with selective agonism of EP1, EP2 or EP3 receptors was as potent as PGE2. The rank order of potency was as follows: PGE2 (EC50 = 0.36 nM [95% confidence intervals [CI] = 0.32-0.41, n = 44], EMR = unity) >> misoprostol (EMR 145, 95% CI 73-217) > [1R-[1 alpha (Z),2 beta (R*),3 alpha]]-4-(benzoyl-amino)phenyl 7-[3 hydroxy-2(2-hydroxy-3-phenoxypropoxy)-5- oxocyclopentyl]-4-heptenoate, single enantiomer (GR 63799K) (EMR 685, 95% CI 427-944) >> ((+/-)-trans-2-[4[(1-hydroxphenyl) phenyl]-5-oxocylopentaneheptanoic acid (AH13205) (EMR > 100,000) > or = sulprostone (EMR > 10,000) > or = 0. The EP1 antagonists, 6-isopropoxy-9-oxoxanthine-2-carboxylic acid (AH6809) (10 microM) and 8-clorodi-benz[b,f][1,4]oxazepine-10 (11H)-carboxylic acid, 2-acetylhydrazide (SC19220) (30 microM), had no significant effect on the sensitivity of the ductus to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Using a range of natural and synthetic prostanoid receptor agonists and antagonists, we have shown that the rat isolated trachea contains a heterogeneous population of prostaglandin receptor sub-types mediating both relaxation and contraction of the smooth muscle. Prostaglandin E2 (PGE2) elicits smooth muscle relaxation of pre-contracted preparations, the responses being well defined, with a mean potency (p[A50]) of 7.81 +/- 0.05. 2. 11-deoxy PGE1 16,16-dimethyl PGE2 and misoprostol were all full agonists at this receptor, whilst AH13205 was a low potency agonist, and sulprostone was inactive. 3. The EP1 receptor antagonist, AH6809 (5 microM), and the selective DP receptor antagonist, BW A868C (0.1 microM), had no significant effect on the concentration-effect (E/[A]) curves to PGE2. 4. The putative EP4-receptor antagonist, AH23848B, produced non-competitive antagonism of the PGE2 response curves; pA2 values of 5.07 +/- 0.15 and 5.24 +/- 0.19 were obtained at concentrations of 30 microM and 100 microM respectively. 5. The synthetic thromboxane A2 mimetic, U46619, caused smooth muscle contractions, with a mean p[A50] of 6.90 +/- 0.11. This response was antagonized by the TP receptor antagonist, GR32191B, yielding a mean pA2 of 8.31. 6. At concentrations of 1 microM and above, prostaglandin D2 (PGD2) and the IP-receptor agonist, cicaprost, generally elicited concentration-dependent relaxations of the rat trachea. Prostaglandin F2 alpha (PGF2 alpha) was without affinity or efficacy. 7. These data suggest that the rat isolated trachea contains EP-receptors, TP-receptors, and few, if any DP, IP or FP-receptors. The inactivity of sulprostone (EP3/EPj receptor selective) and the low potency of AH1 3205 (EP2-receptor selective) suggest that the rat trachea contains an atypical EP-receptor that does not conform to the current classification system.

The vascular contractile mechanism of prostacyclin (PGI2) was investigated using beraprost sodium (BPS), a stable PGI2 analog. Ring strips without endothelium isolated from canine femoral veins and arteries were used. BPS induced a dose-dependent contraction without precontraction and after precontraction with norepinephrine (NE) or 60 mM K+ in the veins. In contrast, BPS induced a dose-dependent relaxation after precontraction with U46619, a thromboxane A2 (TXA2) analog, or prostaglandin F2 alpha (PGF2 alpha) in the veins. In the arteries, BPS induced contraction at higher concentrations after precontraction with NE. However, BPS relaxed arteries dose-dependently after precontraction with PGF2 alpha. By pretreatment with 13-azaprostanoic acid (13-APA), a TXA2/endoperoxide receptor antagonist, the dose-response curve of BPS in the veins was shifted to the right. Schild plot analysis resulted in a linear regression with a slope of 0.86 +/- 0.13, which was not significantly different from unity, and the pA2 value for 13-APA against BPS was 7.10 +/- 0.06. By pretreatment with BPS, the dose-response curve of U46619 in the veins was shifted to the right. Kaumann plot analysis resulted in a linear regression with a slope of 0.89 +/- 0.09, which was not significantly different from unity, and the pA2 value for BPS against U46619 was 5.68 +/- 0.04. These findings indicate that BPS is a partial agonist for the TXA2/endoperoxide receptors.

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.

Some 13-azaprostanoic acid derivatives were shown to have a high cytoprotective activity against the damaging effect of ethanol on the gastric mucosa in non-strain rats and affect ATP- and arachidonate-induced platelet aggregation in the whole blood of rabbits and rats in a different way. The gastroprotective activity that is common to natural and synthetic prostanoids is not closely related to the chemical structure of the compounds. On the contrary, the trends of these compounds to affect the induced platelet aggregation depend on the modification of both the cyclic moiety and both chains of a prostanoid molecule.

A vasoconstrictor-induced prostacyclin (PGI2) production in a perfused rat heart was found, suggesting a mitigating role of PGI2 on coronary vasoconstriction. Treatment of the heart with cyclooxygenase inhibitors (aspirin or indomethacin) decreased PGI2 production by more than 90% and paradoxically reduced the vasoconstriction response. The attenuating effect of cyclooxygenase blockade suggested that endogenous prostanoids contribute to serotonin-, vasopressin- or U46619-induced vasoconstriction. Two prostaglandin (PG) H2/thromboxane A2 (TXA2) receptor antagonists, i.e., 13-azaprostanoic acid (13-APA) and SQ 29,548 were used to investigate putative endogenous vasoconstrictor prostanoids on the exogenously induced vasoconstriction. Retrogradely perfused (5-6 ml/min) rat hearts were rendered guiescent, yet responsive to stimuli, by local injection of lidocaine to the atrioventricular node. Changes in coronary vascular resistance (i.e., perfusion pressure at constant flow) were monitored and the cardiac effluent was collected for analysis of 6-keto PGF1 alpha (the stable metabolite of PGI2) as well as PGF2 alpha by radioimmunoassay. Three vasoconstrictors, i.e., serotonin, vasopressin and the TXA2/PGH2 analog U46619, as well as authentic PGD2, PGE2 and PGF2 alpha were infused. PGD2, PGE2 and PGF2 alpha exerted a dose-related coronary vasoconstriction, as did U46619, serotonin and vasopressin. Treatment with 13-APA (100 microM) or SQ 29,548 (100 nM) almost abolished U46619-induced vasoconstriction. The addition of PGH2/TXA2 receptor antagonists also significantly reduced the pressor effect of exogenously administered PGs, serotonin and vasopressin, with the exception that SQ 29,548 did not significantly antagonize PGE2-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effect of a new prostaglandin analogue, 9-hydroxy-19,20-bis-norprostanoic acid (rosaprostol), on the duodenal mucosa of humans after administration of 40% ethanol. Eighteen healthy volunteers entered the study, which followed a blind cross-over design. At time 0 all the volunteers received rosaprostol or placebo; 5 minutes later, 40% ethanol (50 ml dose) was given. The mucosa was examined for lesions 3, 60 and 180 min later under endoscopy, light and scanning microscopy. It was found that: (i) 40% ethanol damaged the duodenum, with blood extravasation, inflammation and necrosis of the mucosa, (ii) rosaprostol significantly protected the mucosa 3 min after 40% ethanol, and (iii) the damage became worse after 3 h when placebo was given, whereas it was reduced after 1 h when PG was administered. The results suggest that PGs not only protect the mucosa against ethanol damage, but also stimulate its recovery.

The use of enzyme inhibitors to clarify the role of thromboxane A2 in vasoocclusive disease has been complicated by their non-specific action. To address this problem we have examined the effects of thromboxane A2/prostaglandin endoperoxide receptor antagonism in a canine model of platelet-dependent coronary occlusion. Two structurally distinct thromboxane A2/prostaglandin endoperoxide receptor antagonists, 3-carboxyl-dibenzo (b, f) thiepin-5,5-dioxide (L636,499) and (IS-(1 alpha,2 beta(5Z),3 beta,4 alpha))-7-(3-((2-((phenylamino)-carbonyl)hydrazino)methyl)-7- oxabicy-clo(2.2.1)-hept-2-yl)-5-heptenoic acid (SQ 29,548), were studied to ensure that the effects seen in vivo were mediated by receptor antagonism and did not reflect a nonspecific drug effect. Both compounds specifically inhibited platelet aggregation induced by arachidonic acid and by the prostaglandin endoperoxide analogue, U46619, in vitro and ex vivo, and increased the time to thrombotic vascular occlusion in vivo. When an antagonist (L636,499) was administered at the time of occlusion in vehicle-treated dogs, coronary blood flow was restored. In vitro L636,499 and a third antagonist, 13-azaprostanoic acid, specifically reversed endoperoxide-induced platelet aggregation and vascular smooth muscle contraction. Neither compound altered cyclic AMP in platelet-rich plasma before or during disaggregation. Therefore, reversal of coronary occlusion may reflect disaggregation of platelets and/or relaxation of vascular smooth muscle at the site of thrombus formation through specific antagonism of the thromboxane A2/prostaglandin endoperoxide receptor. Thromboxane A2/prostaglandin endoperoxide receptor antagonists are compounds with therapeutic potential which represent a novel approach to defining the importance of thromboxane A2 and/or endoperoxide formation in vivo.

The mechanism of antiplatelet potency of prostanoids and pharmacological methods that regulate their endogenous release are discussed. In addition the role of antiaggregatory prostanoids in atherosclerosis and some clinical trials with prostanoids are reviewed.

The potential deleterious role of the proaggregatory vasoconstrictor, thromboxane A(2), in endotoxic shock was investigated in rats. Plasma thromboxane A(2) was determined by radioimmunoassay of its stable metabolite thromboxane B(2). After intravenous administration of Salmonella enteritidis endotoxin (20 mg/kg), plasma thromboxane B(2) levels increased from nondetectable levels (<375 pg/ml) in normal control rats to 2,054+/-524 pg/ml (n = 8), within 30 min to 2,071+/-429 at 60 min, and decreased to 1,119+/-319 pg/ml, at 120 min. Plasma levels of prostaglandin E also increased from 146+/-33 pg/ml in normal controls (n = 5) to 2,161+/-606 pg/ml 30 min after endotoxin (n = 5). In contrast to shocked controls, rats pretreated with imidazole, a thromboxane synthetase inhibitor, or essential fatty acid-deficient rats, which are deficient in arachidonate and its metabolites, did not exhibit significant elevations in plasma levels of thromboxane B(2). Imidazole did not however inhibit endotoxin-induced elevations in plasma prostaglandin E. Essential fatty acid deficiency significantly reduced mortality to lethal endotoxic shock. This refractoriness could be duplicated in normal rats pretreated with the fatty acid cyclo-oxygenase inhibitor, indomethacin (10 mg/kg), intravenously 30 min before endotoxin injection. Imidazole (30 mg/kg) administered intraperitoneally 1 h before or intravenously 30 min before endotoxin, also significantly (P < 0.01) reduced mortality from lethal endotoxin shock to 40% compared to a control mortality of 95% at 24 h. Likewise pretreatment with 13-azaprostanoic acid (30 mg/kg), a thromboxane antagonist, reduced mortality from endotoxic shock at 24 h from 100% in control rats to only 50% (P < 0.01). The results suggest that endotoxin induces increased synthesis of thromboxane A(2) that may contribute to the pathogenesis of endotoxic shock.

In examining the structure-activity relationship of a diverse group of chemicals reported to prevent cercarial penetration after topical application, we noticed a moiety that was common to free fatty acids and prostaglandins. Because unsaturated fatty acids have been reported to stimulate cercarial penetration, we hypothesized that cercarial stimulation by skin and fatty acids may invoke prostaglandin mechanisms in cercariae, skin, or both. Thus we compared the stimulation of cercariae by a series of essential and nonessential fatty acids and demonstrated an inhibition of this response by ibuprofen and aspirin, known cyclo-oxygenase inhibitors, and by 13-azaprostanoic acid, a potent antagonist of the thromboxane/endoperoxide receptor. These data led us to postulate a major role for prostaglandins in the cercarial penetration response.

9-Hydroxy-19,20-bis-nor-prostanoic acid (IBI-C83) was evaluated on gastric acid secretion and gastric lesions induced in laboratory animals by a variety of experimental conditions: compound IBI-C83 is proved effective in decreasing basal, histamine- and pentagastrin-stimulated total acid output in rats and in pentagastrin perfused dogs. The concentration of N-acetylneuraminic acid in the gastric fluid, a marker of mucus secretion, is enhanced in rats by IBI-C83. This drug prevents gastric damage induced by non-steroidal antiinflammatory compounds such as acetylsalicylic acid, indometacin and phenylbutazone, gastric ulcers following pylorus ligation, and facilitates healing of the gastric ulcers evoked by subserosal injection of acetic acid. A prominent feature of IBI-C83 is its capacity to protect the rat from gastric damage elicited by necrotizing agents such as absolute ethanol, hydrochloric acid and hypertonic saline. This property, called "cytoprotection" and common to naturally occuring prostaglandins, is independent on the antisecretory activity of IBI-C83 and is not shared, at least in the reported experimental models, by the H2-receptor antagonist cimetidine. In spite of the prostaglandin-like properties displayed in its cytoprotective activity, compound IBI-C83 does not affect cardiovascular functions, gastrointestinal transit and uterine motility.

The anti-aggregatory prostanoid, prostaglandin D2 (PGD2) does not completely inhibit ADP-induced aggregation of guinea-pig platelets and thus produces a bell-shaped dose-inhibition curve. The nature of this bell-shaped curve has now been investigated in guinea-pig platelet-rich plasma. Two selective thromboxane receptor antagonists, 13-aza-prostanoic acid (13-AZA; 16-64.4 microM) and BM 13.177 (5.9-29.8 microM), converted PGD2 to a full inhibitor of aggregation in a dose-related manner. The putative platelet PGD2 receptor antagonist, N-0164 (75 microM) also converted PGD2 to a full inhibitor of platelet aggregation. In contrast to 13-AZA and BM 13.177, higher concentrations of N-0164 (380 and 760 microM) caused a dose-related rightward shift of the PGD2 dose-inhibition curve. The thromboxane receptor antagonism of N-0164 was confirmed in studies in which the dose-aggregation curve to U-46619, a thromboxane mimetic, was competitively antagonized with a pA2 value of 4.67 and a slope of 1.13, comparable to that of 13-AZA. The results show that N-0164 acts as both a platelet PGD2 and thromboxane-receptor antagonist in both human and guinea-pig platelet-rich plasma. The results further indicate that PGD2 can interact at thromboxane receptors in guinea-pig platelets.

Platelets obtained from some diabetic patients show enhanced in vitro platelet aggregation. This study sought to determine whether platelets obtained from diabetic subjects synthesize increased quantities of the labile aggregating substance. TXA2, and whether it may play a role in the enhanced platelet aggregation. Arachidonic acid (1 mM)-stimulated TXA2 synthesis, as determined via radioimmunoassay of its stable metabolite TXB2, was significantly greater (p < 0.01, n = 12) in platelet-rich plasma obtained from diabetic patients than in matched controls. Arachidonic acid-stimulated TXB2 synthesis in the diabetic platelet-rich plasma was positively correlated with the ambient fasting plasma glucose (r = 0.61, p < 0.02, n = 15). Platelet aggregation induced by arachidonic acid (0.4 to 0.8 mM) was inhibited significantly less than in matched controls by imidazole, a thromboxane synthetase inhibitor (p < 0.01, n = 11), and 13-azaprostanoic acid, an antagonist of the actions of PGH2 or TXA2 on platelets (p < 0.04, n = 14). We conclude that platelets obtained from some diabetic subjects manifest increased synthesis of TXA2, which may contribute to the enhanced platelet aggregation.

Vasopressin enhances osmotic water flow and sodium transport across the toad urinary bladder by mechanisms involving cyclic AMP and calcium. It is believed that changes in intracellular calcium concentration or in its binding to membranes may in part mediate the effects of vasopressin. In addition, several agents which alter the response of the toad bladder to vasopressin may also act by altering cellular calcium metabolism. The effects of vasopressin and several agents which modify its effects in the toad bladder were studied on 45Ca fluxes in isolated epithelial cells from the toad bladder. Compartmental analysis of 45Ca exchange revealed three components. Vasopressin reduced the amount of calcium in the most rapidly exchanging pool from 1.67 +/- 0.20 to 0.86 +/- 0.12 nmol/mg of protein (P less than .025) and the most slowly exchanging pool from 2.72 +/- 0.26 to 1.90 +/- 0.34 nmol/mg of protein (P less than .001), while not affecting the intermediate pool. Theophylline, which mimics the natriferic and hydroosmotic effects of vasopressin, also mimicked the effects on 45Ca exchange by vasopressin. Exogenous cyclic AMP and the prostaglandin endoperoxide analog U46619, which mimic the hydroosmotic effect of vasopressin, also reduced the amount of calcium in the most slowly exchanging pool. Prostaglandin E1, which inhibits the hydroosmotic effect of vasopressin at the concentrations used increased the size of the most slowly exchanging pool. These studies suggest that prostaglandins and other agents which alter the effect of vasopressin in the isolated toad bladder may elicit their effects in part by influencing the calcium concentration at some critical site.

Incubation of human washed platelets with 9,11-epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA2 stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA2 necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca2+ concentrations higher than those required for MLC kinase activation by the action of STA2, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca2+.

Platelets obtained from some diabetic patients show enhanced in vitro platelet aggregation. This study sought to determine if platelet obtained from insulin-dependent diabetic subjects synthesize increased quantities of the labile aggregating substance, thromboxane A2 (TXA2), and if it may play a role in the enhanced platelet aggregation. Arachidonic acid (1 mM)-stimulated TXA2 synthesis, as determined via radioimmunoassay of its stable metabolite TXB2, was significantly greater (P less than 0.01, N = 12) in platelet-rich plasma obtained from diabetics compared with matched controls. Arachidonic acid-stimulated TXB2 synthesis in the diabetic platelet-rich plasma was positively correlated with the ambient fasting plasma glucose (r = 0.61, P less than 0.02, N = 15). Platelet aggregation induced by arachidonic acid (0.4-0.8 mM) was inhibited significantly less by 13-azaprostanoic acid (P less than 0.04, N = 14), a competitive antagonist of the actions of prostaglandin H2 or TXA2 on platelets, compared with matched controls. The results support the notion that platelets obtained from some insulin-dependent diabetic subjects manifest increased synthesis of TXA2, which may contribute to the enhanced platelet aggregation.

A reproducible method for dissociation and culture of rat luteal cells is described. The concentration of LH required to produce half-maximal stimulation of progesterone secretion was 50 ng/ml. The effects of prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) on basal and luteinizing hormone (LH)-stimulated progesterone production were examined. Both prostaglandins stimulated basal progesterone production but PGE(2) was about twice as active, showing a 2-fold maximal stimulation at 0.75 muM. When either prostaglandin was incubated simultaneously with LH, a dose-dependent inhibition of progesterone secretion occurred; PGF(2alpha) was 4 times more active than PGE(2), showing 50% inhibition at a concentration of 40 x nM. Thus, both prostaglandins are more active as antagonists than as agonists of LH with respect to progesterone secretion. PGF(2alpha) also inhibited LH-stimulated adenylate cyclase activity and cyclic AMP accumulation. The block in progesterone secretion was reversed by addition of dibutyryl cyclic AMP (1 mM) but not by theophylline (5 mM) alone. These data and the finding that PGF(2alpha) did not affect the specific binding activity of the LH receptor in intact luteal cells indicate that the rapid action of prostaglandins in luteal cells is due to a block of LH-dependent production of cyclic AMP which results in a decrease in progesterone secretion.

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.

Experiments were designed to examine the possibility that nonprotein sulfhydryl groups of the gastric mucosa could participate in the protection of rat gastric mucosa by rosaprostol (the Na salt of 9-hydroxy-8,12 trans-19,20-bis-nor-prostanoic acid). Gastric mucosal lesions and the content of nonprotein sulfhydryls were evaluated after orally administered absolute ethanol. Pretreatment with rosaprostol by gavage prevented gastric lesions and reduced or prevented the decrease of mucosal nonprotein thiols. N-ethylmaleimide, a sulfhydryl blocker, worsened the ethanol-induced gastric lesions and lowered further the non protein thiols. Both variables were improved by the PG analogue and by PGE2. These results suggest a possible role of endogenous nonprotein sulfhydryl groups in the gastric protective effect of rosaprostol.

Selective pharmacological blockade of thromboxane-synthase in human platelets by dazoxiben resulted in the reorientation of cyclic-endoperoxides towards PGE2, PGD2 and PGF2 alpha. At concentrations which can be reached when thromboxane-synthase is inhibited, PGE2 (100-500 nM) exerted a marked, concentration-dependent pro-aggregatory effect. This required the formation of endogenous or the addition of exogenous endoperoxides and was prevented by PGD2 or 13-aza-prostanoic acid, a selective antagonist of PGH2/TxA2 receptors. The anti-aggregating effect of PGD2 was evident at concentrations lower than those obtained in dazoxiben-treated platelets. It is proposed that in the absence of TxA2 generation, a combination of endoperoxides and PGE2 may result in normal aggregation. The latter may be inhibited by PGD2. No interference of PGF2 alpha on platelet function could be shown.

A method is described for characterizing disaggregatory and proaggregatory prostanoids in a single assay. This "post-addition assay" consists of adding the test agent to PRP containing PGI2 (4-6 nM) 90 sec after an ADP challenge. The relative potencies of known inhibitors of ADP-induced aggregation were identical to the relative disaggregatory potencies determined by post-addition. The proaggregatory relative potencies for 16, 16-Me2-PGE2,PGE2,PGH2 and 11 alpha,9 alpha-epoxymethano-15 alpha-hydroxy-prostadienoic acid were 5.8 :2 : 1.8 : 1, respectively, revealing the significant thrombosis promoting activity of PGE2 and its analogs.

A series of 13-azaprostanoic acids (4a-h) and a 15-azaprostanoic acid (11a) have been prepared. Synthesis of the 15-aza derivative is based on a novel transformation of a ketone to an N-substituted ethylenamine using a formylmethylimino phosphate derivative. Several of the azaprostanoic acid derivatives were found to be potent inhibitors of platelet aggregation induced by arachidonic acid, whereas no effect was observed on ADP-induced primary aggregation, indicating blockade of the platelet arachidonic acid cascade. The compounds do not inhibit bovine cyclooxygenase activity and are postulated as acting beyond the synthesis of the prostaglandin endoperoxides. The inhibitory effect of the 13-aza series is highly sensitive to both stereochemistry and length of the amino side chain. Any deviation from the natural prostaglandin skeletal arrangement results in decreased biological activity.

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A2/prostaglandin H2 (TXA2/PGH2) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid , 2-(6- carboxyhexyl ) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 microM CPH. On the other hand, CPH was not an effective inhibitor of PGH2-induced aggregation. Furthermore, 100 microM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA2/PGH2 receptor). Measurement of platelet thromboxane B2 (TXB2) production demonstrated tha the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CP inhibited TXB2 formation from AA in a dose-dependent manner (0.1 microM-100 microM CPH)2. In contrast, CPH blocked TXB2 production from PGH2 only at the highest CPH concentration tested, i.e., 100 microM. These results indicate that relative to 13-APA, addition of a second nitrogen at C14 and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.

The specific thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid (13-APA) reverses platelet aggregation stimulated by TXA2/PGH2 and the prostaglandin endoperoxide analog U46619. The present report demonstrates that the deaggregatory properties of 13-APA are potentiated by prostacyclin (PGI2). Human platelet-rich plasma was aggregated with U46619. Deaggregation was induced 2 min subsequent to the addition of the aggregating agent. Concentrations of 13-APA or PGI2 which induced 20 percent deaggregation were determined. Simultaneous addition of half of these concentrations resulted in 60 percent deaggregation, demonstrating that the observed response was supraadditive. Measurement of cyclic adenosine 3':5' monophosphate (cAMP) in resting or deaggregating platelets demonstrated that 13-APA itself did not stimulate cAMP production nor did 13-APA facilitate PGI2-induced increases in cAMP. In separate studies PGI2 and 13-APA were added to PRP prior to the induction of aggregation by U46619. Under these conditions, additive inhibition of aggregation was observed. Thus, it is clear that the pharmacological interaction between PGI2 and 13-APA depends upon the relative state of platelet activation.

The present study evaluated the direct involvement of thromboxane A2/prostaglandin H2 (TXA2/PGH2) in the process of thrombus formation at a site of vascular damage. De-endothelialization of the rabbit aorta was performed by a balloon catheter technique. Platelet deposition to the injured vessel was measured using 111Indium-labeled autologous platelets. Studies using the radiolabeled TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA), indicated that 13-APA has an in vivo half-life of approximately 35 min and is excreted by the kidney in the metabolized form. Addition of 13-APA to rabbit plasma samples in vitro produced a dose-dependent inhibition of arachidonic acid-induced platelet aggregation. When comparable plasma levels of 13-APA were achieved by infusion of 13-APA (300 micrograms/kg/min for 90 min), a similar dose-dependency of inhibition of ex vivo aggregation was observed. Furthermore, at a plasma concentration of 40 microM, 13-APA was found to inhibit platelet deposition to the de-endothelialized rabbit aorta by 45%. Because 13-APA does not interfere with arachidonic acid metabolism, the ability of 13-APA to suppress thrombus formation is presumably due to direct antagonism of TXA2/PGH2 at the platelet receptor level. These findings, therefore, provide evidence that TXA2 and/or PGH2 have a major role in platelet deposition at a site of vascular damage.

The effects of 13-azaprostanoic acid (13-APA) were studied during acute myocardial ischemia in cats and in rabbit sudden death induced by sodium arachidonate (Na-Ar). To more clearly define the mechanism of action of 13-APA, we also examined its effects on isolated cat and rabbit coronary arteries, in vitro aggregation of cat and rabbit platelet-rich plasma (PRP) and circulating rabbit platelet count measured in vivo. 13-APA provided minimal protection during myocardial ischemia in cats, partially reversing ischemia-induced ST segment elevations by 3-5 hours after coronary artery occlusion. However, 13-APA was ineffective in inhibiting the rise in plasma creatine kinase (CK) activity or the loss of CK from ischemic myocardial tissue. 13-APA (1.0 - 100 microM) did not inhibit contraction of cat coronary arteries produced by a stable thromboxane A2 analog. However, 13-APA (100 microM) inhibited aggregation of cat PRP induced by AA (1.0 microM). 13-APA also provided significant protection against sudden death induced by Na-Ar in rabbits. While this agent was ineffective in reducing vasoconstriction of rabbit coronary arteries or inhibiting platelet aggregation in response to 500 microM AA, aggregation of rabbit PRP by 250 microM AA was completely inhibited. AA injection produced a significant decrease in circulating platelet count in vehicle-treated rabbits. However, 13-APA reduced the decrease in circulating platelet count in rabbits which survived AA injection during the 13-APA infusion. These results indicate that antagonism of thromboxane A2 receptors in platelets may be an important feature in protecting against sudden death. The difference in sensitivities of vascular and platelet thromboxane receptors as well as the accessability of 13-APA to these receptors may explain the lack of protection of 13-APA in myocardial ischemia.

The effects of prostaglandin (PG) F2 alpha and 9,11-epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of thromboxane A2, on the cytosolic free calcium concentration ([Ca2+]i) in vascular smooth muscle cells were studied with a new fluorescent Ca2+ indicator fura 2. PGF2 alpha and STA2, which are strong vasoconstrictors, caused rapid phasic and subsequent tonic increases in [Ca2+]i. PGF2 alpha caused dose-dependent elevation of [Ca2+]i not only in control solution but also in the calcium-free solution. A first stimulation with PGF2 alpha caused dose-dependent decrease in the response of [Ca2+]i to a second stimulation with PGF2 alpha. Pretreatment with 13-Azaprostanoic acid, a receptor level antagonist of thromboxane A2 inhibited the increase of [Ca2+]i induced by STA2. These results suggest that PGF2 alpha induces calcium mobilization followed by smooth muscle contraction through its specific receptors.

Acetyl glyceryl ether phosphorylcholine (AGEPC) is a mediator of inflammation and anaphylaxis, released from IgE-sensitized basophils when exposed to antigens. Convincing evidence shows that AGEPC is synthesized by inflammatory cells, and its mechanism of action has been extensively studied with particular interest focusing on platelet stimulation, the key event initiating the inflammatory cascade. However, widely different results have been reported so that the mode of action of AGEPC in activating platelets is still largely obscure. The objective of this investigation was to elucidate whether AGEPC stimulates human platelets independently from arachidonate metabolism, as previously suggested, and to define the role of nonsteroidal antiinflammatory drugs in inhibiting AGEPC-mediated platelet activation. Irreversible platelet aggregation in vitro occurred at low AGEPC concentrations, was dose dependent, and was accompanied by thromboxane A2 formation. Aspirin inhibited irreversible aggregation induced by low AGEPC concentrations. A thromboxane inhibitor, UK-37, 248-01, failed to affect AGEPC aggregation, suggesting a crucial role for cyclic endoperoxides in AGEPC's mechanism of action. A thromboxane/endoperoxide receptor antagonist 13-azaprostanoic acid prevented irreversible platelet aggregation induced by low AGEPC concentrations. The inhibiting effect of aspirin on AGEPC aggregation could be overcome by increasing the concentration of the agonist by a factor of 10, unless a lipoxygenase inhibitor, nordihydroguaiaretic acid, or 5,8,11,14-eicosatetraynoic acid was previously added to platelet-rich plasmas. The phospholipase inhibitor, mepacrine, completely suppressed human platelet aggregation induced by AGEPC. These results indicate that AGEPC induces irreversible aggregation in human platelets by two distinct pathways, both dependent upon arachidonic acid metabolism: a cyclooxygenase-dependent one at low AGEPC concentrations and a cyclooxygenase-independent one, possibly mediated through the formation of lipoxygenase products, at high AGEPC concentrations.

It has been postulated that metabolites of the arachidonic acid pathway exert an important influence on hemostasis and thrombosis. This notion is based on in vitro experiments. We have utilized two experimental models to elucidate the physiologic roles of thromboxane A2 (TxA2) and prostacyclin (PGI2) in the modulation of thrombus formation. The role of TxA2 in promoting thrombus formation was evaluated in a rabbit model where the aorta was deendothelialized by a balloon catheter technique and indium-111-labeled platelets were used as a marker for quantifying platelet deposition. Both 1-benzylimidazole, a thromboxane synthase inhibitor, and 13-azaprostanoic acid, an antagonist of thromboxane/endoperoxide receptors significantly reduced the platelet deposition onto the damaged vessel wall. The data indicate the TxA2 plays an important role in thrombosis and hemostasis. The influence of PGI2 insufficiency due to accelerated PGI2 degradation on microvascular thrombosis was evaluated in a unique clinical disease, i.e. thrombotic thrombocytopenic purpura (TTP). Accelerated PGI2 degradation was observed in several patients with chronic TTP. The degradation abnormalities were corrected by plasma infusion in vivo or serum supplement in vitro. To test the hypothesis that PGI2 must be bound to serum macromolecules to prevent rapid hydrolysis, serum binding capacity for PGI2 was measured by Sephadex G-25 gel filtration. The binding capacity was significantly reduced in the patients and was corrected by serum supplement. Abnormalities of PGI2 binding were also noted in a group of patients with ischemic stroke. Our findings suggest that there exist in the serum certain constituents which bind and stabilize PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)