P.P.A. Humphrey

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.

5‐HT1‐like receptors mediate 5‐hydroxytryptamine‐induced contraction of human isolated basilar artery

1 The 5‐hydroxytryptamine (5‐HT) receptor mediating contraction of endothelium denuded human basilar artery has been characterized in vitro. 2 5‐HT and a variety of 5‐HT agonists contracted human isolated basilar artery with a rank order of agonist potency, 5‐carboxamidotryptamine (5‐CT) > 5‐HT = methysergide > GR43175 > 8‐OHDPAT > 2‐methyl‐5‐HT. The maximum response produced by these agonists differed. 3 None of the agonists relaxed human basilar artery when tone was elevated with prostaglandin F2α, indeed further contraction was seen. 4 The contractile responses of human basilar artery to 5‐HT and the selective 5‐HT1‐like agonist GR43175 were highly reproducible whilst those to 5‐CT were not. 5 The contractile response to both 5‐HT and GR43175 was resistant to antagonism by ketanserin and GR38032, thus excluding activation of 5‐HT2 and 5‐HT3 receptors. The contractile action of 5‐HT and GR43175 was also not antagonized by (±)‐cyanopindolol, excluding the activation of receptors similar to 5‐HT1A and 5‐HT1B recognition sites identified in ligand binding studies. 6 In marked contrast, methiothepin was a potent antagonist of the contractile actions of both 5‐HT and GR43175, with a pA2 value of 8.8 against both agonists. Methiothepin (100 nm) had no effect on the contractile response to the thromboxane A2‐mimetic U46619. 7 We conclude that 5‐HT and GR43175 contract the human isolated basilar artery by activating the same receptor type. This receptor appears identical to the 5‐HT1‐like receptor causing contraction of the dog isolated saphenous vein and cerebral blood vessels from the dog and primate.

GR32191, a highly potent and specific thromboxane A2 receptor blocking drug on platelets and vascular and airways smooth muscle in vitro.

1 The thromboxane A2 (TP)‐receptor blocking activity and specificity of action of GR32191 ([1R‐[1α(Z),2β,3β,5α]]‐(+)−7‐[5‐([1,1′‐biphenyl]‐4‐ylmethoxy)−3‐hydroxy‐2‐(1‐piperidinyl)cyclopentyl]‐4‐heptonoic acid has been evaluated in human platelets and various smooth muscle preparations, both vascular and non‐vascular, from a range of species including man. 2 Utilising a platelet counting method to assess aggregation the drug was found to antagonise, in a surmountable manner, human platelet aggregation produced by the TP‐receptor agonists, U‐46619, EP171 and SQ26655, in whole blood and physiological buffer, with pA2 values of approximately 8.3 and 8.7 in the two media respectively. In the presence of GR32191 the rate of aggregation induced by U‐46619 was slowed. 3 The effect of GR32191 upon U‐46619‐induced platelet shape change and aggregation in plateletrich plasma was evaluated utilising a turbidometric technique. Both shape change and aggregation were antagonised by GR32191. At relatively high concentrations of the drug a slowing of aggregation and shape change to U‐44619 was seen and an unsurmountable antagonism became apparent. 4 The action of GR32191 upon human platelets was specific with platelet aggregation induced by adenosine 5′‐diphosphate, platelet activating factor, vasopressin and adrenaline and the inhibitory effects of prostacylin (PGI2), prostaglandin D2 (PGD2) and N‐ethylcarboxamide‐adenosine (NECA) being unaffected by concentrations of the drug as high as 10 μm. Furthermore, at concentrations of up to 100 μm, the drug itself produced no shape change or aggregation, of human platelets. 5 GR32191 also specifically and potently antagonised in a competitive, surmountable manner the contractile actions of U‐46619 upon human vascular smooth muscle and antagonised U‐46619‐induced contractions of vascular and airways smooth muscle preparations from rat, dog, guinea‐pig and rabbit with varying potency. This is discussed in terms of possible heterogeneity of TP‐receptors. 6 GR32191 therefore represents a highly potent and specific TP‐receptor blocking drug. This profile of action, coupled to its long duration of effect in man described elsewhere, make it an ideal drug tool for elucidating the physiological and pathophysiological role of thromboxane A2.

A comparison of 5‐hydroxytryptamine receptors mediating contraction in rabbit aorta and dog saphenous vein: evidence for different receptor types obtained by use of selective agonists and antagonists

1 Using recently available selective agonists and antagonists we have examined further our postulate (Apperley et al., 1980) that 5‐hydroxytryptamine (5‐HT) mediates contraction of dog saphenous vein via a different 5‐HT receptor type from that in the rabbit aorta. 2 In the rabbit isolated aorta, ketanserin and spiperone were potent, specific, competitively‐acting antagonists of the contractile effects of 5‐HT. 3 In contrast, in the dog isolated saphenous vein neither ketanserin nor spiperone caused any rightward displacement of concentration‐response curves to 5‐HT although the maximum response was reduced by about 10%. 4 In the rabbit aorta 5‐carboxamidotryptamine (5‐CONH2‐T) was a weak agonist whilst the 5‐N,N‐dimethyl and 5‐N‐ethyl derivatives were even weaker or inactive. The contractile effect of 5‐CONH2‐T in the rabbit aorta was potently and competitively antagonized by ketanserin. 5 In contrast, in the dog saphenous vein 5‐CONH2‐T and its 5‐N,N‐dimethyl and 5‐N‐ethyl derivatives were all potent agonists. The contractile effect of 5‐CONH2‐T was not markedly affected by ketanserin. 6 The profile of action of ketanserin and spiperone in the rabbit aorta is consistent with the view that 5‐HT2 receptors mediate contraction in this preparation. However, the 5‐HT receptor mediating contraction in the dog saphenous vein appears to be ‘5‐HT1‐like’, sharing a number of characteristics with the 5‐HT1 recognition site identified from [3H]‐5‐HT ligand binding studies in brain tissue. tissue.

Identification of putative 5-HT4 receptors in guinea-pig ascending colon

Experiments were carried out to characterise pharmacologically a neuronal, non-5-HT3, 5-hydroxytryptamine (5-HT) receptor in guinea-pig isolated ascending colon. In preparations pretreated with methysergide (1 microM) and ondansetron (10 microM), 5-HT (0.003-1 microM) produced repeatable concentration-related contractions of guinea-pig ascending colon with an EC50 value of 29 (20-41) nM. The responses to 5-HT could be antagonised substantially by tetrodotoxin (0.3 microM) and atropine (1 microM) indicating a neuronal cholinergically mediated effect. The 5-HT-induced response was mimicked by 5-methoxytryptamine and alpha-methyl-5-HT with equipotent concentration ratios of 26 and 28, respectively. In contrast, 2-methyl-5-HT, 8-OH-DPAT, sumatriptan, phenylbiguanide and 5-hydroxyindalpine had no agonist activity up to 10 microM. The benzamides, metoclopramide, cisapride, R,S-zacopride and renzapride, mimicked the contractile action of 5-HT, acting like partial agonists. ICS205-930 (3 microM) acted as a competitive antagonist against 5-HT and 5-methoxytryptamine with estimated pKB values of 6.4 and 6.7 respectively. ICS205-930 (1 microM) also antagonised responses to R,S-zacopride and renzapride. Ketanserin (1 microM), phenylbiguanide (10 microM) and sulpiride (1 microM) had no effect on responses to 5-HT. We conclude that the pharmacological characteristics of the receptor, which mediates contraction of guinea-pig ascending colon by activation of cholinergic nerves, are consistent with it being of the putative 5-HT4 receptor type.

Role of 5‐HT1‐like receptors in the reduction of porcine cranial arteriovenous anastomotic shunting by sumatriptan

1 The new tryptamine derivative sumatriptan (GR43175) is effective in the treatment of migraine. Since several antimigraine agents reduce cranial arteriovenous anastomotic blood flow in the anaesthetized pig, we have investigated the carotid haemodynamic effects of sumatriptan. 2 Sumatriptan (10, 30, 100 and 300 μg kg−1, i.v.) reduced total common carotid blood flow, exclusively by affecting its arteriovenous anastomotic fraction; the capillary fraction even increased with the highest doses. 3 These reductions in the carotid arteriovenous anastomotic (‘shunt’) blood flow were mediated by a 5‐HT1‐like receptor, as methiothepin, but not ketanserin, antagonized the responses to sumatriptan. 4 Sumatriptan increased the difference in oxygen saturation between arterial and jugular venous blood, which is likely to be a consequence of the reduction of the carotid shunt blood flow. 5 The selective reduction in arteriovenous anastomotic blood flow produced by sumatriptan may reflect its antimigraine action, thought to involve vasoconstriction of those cranial vessels, be they ‘shunt’ vessels or not, which are distended and inflamed during a migraine attack.

Investigation into the 5-hydroxytryptamine receptor mediating smooth muscle relaxation in the rat oesophagus

1 An investigation has been made into the 5‐hydroxytryptamine (5‐HT) receptor mediating relaxation of rat oesophagus in preparations precontracted with carbachol. 2 In tissues treated with pargyline (100 μm) and in the presence of corticosterone (30 μm) and cocaine (30 μm) the potency of 5‐HT and 5‐methoxytyramine (5‐MeOT) was not changed but the maximum response to these agonists was reduced. Thus there was no evidence of metabolism and/or uptake through an amine depleting mechanism. 3 The relaxant concentration‐effect curves to 5‐HT were shifted to the left in a concentration‐related manner by isobutylmethylxanthine (1 and 10 μm), suggesting the involvement of adenosine 3′:5′‐cyclic monophosphate in these responses. 4 5‐HT produced concentration‐related relaxations of rat oesophagus with an EC50 value of 0.24 μm. Several indole agonists were tested and the following rank order of potency of key agonists obtained: 5‐HT > α‐methyl‐5‐hydroxytryptamine = 5‐carboxamidotryptamine (5‐CT) > 5‐MeOT. In contrast, 2‐methyl‐5‐hydroxytryptamine, sumatriptan and 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin were weak or inactive. 5 The substituted benzamides, metoclopramide, cisapride, renzapride and R,S‐zacopride acted as partial agonists, producing 60–70% of the 5‐HT maximum. 6 The relaxation responses to 5‐HT were neither inhibited by antagonists selective for 5‐HT1 or 5‐HT2 receptors nor by the 5‐HT3 receptor antagonists, ondansetron, granisetron or MDL 72222. 7 The relaxation responses induced by 5‐HT, 5‐CT, 5‐MeOT and renzapride were selectively inhibited by high concentrations of ICS 205–930 with pKB values of approximately 6. 8 The 5‐HT receptor mediating relaxation in rat oesophagus cannot be designated 5‐HT1, 5‐HT2 or 5‐HT3 under the current 5‐HT classification, but the observed effects are consistent with stimulation of the putative 5‐HT4 receptor.

5‐Carboxamidotryptamine is a selective agonist at 5‐hydroxytryptamine receptors mediating vasodilatation and tachycardia in anaesthetized cats

1 We have attempted to characterize the 5‐hydroxytryptamine (5‐HT) receptors mediating bronchoconstriction, vasodilatation, vasodepression and tachycardia in anaesthetized cats following bilateral vagosympathectomy and β‐adrenoceptor blockade with propranolol. 2 5‐HT (1–100 μg kg−1 i.v.) caused dose‐related bronchoconstriction and tachycardia but variable and complex effects on diastolic blood pressure and carotid arterial vascular resistance. 3 In contrast, 5‐carboxamidotryptamine (5‐CT; 0.01–1 μg kg−1 i.v.) caused consistent, dose‐related decreases in diastolic blood pressure and carotid arterial vascular resistance and increases in heart rate. 5‐CT did not cause bronchoconstriction. 4 The 5‐HT‐induced bronchoconstriction was dose‐dependently antagonized by methiothepin, methysergide and ketanserin (10–100 μg kg−1 i.v.). The highest doses used of these antagonists did not antagonize bronchoconstriction induced by prostaglandin F2α. The high potency of all three antagonists indicate a 5‐HT2‐receptor mediated effect. 5 The 5‐HT‐ and 5‐CT‐induced tachycardia as well as the 5‐CT‐induced vasodepressor and carotid arterial vasodilator responses were dose‐dependently antagonized by low doses of methiothepin (10–100 μg kg−1 i.v.) and by high doses of methysergide (100–1000 μg kg−1 i.v.) but were little affected by ketanserin in doses up to 1000 μg kg−1 i.v. These selective effects of 5‐CT appear to be mediated by ‘5‐HT1‐like’ receptors.

Effects of intravenous infusions of prostaglandin D2 in man

Prostaglandin D2 (PGD2) was infused intravenously into normal male volunteers. Seven subjects received infusions of 16, 32, 64 ng/kg/min and six of these a further dose of 128 ng/kg/min. Each individuals maximum dose was limited by discomfort caused by intense facial flushing and nasal congestion. At these doses there was no significant effect on systolic or diastolic blood pressure nor on spirometric measurements. There was a small but statistically significant tachycardia at 64 and 128 ng/kg/min. Collagen- and adenosine diphosphate (ADP)-induced platelet aggregation ex vivo was not affected at any of the infusion rates. Infused PGD2 is unlikely to be a useful antithrombotic agent.