Robert A. Coleman

COMPARISON OF THE ACTIONS OF U‐46619, A PROSTAGLANDIN H2‐ANALOGUE, WITH THOSE OF PROSTAGLANDIN H2 AND THROMBOXANE A2 ON SOME ISOLATED SMOOTH MUSCLE PREPARATIONS

1 The actions of the prostaglandin H2 (PGH2) analogue, U‐46619, have been compared with those of PGH2 and thromboxane A2 (TxA2) on a range of isolated smooth muscle preparations in a superfusion cascade system. 2 U‐46619 was a potent agonist on guinea‐pig lung strip, dog saphenous vein and rat and rabbit aortae. In contrast, U‐46619 was weak or inactive on guinea‐pig ileum and fundic strip, cat trachea and dog and cat iris sphincter muscles, preparations on which either PGE2 or PGF2α was the most potent agonist studied. 3 PGH2 was active on all of the preparations and displayed little selectivity. On some of the preparations, the actions of PGH2 may have been mediated indirectly by conversion to other prostanoids. 4 In contrast, TxA2 displayed the same pattern of selectivity as U‐46619, being a potent agonist on the lung strip and vascular preparations but weak or inactive on the others. 5 It is suggested that U‐46619 is a selective TxA2‐mimetic and that it should therefore be a valuable tool in the study of the actions of TxA2

Studies of the characterisation of prostanoid receptors: A proposed classification

Comparison of rank orders of agonist potency of the naturally occurring prostanoids, PGD2, PGE2, PGF2 alpha and PGI2 as well as the stable TxA2 mimetic, U-46619, on a range of smooth muscle preparations provides evidence for the existence of distinct receptors for PGE2, PGF2 alpha and TxA2. Since others have provided evidence for the existence of distinct receptors for PGD2 and PGI2, we suggest that receptors exist for each of these naturally occurring 2-series prostanoids. Results obtained with two specific prostanoid receptor blocking drugs, SC-19220 and AH 19437, support and extend these conclusions. SC-19220 selectively blocks some but not all PGE-sensitive receptors, while AH 19437 selectively blocks all U-46619/TxA2-sensitive receptors. A nomenclature for prostanoid receptors is proposed, in which each receptor is designated the letter P preceded by a letter signifying the most potent natural prostanoid agonist at that receptor, such that receptors sensitive to PGs D2, E2, F2 alpha, I2 and TxA2 become DP-, EP-, FP-, IP and TP- receptors respectively. Where some sub-division is required within a receptor group, e.g. EP-receptors (SC-19220-sensitive and SC-19220-insensitive), subscript numbers may be used such that these are EP1 and EP2 subtypes. The resulting scheme is a working hypothesis and its confirmation requires the development of potent selective prostanoid receptor blocking drugs for each postulated type.

A novel inhibitory prostanoid receptor in piglet saphenous vein

A range of prostanoid agonists were tested for activity on isolated ring preparations of piglet saphenous vein. The selective TxA2-mimetic (TP-receptor agonist), U-46619, contracted the preparation in a concentration-related fashion. These contractions were inhibited by the TP-receptor blocking drug, GR32191B, producing a pA2 of 7.8 (slope = 1.6). Prostanoid-induced relaxant responses were studied on preparations which had been pre-contracted using an EC60 concentration of phenylephrine (mean EC60 = 0.97 microM), in the presence of GR32191B (1 microM), to block contractile TP-receptors. Under these conditions, PGD2, PGE2, PGF2 alpha, PGI2, and U-46619, all caused concentration-related relaxation. PGE2 was the most potent agonist (EC50 = 0.23nM), whereas, all of the other agonists were at least 1,000-fold weaker, providing strong evidence for the presence of inhibitory EP-receptors. The selective synthetic EP-agonists, sulprostone (EP1/EP3) and AH13205X (EP2), were next tested for relaxant activity. While both compounds caused concentration-related relaxant activity, they were respectively 6,000 and 11,000-fold less potent than PGE2. The potent TP-receptor blocking drugs, AH22921X and AH23848B, were both weak antagonists of PGE2 but not isoproterenol-induced relaxant responses of piglet saphenous vein in a concentration-related fashion. These two compounds had pA2 values against PGE2 of 5.3 and 5.4 respectively, with regression slopes not significantly different from unity. In contrast, neither compound at a concentration of 30 microM had any antagonist activity against prostanoid-induced effects on guinea-pig fundus (EP1), rabbit ear artery (EP2) or guinea-pig vas deferens (EP3). In conclusion, the piglet saphenous vein contains TP-receptors mediating smooth muscle contraction, and a PGE2-specific (EP) receptor mediating relaxation. The inhibitory EP-receptor does not appear to be of the EP1, EP2 or EP3-subtypes, and appears therefore to be a novel subtype which we tentatively term EP4, and the potent TP-receptor blocking drugs, AH22921X and AH23848B, appear to be weak, but specific EP4-receptor blocking drugs.

Salmeterol, a novel, long-acting β2-adrenoceptor agonist: characterization of pharmacological activity in vitro and in vivo

1 Salmeterol, a novel, long‐acting β‐adrenoceptor agonist, has been compared with isoprenaline and salbutamol for activity in vitro on a range of β‐adrenoceptor containing preparations from laboratory animals and man, and in vivo for bronchodilator activity in the conscious guinea‐pig. 2 Salmeterol, like isoprenaline and salbutamol, relaxed preparations of both guinea‐pig trachea (contracted by prostaglandin (PG)F2α or electrical stimulation) and human bronchus (contracted by PGF2α) in a concentration‐related fashion. Salmeterol was of similar potency to isoprenaline and more potent than salbutamol on both airway preparations. 3 Relaxant responses of superfused guinea‐pig trachea and human bronchus to isoprenaline and salbutamol declined rapidly when the agonists were washed from the tissues, with complete recovery within 10 min, whereas responses to salmeterol were more persistent. In electrically‐stimulated guinea‐pig trachea preparations, inhibition by salmeterol persisted for periods of up to 12 h, despite continuous superfusion with agonist‐free medium. However, these persistent responses were rapidly and fully reversed by the β‐adrenoceptor blocking drug, propranolol (0.1 μm). In further studies, on guinea‐pig trachea, propranolol caused concentration‐related parallel, rightward shifts of salmeterol concentration‐effect curves, yielding a pA2 of 9.0. The slope of the Schild plot was 1.02. 4 Another β‐adrenoceptor blocking drug, sotalol (10 μm), also fully and rapidly reversed established submaximal responses to salmeterol in superfused guinea‐pig trachea. However, after administration of sotalol was stopped, the antagonism waned, and salmeterol responses were reasserted without the addition of further agonist. 5 In the β1‐adrenoceptor containing preparation, rat left atria, isoprenaline exhibited potent, concentration‐related, positive inotropic activity, whereas salbutamol and salmeterol were at least 2000–5000 fold less potent, and appeared to be partial agonists. At a concentration of 72μm, salmeterol exhibited weak antagonism of isoprenaline‐induced increases in atrial force of contraction. This antagonism was less marked than that caused by salbutamol (42 μm). 6 On the guinea‐pig isolated gastric fundus strip, a putative β3‐adrenoceptor containing preparation, salbutamol and salmeterol had only modest agonist activity, being 20–30 fold less potent than isoprenaline and the selective β3‐adrenoceptor agonist, BRL 35135. 7 In conscious guinea‐pigs, inhaled salmeterol and salbutamol were approximately equipotent in causing dose‐related bronchodilatation. Whereas the duration of action of salbutamol at its threshold‐effective dose was less than 90 min, the responses to a similarly effective dose of salmeterol were well‐maintained for at least 6 h. 8 Salmeterol is therefore a potent and selective β2‐adrenoceptor agonist with a remarkably long duration of action in isolated superfused airways smooth muscle. It also causes persistent bronchodilatation in vivo, in the guinea‐pig, when administered by the inhaled route.

Prostanoid-induced contraction of human bronchial smooth muscle is mediated by TP-receptors.

1 A range of naturally‐occurring prostaglandins sulprostone, 16,16‐dimethyl prostaglandin E2 (DME2) and the thromboxane A2 (TXA2)‐mimetic, 11α,9α‐epoxymethano prostaglandin H2 (U‐46619) have been tested for contractile agonist activity on human isolated bronchial smooth muscle. 2 Prostaglandin D2 (PGD2), PGF2α, 9α,11β‐PGF2 (11β‐PGF2) and U‐46619 all caused concentration‐related contractions. U46619 was at least 300 fold more potent than the other prostanoids with a mean EC50 of 12 nm. Sulprostone caused contraction only at the highest concentration tested (30 μm). PGE2 and PGI2 caused relaxations at low concentrations, and only caused contractile responses at high concentrations (≥ 10 μm). In contrast, DME2 caused small contractions at low concentrations but relaxation at the highest concentration tested (30 μm). 3 The rank order of contractile agonist potency was: U‐46619 > 11β‐PGF2 > PGF2α > PGD2 > PGE2 > PGI2 > sulprostone > DME2. 4 The TP‐receptor blocking drug, AH23848 (1 μm) antagonized the contractile effects of U‐46619, PGD2, PGF2α and 11β‐PGF2, but had no effect against contractions to carbachol. In a single experiment, a pA2 of 8.3 (slope = 1.2) was obtained for AH23848 against U‐46619. 5 In most preparations, administration of AH23848 (1 μm) to human bronchus resulted in small, transient contractile responses. 6 The results obtained with both the agonists and the antagonist, AH23848 are therefore consistent with prostanoid‐induced contractions of human bronchial smooth muscle being mediated by TP‐receptors.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. In the presence of indomethacin (3 μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration‐dependent manner (mean pEC50 8.0±0.1, n=5). Establishment of a rank order of potency using the EP4>EP2 agonist 11‐deoxy PGE1, and the EP2>EP4 agonist PGE1‐OH (mean pEC50 of 7.6±0.1 (n=6) and 6.4±0.1 (n=4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations <1 μM failed to relax the tissues. Blockade of EP4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration–response curves, exhibiting pA2 and pKB values of 5.7±0.1, n=3, and 8.4, n=3, respectively. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3±0.1 (n=4) and 8.1±0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGF2α failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration‐dependent contraction of cerebral artery (mean pEC50 7.4±0.3, n=3). These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

Effects of β‐adrenoceptor agonists in human bronchial smooth muscle

1 We have investigated the potency and duration of action of isoprenaline and a range of β‐adrenoceptor agonists as relaxants of inherent tone in human superfused, isolated bronchial smooth muscle, a tissue reported to contain a homogeneous population of β2‐adrenoceptors. 2 All of the β‐adrenoceptor agonists caused concentration‐related inhibition of inherent tone, with isoprenaline having an EC50 of 27 nm. The rank order of agonist potency was: formoterol ≥‐salmeterol ≥ clenbuterol > fenoterol = isoprenaline > terbutaline ≥ salbutamol > quinprenaline. 3 Relaxant responses to salmeterol were fully reversed by the selective β2‐adrenoceptor blocking drug, ICI 118551, demonstrating the involvement of β2‐adrenoceptors. 4 Rt50, i.e. the time taken for 50% recovery from the effects of an EC50 concentration of agonist, differed considerably between the different β2‐adrenoceptor agonists. Most agonists were short‐acting, having Rt50 values less than 13 min. Quinprenaline was of moderate duration, with an Rt50 value of ≥ 20 min. In contrast, salmeterol was extremely long‐acting, with no sign of recovery within 4 h. 5 Estimates of relative potency and duration of action were similar to those previously determined for these agonists in the guinea‐pig isolated trachea. These results suggest, therefore, that guinea‐pig trachea is a suitable alternative to human bronchus for the evaluation of the actions of β‐adrenoceptor agonists on airways smooth muscle.

Characterization of the prostanoid receptors mediating constriction and relaxation of human isolated uterine artery

1 This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating constriction and relaxation of human isolated uterine artery. 2 U‐46619 was a potent constrictor agonist on human uterine artery (EC50 [95% CL] = 3.5 [1.8‐6.7] μM). Prostaglandin E2 (PGE2α), PGF2, PGD2 and PGI2 only weakly constricted the uterine artery, being at least 100 times less potent than U‐46619. The PGE2 and PGI2 constrictor effects may be modified by the potent dilator effects of these compounds. A number of agonists which show selectivity for FP‐, DP‐ and EP‐receptors including ICI 81008, BW 245C, sulprostone, rioprostil and butaprost, failed to cause any constriction at concentrations up to 30 μM. 3 Constrictor responses induced by all agonists tested were reduced or abolished by the TP‐receptor blocking drugs, GR 32191 and EP 092. pA2 estimates for both antagonists versus U‐46619 were 8.50, values which are consistent with their affinities at TP‐receptors. 4 In preparations pre‐constricted with phenylephrine (1 μM) both PGI2 and PGE2 were potent relaxant agonists. The selective IP‐receptor agonists, cicaprost and iloprost, also dilated human uterine artery and were approximately 10 fold more potent than PGI2. The EP2‐receptor agonists, butaprost and rioprostil and the selective DP‐agonist, BW 245C, were at least 100 fold weaker than PGI2 and PGE2 suggesting that neither DP‐ nor EP2 receptors were involved. 5 We conclude that TP‐receptors mediate constriction, whereas IP‐ and possibly EP4‐receptors mediate relaxation of human uterine artery.

CONTRACTILE AND RELAXANT ACTIONS OF PROSTAGLANDINS ON GUINEA-PIG ISOLATED TRACHEA

1 The effects of 12 prostaglandins on guinea‐pig isolated trachea have been examined in the presence of indomethacin. Two series of experiments were carried out, the first on preparations without tone (‘zero tone’), and the second on preparations with tone induced with acetylcholine (‘high tone’). 2 The compounds tested fell into two groups. The first, comprising prostaglandins F1α, F2α F2α acetal, I2 and Wy 17186, contracted both zero and high tone preparations. The second, comprising prostaglandins A1, A2, B1, B2, E1 E2 and F2β, contracted zero, but relaxed high tone preparations. Responses to the second group of compounds are probably the resultant of their contractile and relaxant actions. 3 The order of potency for contracting zero tone preparations was prostaglandin E (PGE) > F = I = Wy 17186 > B > A, 2‐series compounds being 5 to 18 times more potent than 1‐series compounds. 4 The order of potency for relaxing high tone preparations was PGE > Fβ > B > A > Wy 17186 > Fα = I = 0. There was little difference between the potency of 1‐ and 2‐series compounds. 5 The possible relevance of these results to the interpretation of the effects of prostaglandins on human airways is discussed.

Novel and versatile superfusion system. Its use in the evaluation of some spasmogenic and spasmolytic agents using guinea-pig isolated tracheal smooth muscle

We have developed a novel, eight-chamber superfusion system that is suitable for a variety of applications involving the study of both contraction and relaxation of smooth muscle preparations, and the effect of agents that interfere with these actions. The system allows electrical stimulation of preparations, and thus neuronally mediated responses and agents that interfere with neurotransmission may also be studied. To demonstrate some of the applications of the system, we have evaluated both spasmogenic and spasmolytic agents on the guinea-pig isolated tracheal strip preparation. The potency and the times for onset and offset of action of the spasmogens, acetylcholine, histamine, and prostaglandin F2 alpha, and the spasmolytics, isoprenaline, clenbuterol, salbutamol, papaverine, N-ethylcarboxamide adenosine, theophylline, and verapamil, have been investigated. The spasmolytic agents have been tested against both prostaglandin F2 alpha-induced tone and electrically induced contractile responses of the guinea-pig trachea. This superfusion system has several advantages over previously described superfusion or immersion techniques. It is compact and allows simultaneous study of up to eight preparations. It is suitable for a wide range of tissues, and the use of this system avoids the necessity of repeatedly washing drugs from organ baths. However, one of the most important applications of the system is its use in the study of rates of onset and offset of drug action. We believe, therefore, that this system represents an important alternative to the classical organ bath for in vitro pharmacological experimentation.