Helen E. Connor

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.

Naratriptan: Biological Profile in Animal Models Relevant to Migraine

The biological profile of naratriptan (N-methyl-3-(1-methyl-4-piperidinyl)-1H-indole-5-ethane-sulphona-mide), a novel 5HT1B/1D receptor agonist, was investigated in a variety of experimental models of relevance to migraine. Naratriptan has high affinity for human recombinant 5HT1B and 5HT1D receptors (pKi = 8.70.03 and 8.30.1, respectively) and causes contractions of dog isolated basilar and middle cerebral artery (EC50 values of 0.11 and 0.07 M, respectively). Naratriptan causes small contractions of human isolated coronary arteries (EC50 value of 0.17 M; maximum contraction equivalent to 33% of 5HT maximum). In anaesthetized dogs, naratriptan causes selective vasoconstriction of the carotid arterial bed (CD50 dose = 193 g kg-1) and, in anaesthetized rats, naratriptan selectively inhibits neurogenic plasma protein extravasation in the dura (ID50 = 4.1 g kg-1). In a variety of antinociceptive tests, naratriptan has no effect even at high doses. In conscious rats and dogs, naratriptan has high oral bioavailability (71% and 95%, respectively). The data show that naratriptan is a selective agonist at 5HT1B/1D receptors, with a pharmacological profile very similar to that of sumatriptan, albeit 2–3 fold more potent. These observations, coupled with high oral bioavailability in animals, suggest that naratriptan has the profile of an orally effective anti-migraine drug.

The pharmacology of the novel 5-HT1-like receptor agonist, GR43175.

We describe the identification of a novel drug, GR43175, for the acute treatment of migraine. GR43175 is a tryptamine analogue with a very selective agonist action at a 5-HT1-like receptor subtype first identified in the dog saphenous vein. Using this drug as a research probe, we have now shown that this 5-HT receptor type predominates in the carotid circulation, which explains the remarkably selective vasoconstrictor action of GR43175 in vivo in the carotid arterial bed of dogs and cats. Its vasoconstrictor action can be shown to be localized even further to arteriovenous anastomoses (shunts) within the carotid circulation, in such a way that blood flow to the brain as well as to extracerebral capillary beds remains unaffected or may even be increased. In the treatment of migraine demonstrated to date, the impressive effectiveness of GR43175 must reinforce the evidence in favour of an important vascular component being involved in the aetiology of the disease. The question is again raised as to whether the opening of carotid shunts is involved, as suggested by Heyck. If not, an alternative vascular locus needs to be identified.

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.

The pharmacology of GR203040, a novel, potent and selective non‐peptide tachykinin NK1 receptor antagonist

1 The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)‐2‐methoxy‐5‐tetrazol‐1‐yl‐benzyl‐(2‐phenyl‐piperidin‐3‐yl)‐amine), a novel, highly potent and selective non‐peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2 GR203040 potently inhibited [3H]‐substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea‐pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5), GR203040 had little affinity (pIC50 < 6.0) at all non‐NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH‐SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca2+‐evoked contractions of rat isolated portal vein (pKB = 4.1). The enantiomer of GR203040, GR205608 ((2R, 3R)‐2‐methoxy‐5‐tetrazol‐1‐yl‐benzyl‐(2‐phenyl‐piperidin‐3‐yl)‐amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3 In gerbil ex vivo binding experiments, GR203040 produced a dose‐dependent inhibition of the binding of [3H]‐substance P to cerebral cortical membranes (ED50 = 15 μg kg−1 s.c. and 0.42 mg kg−1 p.o.). At 10 μg kg−1 s.c., the inhibition of [3H]‐substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg−1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4 In guinea‐pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration‐effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5 In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose‐ratio of 10)= 1.1 μg kg−1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 μg kg−1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6 In anaesthetized rats, GR203040 (3 and 10 mg kg−1, i.v.) produced a dose‐dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7 It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

Influence of the endothelium on contractile effects of 5-hydroxytryptamine and selective 5-HT agonists in canine basilar artery.

1 We have investigated the influence of endothelial damage on the cerebrovascular reactivity to 5‐hydroxytryptamine (5‐HT) and some selective 5‐HT agonists in canine basilar artery. 2 5‐HT, α‐methyl 5‐HT, GR 43175 (3‐[2‐dimethyl amino] ethyl‐N‐methyl‐lH‐indole‐5‐methane sulphonamide) and 5‐carboxamidotryptamine (5‐CT) produced concentration‐dependent contractions of untreated dog basilar artery with a functional endothelium. Following endothelial damage by perfusion with Triton X‐100 (0.1%), which abolished the relaxant response to substance P, the maximum contractile effect of 5‐HT, α‐methyl 5‐HT, GR 43175 and 5‐CT was markedly enhanced although there was little change in the EC50 values. Endothelial damage did not modify the vasoconstrictor effect of the thromboxane agonist, U46619, or potassium chloride. 3 Neither 5‐HT nor 5‐CT caused relaxation of untreated canine basilar arteries contracted with prostaglandin F2α, U46619, uridine triphosphate or potassium chloride. 4 These results suggest that canine basilar artery spontaneously releases endothelium‐derived relaxing factor which can attenuate the vasoconstrictor effect of 5‐HT and selective 5‐HT agonists. This effect appeared to be specific since the vasoconstrictor response to U46619 was not modified. 5 These results demonstrate that the cerebrovascular endothelium can markedly influence the reactivity of the vascular smooth muscle of canine basilar artery to 5‐HT and 5‐HT1‐like receptor agonists. However we could find no evidence that 5‐HT receptor activation stimulates endothelial cell function as it does in some other blood vessels.

The activity of 5-HT1D receptor ligands at cloned human 5-HT1Dα and 5-HT1Dβ receptors

Abstract The present study has examined the functional activity of the 5-HT 1D receptor agonist, sumatriptan, and antagonists, GR127935 (2′-methyl-4′-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide), GR55562 (3-[3-(dimethylamino)propyl]-4-hydroxy- N -[4-(4-pyridinyl) phenyl]benzamide), metergoline and methiothepin in HeLa cells, stably transfected with either 5-HT 1Dα or 5-HT 1Dβ receptor subtypes. Sumatriptan, GR127935 and metergoline (each 0.01–1 μM) behaved as agonists, producing a concentration-dependent inhibition of forskolin-stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) production at both 5-HT 1Dα and 5-HT 1Dβ receptor subtypes (mean pIC 50 values of 8.4 and 8.3 for sumatriptan, 7.9 and 8.0 for GR127935, and 7.9 and 8.3 for metergoline, respectively). In contrast, GR55562 and methiothepin behaved as competitive 5-HT 1D receptor antagonists and were devoid of any agonist activity. GR55562 (10 μM) caused a rightward displacement of the GR127935 and metergoline concentration-response curves. The agonist activity of GR127935 and metergoline, observed in the present study, contrasts with their recognised 5-HT 1D receptor antagonist profiles in animal isolated tissue and behavioural models. Unlike GR127935, GR55562 behaved as a silent antagonist at the cloned human 5-HT 1Dα and 5-HT 1Dβ receptors in the study.

Role of endothelium in haemoglobin-induced contraction of dog basilar artery

Haemoglobin (10 nM-30 microM) caused contraction of dog isolated basilar artery. In preparations which were perfused with Triton X-100 (0.1%) for one min to remove the endothelium, substance P relaxation was abolished but haemoglobin-induced contractions were unchanged. Endothelium removal or damage was confirmed histologically. These results suggest that the integrity of the endothelium is not essential for haemoglobin to produce contraction in dog basilar artery.

The activity of GR205171, a potent non-peptide tachykinin NK1 receptor antagonist, in the trigeminovascular system

The in vivo activity of GR205171, a novel, highly potent non-peptide tachykinin NK1 receptor antagonist, has been investigated in the trigeminovascular system in order to assess its potential as an acute therapy for migraine headache. In anaesthetised rabbits, GR205171 attenuated reductions in carotid arterial vascular resistance evoked by the tachykinin NK1 receptor agonist, substance P methyl ester (SPOMe), injected via the lingual artery (DR30 (i.e., the dose producing a dose-ratio of 30) = 0.4 microgram/kg, i.v.). In anaesthetised rats, GR205171 (0.1 and 1 mg/kg, i.v.) produced a dose-dependent inhibition of plasma protein extravasation (PPE) in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. In anaesthetised guinea-pigs, GR205171 (1.10 and 100 micrograms/kg, i.v.) inhibited, by up to approximately 60%, expression of c-fos in the trigeminal nucleus caudalis in response to electrical stimulation of the trigeminal ganglion. It is concluded that GR205171 is a potent antagonist of NK1 receptor-mediated cranial vasodilatation, dural PPE and expression of c-fos in the trigeminal nucleus caudalis. Such a profile of action suggests that GR205171 may have potential as a novel therapeutic agent in the treatment of migraine headache.

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

1 The effects of selective neurokinin agents on pial artery diameter, measured with an on‐line image analyser, have been studied in anaesthetized guinea‐pigs in order to characterize the neurokinin receptors present on pial arteries. 2 Perivascular injection of either substance P (0.01–1 μm) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01–1 μm) and GR73632 (0.1 μm), increased pial artery diameter. 3 In contrast, the selective NK2 receptor agonist, GR64349 (1 μm), produced a small vasoconstriction while the NK3 receptor‐selective agonist, senktide (1 μm) was inactive. 4 Co‐administration of GR82334 (1 μm), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 μm) but not that caused by calcitonin gene‐related peptide (CGRP, 0.01 μm). 5 The results are consistent with an involvement of NK1 receptors in the neurokinin‐induced increase in guinea‐pig pial artery diameter.