Roma A. Armstrong

Investigation of the inhibitory effects of PGE2 and selective EP agonists on chemotaxis of human neutrophils

1 The aims of this study were to investigate the inhibitory effects of prostaglandin E2 (PGE2) on chemotaxis of N‐formyl‐methionyl‐leucine‐phenylalahine (FMLP)‐stimulated human neutrophils, and to test the hypothesis that cyclic AMP is the second messenger involved. For this purpose, the inhibitory effect of selective EP agonists, and the modulatory effects of the adenylate cyclase inhibitor, SQ 22536, the protein kinase A (PKA) inhibitors H‐89 and Rp‐cAMPS, and the type IV phosphodiesterase (PDE) inhibitors, rolipram and Ro20‐1724 have been examined. 2 Chemotaxis has been measured using blindwell chambers. When human neutrophils were stimulated with FMLP (100 nM), PGE2 inhibited chemotaxis in a concentration‐dependent manner (0.01–10 μm), with an EC50 of 90±24.5 nM, a maximum effect ranging from 45–75% and a mean inhibition of 64.5 ±2.4%. 3 The EP2‐receptor agonists, 11‐deoxy PGE1, butaprost and AH 13205 also inhibited chemotaxis. The order of potency of these agonists was PGE2 > butaprost (EC50= 106.4 ± 63 nM)> 11‐deoxy PGE1 (EC50= 140.9±64.7 nM)>AH 13205(EC50= 1.58±0.73 μm). Correlation of the ability of EP2 agonists to increase cyclic AMP and to inhibit chemotaxis was poor (r=0.38). 4 The IP agonist, cicaprost gave similar increases in cyclic AMP to those achieved with PGE2, yet produced 50% of the maximum inhibition of chemotaxis observed with PGE2. 5 Slight potentiation of the inhibitory effects of PGE2 after type IV PDE block was observed with rolipram (EC50 for PGE2=57.2 ±5.9; 35.2 ±6.8 nM) but not Ro20‐1724 (EC50 for PGE2 216.0 ±59.7; 97.8 ±50.6 nM). Type IV PDE inhibitors are themselves potent inhibitors of chemotaxis with EC50 values of 23.0 ±2.3 and 73.6 ±10.3 nM for rolipram and Ro20‐1724, respectively. 6 Inhibition of cyclic AMP production with the adenylate cyclase inhibitor SQ 22,536 (0.1 mm) failed to antagonize inhibition of chemotaxis by PGE2 (EC50S for PGE2 of 57.2 ±5.9 and 56.8 ±27.3 nM, in the absence and presence of SQ 22,536, respectively) despite a reduction in the increase in cyclic AMP induced by PGE2. 7 Inhibition of PKA with either H‐89 (10 μm) or Rp cyclic AMPS (10 μm) similarly failed to antagonize inhibition of chemotaxis by PGE2; EC50 for PGE2 of 90 ±40 and PGE2+H‐89 60 ± 17 nM; PGE2 216.0±58.7 and PGE2Rp cyclic AMP 76.9±14.7nM. 8 Of the two PKA inhibitors tested, H‐89 (10 μm) and Rp cyclic AMPS (10 /zm), the more effective inhibitor of PGE2‐induced inhibition of neutrophil superoxide anion generation was H‐89 (EC50S for PGE2 were 0.36 ±0.1 and >10 μm, respectively). We have previously shown this to be a cyclic AMP‐dependent effect of PGE2. 9 Confirmation of block of PKA by H‐89 was suggested by the finding that H‐89 blocked inhibition of superoxide anion generation observed with the type IV PDE inhibitors rolipram and Ro20‐1724; EC50S of 12.9 ±8.9 nM for rolipram alone and rolipram+H‐89 >1 μm; Ro20‐1724 alone 59.5 ±28.1 nM and Ro20‐1724+H‐89 >1μm. 10 The results suggest that inhibition of chemotaxis by PGE2 and EP2 agonists is not mediated by increased neutrophil cyclic AMP levels.

Ligand binding to thromboxane receptors on human platelets: correlation with biological activity

1 The preparation of enantiomerically pure [3H]‐15 (S) 9, 11‐epoxymethano PGH2 (a thromboxane A2‐like agonist) has enabled the binding of ligands to the thromboxane receptor of the human platelet to be studied. 2 The binding of the radio‐ligand to washed human platelets has 3 components. One component is not displaceable by ‘cold’ 9, 11‐epoxymethano PGH2 and its concentration‐binding plot is roughly linear. The other 2 components are displaceable and saturable, and the larger of the two, which is sensitive to the stereochemistry of the C15 secondary alcohol, appears to represent the thromboxane receptor. About 1700 15(S)9, 11‐epoxymethano PGH2 molecules are specifically bound to a single platelet and 50% of this binding is achieved with a concentration of 75 nM. 3 Displacement of [3H]‐15(S)9, 11‐epoxymethano PGH2 is effected by (a) TXA2 and PGH2 and a number of bicyclic stable analogues (e.g. 9,11‐azo PGH2), all of which produce irreversible aggregation of human platelets; (b) analogues of PGF2α with potent thromboxane‐like activity (e.g. ICI 79939); (c) compounds with partial agonist activity on the platelet thromboxane system (e.g. CTA2); (d) Thromboxane/endoperoxide analogues which specifically antagonize thromboxane‐like actions on the human platelet (e.g. PTA2 and EP 045). 4 Displacement is not achieved with the natural prostaglandins PGE2, PGD2 and PGF2α. Neither the thromboxane‐synthetase inhibitor dazoxiben nor R(+)‐trimethoquinol have high displacing activity. 5 The correlation of radio‐ligand displacement with the biological activity of the competing ligands is discussed in relation to the nature of the thromboxane receptor on the human platelet.

Characterization of the PGE receptor subtype mediating inhibition of superoxide production in human neutrophils

1 The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)‐stimulated human neutrophils, and to test the hypothesis that adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2.

Competitive antagonism at thromboxane receptors in human platelets.

1 The inhibitory effects of three prostanoid analogues, EP 045, EP 092 and pinane thromboxane A2 (PTA2), on the aggregation of human platelets in vitro have been investigated. 2 In diluted platelet‐rich plasma (PRP), EP 045 (20 μm) and EP 092 (1 μm) completely inhibited irreversible aggregation responses to thromboxane A2 (TXA2), prostaglandin H2 (PGH2) and five chemically stable thromboxane mimetics, including 11,9‐epoxymethano‐PGH2 and 9,11‐azo‐PGH2. Reversible aggregation produced by the prostanoid analogue, CTA2, was also inhibited. The block of the stable agonist action was surmountable. In plasma‐free platelet suspensions EP 045 and EP 092 were more potent antagonists. Schild analysis indicated a competitive type of antagonism for EP 045 (affinity constant of 1.1 × 107 m−1); the nature of the EP 092 block is not clear. 3 Primary aggregation waves induced by ADP, platelet activating factor (Paf) and adrenaline were unaffected by EP 045 and EP 092, whereas the corresponding second phases of aggregation were suppressed. Aggregation and 5‐hydroxytryptamine (5‐HT) release induced by either PGH2 or 11,9‐epoxymethano‐PGH2 were inhibited in a parallel manner by EP 045. Inhibition of thromboxane biosynthesis is not involved in these effects. 4 EP 045 and EP 092 did not raise adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels in the platelet suspensions. 5 In plasma‐free platelet suspensions PTA2 produced a shape change response which could be blocked by EP 045. PTA2, therefore, has a thromboxane‐like agonist action. The block of the aggregatory action of 11,9‐epoxymethano‐PGH2 by PTA2 appears to be mainly due to competition at the thromboxane receptor. However, PTA2 produced a slight rise in cyclic AMP levels; this could be due to a very weak stimulant action on either PGI2 or PGD2 receptors present in the human platelet. Functional antagonism by PTA2 may therefore augment its thromboxane receptor blocking activity. 6 The results are discussed in terms of (a) the specificity of antagonism produced by EP 045, EP 092 and PTA2, (b) the validity of affinity constant determinations for receptor antagonists when aggregation is the biological response, and (c) the characteristics of the human platelet thromboxane receptor in comparison with those of thromboxane receptors in smooth muscle.

Functional and ligand binding studies suggest heterogeneity of platelet prostacyclin receptors.

1 This study describes attempts to compare prostacyclin (IP‐) receptors in human, pig, horse, rabbit and rat platelets and in circular muscle of human, rabbit and dog mesenteric and pig gastroepiploic arteries. Three stable prostacyclin analogues, iloprost, cicaprost and 6a‐carba‐prostacyclin (6a‐carba‐PGI2) and a prostaglandin endoperoxide analogue EP 157 (previously shown to mimic prostacyclin on human platelets) were used. 2 Our main conclusion is that prostacyclin receptors on human, pig and horse platelets are similar in nature, but distinct from those on rabbit and rat platelets. Functional studies (inhibition of aggregation) showed that iloprost and cicaprost always had similar potencies whereas 6a‐carba PGI2 was much more potent than EP 157 on rabbit and rat platelets (300 and 1000 fold on a molar basis) compared with human, pig and horse platelets (2, 7 and 7 fold respectively). Measurement of initial rates of cyclic AMP production confirmed these orders of potency. 3 Although pig platelets were quite sensitive to inhibition by EP 157 (threshold = 10 nM in some experiments), maximal inhibition of aggregation was not always achieved (20 μm). EP 157 also produced only small elevations of cyclic AMP and inhibited rises in cyclic AMP induced by iloprost. It is possible that EP 157 has a lower efficacy than iloprost at the IP‐receptor and on pig platelets it can sometimes act as a partial agonist. 4 Human, pig and horse platelet membranes bound [3H]‐iloprost at 30°C and this binding was inhibited by the four prostanoids. On human and pig membranes the order of potency was cicaprost = iloprost > 6a‐carba PGI2 > EP 157. The order of potency may be similar on horse platelet membranes, but the analysis is complicated by the presence of a second component of [3H]‐iloprost binding that is inhibited by iloprost and 6a‐carba PGI2 but not by cicaprost. This binding may be due to the presence of an EP1‐receptor, since iloprost and 6a‐carba PGI2 but not cicaprost are known to have potent EP1‐receptor agonist actions on smooth muscle preparations. IC50 values for cicaprost inhibition on human, pig and horse membranes were 110, 90 and 165 nM respectively. The need for IP‐receptor radioligands of greater specificity is apparent from these studies. 5 Minimal binding of [3H]‐iloprost to rabbit and rat platelet membranes was obtained at 30°C. Lowering the incubation temperature to 4°C and ensuring that the temperature did not rise during the filtration process increased binding and allowed inhibition curves to be obtained. The results suggest a lower binding affinity for [3H]‐iloprost, associated with a higher dissociation rate for the radioligand‐receptor complex. IC50 values for cicaprost were 900 nM for rabbit and 640 nM for rat platelets. In a similar manner to horse platelet membranes, the presence of a second binding site for [3H]‐iloprost was detected on rabbit platelet membranes. 6 Sensitivity to the relaxant action of iloprost on the arterial smooth muscle preparations decreased in the order: human mesenteric, dog mesenteric, rabbit mesenteric, pig gastro‐epiploic. Cicaprost was always slightly more potent than iloprost (1.2–2.8 fold). On the pig vessel preparation 6a‐carba PGI2 did not produce complete relaxation. The possibility that this is due to an opposing contractile action mediated via EP1 or EP3 receptors is discussed. 7 EP 157 relaxed the human, pig and rabbit arterial preparations at concentrations 100–200 times those of iloprost. This correlates well with its IP‐receptor agonist potency on human, pig and horse platelets. The results obtained with EP 157 further demonstrate the potential difficulties in separating platelet inhibitory and vasodilator properties of prostacyclin mimetics in man.

Aspects of the thromboxane receptor system

1. The aim of this review is to establish what is known about the thromboxane (TP) receptor, and to identify where future research is headed. In addition, the impact of the recent advances at the molecular level on resolving pharmacological controversies, such as possible subtypes of the TP receptor, is discussed and what molecular information is known about the TP receptor presented. 2. The clinical status of TP receptor antagonists is considered particularly in relation to the potential role of epi prostaglandins. 3. Basic information about TP agonists, antagonists and signal transduction pathways is also given.

Comparison of the EP receptor subtypes mediating relaxation of the rabbit jugular and pig saphenous veins.

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.

Characteristics of the binding of [3H]-GR32191 to the thromboxane (TP-) receptor of human platelets.

1 The interaction of the specific thromboxane (TP‐) receptor blocking drug, [3H]‐GR32191 with human intact platelets and platelet membranes has been investigated in vitro. 2 On intact platelets, association of specific [3H]‐GR32191 binding at 37°C was biphasic, with an initial rapid component and a slower secondary phase. Dissociation experiments indicated displacement from two sites with t½ values of 8.1 and 65.6 minutes. Kd values derived from the kinetic rate constants for the rapid onset/offset and slow onset/offset phases were 0.4 and 0.5 nm respectively. 3 Competition binding of [3H]‐GR32191 and GR32191 on intact platelets gave an IC50 of 2.3 nm. Scatchard analysis indicated a single class of binding site with a Kd of 2.2 nm. Further analysis of the data yielded a Hill slope of − 1.0 again indicating an interaction at a single binding site. Saturation binding experiments gave a similar estimate of the Kd value for [3H]‐GR32191 to that obtained from competition binding experiments. A possible explanation for the biphasic interaction of the GR32191 in intact platelets may lie in restriction of its access to and egress from a population of TP‐receptors. 4 In platelet membranes at 37°C, specific [3H]‐GR32191 binding was complete within 5 min with a calculated association rate constant of 3.2 × 108 M−1 min−1. Dissociation of [3H]‐GR32191 was relatively slow, with measurable specific binding persisting for > 40 min. Analysis of these data yielded a t½ of 17.7 min and a dissociation rate constant of 0.04 min−1 and indicated dissociation from a single site. The t½ for dissociation appeared to be related to the contact time of platelet membranes with [3H]‐GR32191. Derivation of a Kd from the kinetic rate constants gave a value of 0.13 nm. 5 Competition binding of [3H]‐GR32191 and GR32191 to platelet membranes gave an IC50 value of 3.5 nm. Scatchard analysis of these data indicated a single binding site with a Kd of 2.1 nm. Saturation binding experiments with [3H]‐GR32191 yielded similar IC50 and Kd values to those from competition experiments. 6 In further competition binding experiments, the TP‐receptor agonists U‐46619, STA2, EP171 and 9,11‐azo PGH2 and antagonists SQ29,548, BM 13.177 and EP092 all competed with specific [3H]‐GR32191 binding on intact platelets and, where determined, on platelet membranes. All compounds fully displaced specific [3H]‐GR32191 binding. However, where tested, the IC50 values for a particular compound were always greater when [3H]‐GR32191 was the radioligand than when [3H]‐SQ29,548 was used. At the concentrations used in these studies (2 and 5 nm respectively), platelets appeared to bind approximately twice as much [3H]‐GR32191 as [3H]‐SQ29,548. 7 In conclusion, the interaction of [3H]‐GR32191 with human intact platelets was complex but the data were consistent with an action at a single class of binding site; from competition experiments this appears to be the functional TP‐receptor. The interaction of the drug with this binding site is, however, characterized by a slow dissociation. This characteristic was confirmed in studies with platelet membranes and does not therefore appear to be an artefact of diffusion. Estimates of the Kd of the drug differed depending on the method of determination. Because of the slow dissociation of [3H]‐GR32191, those relying upon equilibrium of the radioligand with competing agent may be unreliable. The rate of dissociation also appeared to be related to the contact time of drug with receptor. An explanation for this phenomenon may lie in the ability of GR32191 to induce a change in the conformational state or location of the human platelet TP‐receptor.

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF2 alpha is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF2 alpha. We suggest that the very weak effect of PGF2 alpha on cyclic AMP production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.

COMPARISON OF THE PROSTAGLANDIN E (EP) RECEPTOR OF HUMAN NEUTROPHILS AND HL-60 CELLS DIFFERENTIATED WITH DMSO

Human promyelocytic leukaemic HL-60 cells can be differentiated with DMSO to become neutrophil-like. In this study, the prostanoid receptors linked to adenylate cyclase have been compared in human neutrophils and in differentiated HL-60 cells. Both cell types appear to express EP2 receptors as recognised by the ability of EP2 agonists and not EP1 or EP3 agonists to increase cell cyclic AMP levels, and the finding that the increase in cyclic AMP induced by PGE2 was not blocked by the EP4 receptor antagonist AH 23,848 (30 microM). Neither cell type appears to express receptors for PGI2, but human neutrophils and not differentiated HL-60 cells express receptors for PGD2. In addition, human neutrophils may contain EP3 receptors linked to a reduction in cyclic AMP levels. The lack of other prostanoid receptors coupled to adenylate cyclase in HL-60 cells suggests that these cells may provide a useful starting point for the cloning of the EP2 receptor.