N.H. Wilson

Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues

1 The agonist activities of a range of prostaglandin analogues on smooth muscle preparations sensitive to prostaglandin E2 (PGE2) have been investigated. When necessary thromboxane‐like activity was eliminated using the thromboxane receptor antagonists EP 045 and EP 092. 2 On the bullock iris sphincter, rat stomach fundus and guinea‐pig trachea, (±) ω‐tetranor‐16‐p‐chlorophenoxy PGE2 (ICI 80205) and 16,16‐dimethyl PGE2 were more active contractile agents than PGE2, whereas for relaxant activity on the cat trachea, guinea‐pig trachea and dog hind limb arterial vessels in vivo the order of potency was reversed. 11‐Deoxy PGE, exhibited greater relaxant than contractile activity when compared to PGE2. 3 Iloprost and 6a‐carba‐Δ6,6aPGI1 (potent mimetics of PGI2) showed high contractile activity on the PGE‐sensitive preparations. PGI2 was less active and another potent PGI2 mimetic, ZK 96480, showed only very weak activity. When tested, the dibenzoxazepines SC 19220 and SC 25191 blocked the contractile actions of iloprost and 6a‐carba‐Δ6,6aPGI1 and those of PGE2 and 16,16‐dimethyl PGE2 to similar extents. Each of the PGI2 analogues showed weak activity on the relaxant systems. 4 On the proximal portion of the ascending colon of the rat, PGI2, iloprost, 6a‐carba‐Δ6,6aPGI1 and ZK 96480 always inhibited spontaneous activity at nanomolar concentrations. PGE2 and PGE1 showed weak contractile activity. The distal portion of the ascending colon was more responsive to the contractile action of PGE analogues: both iloprost and 6a‐carba‐Δ6,6aPGI1 showed evidence of contractile activity, whereas PGI2 and ZK 96480 always inhibited spontaneous activity. 5 Evidence was obtained that the rat stomach fundus also contains a PGF receptor; (±) ω‐tetranor‐16‐m‐trifluoromethylphenoxy PGF2α (ICI 81008) acted as a specific agonist. PGF2α and its ω‐tetranor‐16‐p‐fluorophenoxy analogue produced a higher maximum response that ICI 81008 probably due to their additional agonist action at the PGE receptor. 6 The data support the hypothesis that there are two subtypes of the PGE receptor. ZK 96480 has minimal activity on both receptor subtypes and appears to be a highly specific PGI2 mimetic.

Characterization of receptors involved in the direct and indirect actions of prostaglandins E and I on the guinea-pig ileum

1 . A study of the effects of prostaglandin E2 (PGE2) and eleven synthetic analogues on the guinea‐pig isolated ileum preparation has revealed three distinct contractile actions, each associated with a different prostaglandin E (EP‐) receptor subtype. In addition, PGI2 (prostacyclin) and its stable analogues can activate prostaglandin I (IP‐) receptors to elicit both contraction and relaxation of the ileum. 2 . Two of the PGE actions involve direct stimulation of the smooth muscle, being unaffected by 1 μm morphine treatment. One action is blocked by AH 6809 at micromolar concentrations and ICI 80205 and 16,16‐dimethyl PGE2 are particularly potent agonists. Activation of EP1‐receptors appears to be involved. The second action is unaffected by AH 6809; sulprostone and MB 28767 are potent agonists. Comparison with agonist potency rankings on the guinea‐pig vas deferens indicates that EP3‐receptors may be involved. 3 . The third PGE effect and the stimulant PGI effect are blocked by morphine, indicating enteric neurones and/or sensory nerve terminals as sites of action. EP2‐receptors may be involved in the PGE action, in view of the marked effect of morphine on the contractile actions of misoprostol, 11‐deoxy PGE2‐1‐alcohol, 11‐deoxy PGE1 and butaprost, all of which show some selectivity for EP2‐receptors. The PGI action is most easily studied with cicaprost (EC25 = 1.3 nm), since iloprost, carbacyclin and to a lesser extent PGI2 also have agonist activity at EP1‐receptors. 4 . The contractile action of 17‐phenyl‐ω‐trinor PGE2 on the ileum is unaffected by morphine. Since this analogue shows only weak agonist activity on the rabbit jugular vein (EP2 preparation) and guinea‐pig vas deferens (EP3), it may be a more useful standard agonist than PGE2 in EP1‐receptor studies. 5 . In the presence of morphine and AH 6809, cicaprost inhibits histamine‐induced contractions (IC25 = 22 nm). PGI2 and iloprost show mixed inhibitory/potentiating actions, whereas carbacyclin only potentiates histamine contractions. This IP‐receptor‐mediated inhibition may account for the bell‐shaped log concentration‐response curve of cicaprost (no inhibitors present) and the very marked block of iloprost‐induced contractions by AH 6809. 6 . We have found no evidence for either IP‐receptors mediating direct contraction or EP‐receptors mediating inhibition of the ileum longitudinal smooth muscle, as has been suggested in the literature. 7 . In view of the complexity of prostanoid action on the guinea‐pig ileum we feel that the preparation must be used with caution to ascertain the EP1 agonist or antagonist potencies of novel compounds.

ANTAGONISM OF THE THROMBOXANE‐SENSITIVE CONTRACTILE SYSTEMS OF THE RABBIT AORTA, DOG SAPHENOUS VEIN AND GUINEA‐PIG TRACHEA

1 The thromboxane‐sensitive contractile systems in spirally‐cut preparations of the rabbit aorta, dog saphenous vein and guinea‐pig trachea have been compared. The full or partial agonist activities of a range of bicyclic ring analogues were found to be remarkably similar on the three preparations. In addition, EP 045, a prostanoid with a phenylsemicarbazone ω‐chain, blocked the action of both thromboxane A2 (TXA2) and the bicyclic ring analogues. Using 11,9‐epoxymethano prostaglandin H2 as the agonist, linear Schild plots with slopes close to unity were obtained on each preparation; this suggests a competitive type of antagonism. 2 Analogues of prostaglandin D2 (PGD2), PGE2 and PGF2α also contracted the three smooth muscle preparations; those analogues containing a 16‐p‐halophenoxy residue were highly active. On the rabbit aorta, EP 045 completely blocked the contractile actions of these agonists, perhaps indicating a single type of prostanoid receptor in this tissue. On the dog saphenous vein PGD2, PGE2 and 15‐methyl PGE2 exhibited relaxant activity when the tissue was partially contracted with either a thromboxane agonist or noradrenaline. On the guinea‐pig trachea 16,16‐dimethyl PGE2 and the 16‐p‐chlorophenoxy analogue of PGE2 were potent contractile agents whose action was not blocked by EP 045. PGE2 and 15‐methyl PGE2 showed similar properties but exhibited relaxant activity with increasing concentrations in the organ bath. Our results indicate the presence of three types of prostanoid receptors in the guinea‐pig trachea: thromboxane‐ and PGE‐sensitive systems mediating contraction and a PGE‐sensitive system mediating relaxation. 3 The similarity of the thromboxane‐sensitive systems in the three smooth muscle preparations is discussed with particular reference to the differences in the equilibrium dissociation constants for EP 045.

Production of prostaglandins by the pseudopregnant rat uterus, in vitro, and the effect of tamoxifen with the identification of 6-keto-prostaglandin F1alpha as a major product.

1 Prostaglandin production by rat uterus homogenates has been studied, in vitro, on days 2 to 13 of pseudopregnancy. 2 The highest production of prostaglandins occurred on day 5. 3 The amounts of prostaglandins F and D formed were higher than the amounts of prostaglandin E on every day studied. 4 The ratios of prostaglandins F and D to prostaglandin E produced steadily decreased up to day 6. It then increased with the highest values occurring between days 10 and 13. 5 Progesterone levels in peripheral plasma increased rapidly from days 2 to 5, remained high up to day 9, then steadily decreased between days 10 and 13. 6 The anti‐oestrogenic drug, tamoxifen administered on day 2, significantly inhibited the increase of prostaglandin production which occurred on day 5. Prostaglandin E production was inhibited more than the production of prostaglandins F and D. 7 Analysis of the uterine extracts by gas chromatography and mass spectrometry showed prostaglandin F2α, F1α (in trace amounts), E2 and D2 to be present. 8 The major product detected was 6‐keto‐prostaglandin F1α. Its identification forms an addendum to the paper. 9 Also present as a major product was 6(9)‐oxy‐11,15‐dihydroxyprosta‐7,13‐dienoic acid.

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.

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.

Heterogeneity of thromboxane A2 (TP‐) receptors: evidence from antagonist but not agonist potency measurements

1 Thromboxane A2 (TP‐) receptors in human, rat and rabbit platelets and in smooth muscle of guinea‐pig trachea, rat aorta and rabbit aorta have been characterized by measurement of the potencies of agonists and antagonists having considerable variations in chemical structure. 2 On each washed platelet system, eight prostanoids induced maximal irreversible aggregation (full agonists) and the potency ranking was EP 171 > STA2 > 9,11‐azo PGH2 > 9,11‐endoxy‐10a‐homo PGH2 > U‐46619 (standard) > PGH2 = 16‐p‐fluorophenoxy‐ω‐tetranor PGF2α > 16,16‐dimethyl PGF2α. Correlations between the three platelet preparations for both absolute and relative potencies were good. On human platelets, STA2, at concentrations above that required for maximum aggregation, exerted an inhibitory effect which was independent of its interaction with the TP‐receptor. 3 Five prostanoids, EP 109, EP 167, EP 204, PTA2 and 16,20‐methano PTA2, exhibited partial agonist activity on the platelet and smooth muscle preparations. There was good agreement between absolute potencies on the six preparations; on platelets potency was assessed from shape change measurements, since aggregation, when present, always showed a very shallow concentration‐response relationship. The magnitude of the maximum response induced by each compound decreased in the order listed above, to the extent that 16,20‐methano PTA2 could be treated as a pure antagonist. 4 With U‐46619 as agonist, the pA2 values of seven antagonists were found to be very similar on human and rat platelets. The potency ranking was EP 169 > AH 23848 > EP 092 > ONO 11120 > EP 115 = 16,20‐methano PTA2 > BM 13177. There was a similar trend on rabbit platelets but pA2 values were 1.0–1.5 log units smaller; the exception was BM 13177 which had similar affinities. The antagonism produced by EP 169 and AH 23848 was surmountable on rabbit platelets but not on human and rat platelets. 5 None of the antagonists was highly potent on the rabbit aorta (pA2 values < 7.5 by Schild analysis). Affinities on the guinea‐pig trachea and the rat aorta were higher and in the same range as those obtained for human and rat platelets. However the correlations of pA2 values between any pair of smooth muscle preparations and between any pair of platelet/smooth muscle preparations were either weak or not significant (P > 0.05). 6 The excellent agreement for both full and partial agonist potencies between the six preparations provides no evidence for TP‐receptor subtypes and further suggests that the agonist recognition sites of the TP‐receptors could be very similar, if not identical, in nature. In contrast, the different antagonist affinities found in this and other published studies indicate heterogeneity of TP‐receptors. However, classification into TP1‐, TP2‐receptors, etc. on the basis of the limited antagonist data available does not appear appropriate at this time.

EFFECT OF p‐BROMOPHENACYL BROMIDE, AN INHIBITOR OF PHOSPHOLIPASE A2, ON ARACHIDONIC ACID RELEASE AND PROSTAGLANDIN SYNTHESIS BY THE GUINEA‐PIG UTERUS in vitro

1 The synthesis of prostaglandins F2α and E2 by guinea‐pig uterine homogenates was inhibited by p‐bromophenacyl bromide (PBPAB), an inhibitor of phospholipase A2. 2 Metabolism of prostaglandin F2α by uterine homogenates was undetectable; this was not affected by PBPAB. 3 There was no significant difference between the amounts of arachidonic acid released from uterine homogenates on days 7 and 15 of the oestrous cycle. Small amounts of dihomo‐γ‐linolenic acid were detected in the homogenates. 4 The release of arachidonic acid from uterine homogenates was greatly inhibited by PBPAB. 5 Addition of exogenous arachidonic acid to uterine homogenates did not overcome the inhibition of uterine prostaglandin F2α synthesis produced by PBPAB. 6 It is concluded that PBPAB inhibits both the release of arachidonic acid from the guinea‐pig uterus and its subsequent conversion into prostaglandins.

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.