Araceli Sánchez-López

Canine external carotid vasoconstriction to methysergide, ergotamine and dihydroergotamine: role of 5-HT1B/1D receptors and α2-adrenoceptors

The antimigraine drugs methysergide, ergotamine and dihydroergotamine (DHE) produce selective vasoconstriction in the external carotid bed of vagosympathectomized dogs anaesthetized with pentobarbital and artificially respired, but the receptors involved have not yet been completely characterized. Since the above drugs display affinity for several binding sites, including α‐adrenoceptors and several 5‐HT1 and 5‐HT2 receptor subtypes, this study has analysed the mechanisms involved in the above responses. Intracarotid (i.c.) infusions during 1 min of methysergide (31–310 μg min−1), ergotamine (0.56–5.6 μg min−1) or DHE (5.6–31 μg min−1) dose‐dependently reduced external carotid blood flow (ECBF) by up to 46±4, 37±4 and 49±5%, respectively. Blood pressure and heart rate remained unchanged. The reductions in ECBF by methysergide were abolished and even reversed to increases in animals pre‐treated with GR127935 (10 μg kg−1, i.v.). The reductions in ECBF by ergotamine and DHE remained unchanged in animals pre‐treated (i.v.) with prazosin (300 μg kg−1), but were partly antagonized in animals pre‐treated with either GR127935 (10 or 30 μg kg−1) or yohimbine (1000 μg kg−1). Pre‐treatment with a combination of GR127935 (30 μg kg−1) and yohimbine (1000 μg kg−1) abolished the responses to both ergotamine and DHE. The above doses of antagonists were shown to produce selective antagonism at their respective receptors. These results suggest that the external carotid vasoconstrictor responses to methysergide primarily involve 5‐HT1B/1D receptors, whereas those to ergotamine and DHE are mediated by 5‐HT1B/1D receptors as well as α2‐adrenoceptors.

Mediation of 5‐HT‐induced external carotid vasodilatation in GR 127935‐pretreated vagosympathectomized dogs by the putative 5‐HT7 receptor

The vasodilator effects of 5‐hydroxytryptamine (5‐HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho‐inhibitory 5‐HT1B/1D receptors and postjunctional 5‐HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5‐HT with the 5‐HT1B/1D receptor antagonist, GR 127935, unmasks a dose‐dependent vasodilator effect of 5‐HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5‐HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg−1, i.v.). One‐minute intracarotid (i.c.) infusions of 5‐HT (0.330 μg min−1), 5‐carboxamidotryptamine (5‐CT; 0.010.3 μg min−1), 5‐methoxytryptamine (1100 μg min−1) and lisuride (31000 μg min−1) resulted in dose‐dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5‐CT>>5‐HT5‐methoxytryptamine>lisuride, whereas cisapride (1001000 μg min−1, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg−1, i.c.), but not cisapride (mean dose of 67±7 μg kg−1, i.c.), specifically abolished the responses induced by 5‐HT, 5‐CT and 5‐methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8‐OH‐DPAT (33000 μg min−1) produced dose‐dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg−1, i.c.) the agonist‐induced vasodilator responses. The external carotid vasodilator responses to 5‐HT, 5‐CT and 5‐methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)‐pindolol (4 mg kg−1) or ritanserin (100 μg kg−1) plus granisetron (300 μg kg−1), but were dose‐dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg−1, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg−1), metergoline (1 and 3 mg kg−1), methysergide (1 and 3 mg kg−1) or clozapine (0.3 and 1 mg kg−1). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5‐ht7 receptor, our results indicate that the 5‐HT receptor mediating external carotid vasodilatation in GR 127935‐pretreated vagosympathectomized dogs is operationally similar to the putative 5‐HT7 receptor mediating relaxation of vascular and non‐vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea‐pig ileum) as well as tachycardia in the cat.

The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes.

It has been suggested that the inhibition of sympathetically‐induced vasopressor responses produced by 5‐hydroxytryptamine (5‐HT) in pithed rats is mediated by 5‐HT1‐like receptors. The present study has re‐analysed this suggestion with regard to the classification schemes recently proposed by the NC‐IUPHAR subcommittee on 5‐HT receptors. Intravenous (i.v.) continuous infusions of 5‐HT and the 5‐HT1 receptor agonists, 8‐OH‐DPAT (5‐HT1A), indorenate (5‐HT1A), CP 93,129 (5‐HT1B) and sumatriptan (5‐HT1B/1D), resulted in a dose‐dependent inhibition of sympathetically‐induced vasopressor responses. The sympatho‐inhibitory responses induced by 5‐HT, 8‐OH‐DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5‐HT receptor antagonists, WAY 100635 (5‐HT1A), cyanopindolol (5‐HT1A/1B) or GR 127935 (5‐HT1B/1D). Thus, after WAY 100635, the responses to 5‐HT and indorenate, but not to 8‐OH‐DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5‐HT, indorenate and CP 93,129 were abolished, whilst those to 8‐OH‐DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5‐HT, CP 93,129 and sumatriptan, but not to 8‐OH‐DPAT and indorenate, were abolished. In additional experiments, the inhibition induced by 5‐HT was not modified after 5‐HT7 receptor blocking doses of mesulergine. The above results suggest that the 5‐HT1‐like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5‐HT1A, 5‐HT1B and 5‐HT1D, but not that of 5‐HT7, receptors.

Pharmacological profile of the 5‐HT‐induced inhibition of cardioaccelerator sympathetic outflow in pithed rats: correlation with 5‐HT1 and putative 5‐ht5A/5B receptors

Continuous infusions of 5‐hydroxytryptamine (5‐HT) inhibit the tachycardiac responses to preganglionic (C7‐T1) sympathetic stimulation in pithed rats pretreated with desipramine. The present study identified the pharmacological profile of this inhibitory action of 5‐HT. The inhibition induced by intravenous (i.v.) continuous infusions of 5‐HT (5.6 μg kg−1 min−1) on sympathetically induced tachycardiac responses remained unaltered after i.v. treatment with saline or the antagonists GR 127935 (5‐HT1B/1D), the combination of WAY 100635 (5‐HT1A) plus GR 127935, ritanserin (5‐HT2), tropisetron (5‐HT3/4), LY215840 (5‐HT7) or a cocktail of antagonists/inhibitors consisting of yohimbine (α2), prazosin (α1), ritanserin, GR 127935, WAY 100635 and indomethacin (cyclooxygenase), but was abolished by methiothepin (5‐HT1/2/6/7 and recombinant 5‐ht5A/5B). These drugs, used in doses high enough to block their respective receptors/mechanisms, did not modify the sympathetically induced tachycardiac responses per se. I.v. continuous infusions of the agonists 5‐carboxamidotryptamine (5‐CT; 5‐HT1/7 and recombinant 5‐ht5A/5B), CP 93,129 (r5‐HT1B), sumatriptan (5‐HT1B/1D), PNU‐142633 (5‐HT1D) and ergotamine (5‐HT1B/1D and recombinant 5‐ht5A/5B) mimicked the above sympatho‐inhibition to 5‐HT. In contrast, the agonists indorenate (5‐HT1A) and LY344864 (5‐ht1F) were inactive. Interestingly, 5‐CT‐induced cardiac sympatho‐inhibition was abolished by methiothepin, the cocktail of antagonists/inhibitors, GR 127935 or the combination of SB224289 (5‐HT1B) plus BRL15572 (5‐HT1D), but remained unchanged when SB224289 or BRL15572 were given separately. Therefore, 5‐HT‐induced cardiac sympatho‐inhibition, being unrelated to 5‐HT2, 5‐HT3, 5‐HT4, 5‐ht6, 5‐HT7 receptors, α1/2‐adrenoceptor or prostaglandin synthesis, seems to be primarily mediated by (i) 5‐HT1 (probably 5‐HT1B/1D) receptors and (ii) a novel mechanism antagonized by methiothepin that, most likely, involves putative 5‐ht5A/5B receptors.

Operational characteristics of the 5-HT1-like receptors mediating external carotid vasoconstriction in vagosympathectomized dogs

It has recently been shown that the external carotid vasoconstrictor response to 5-HT in the dog is primarily mediated by sumatriptan-sensitive 5-HT1-like receptors; however, the fact that these receptors are not blocked by metergoline, a 5-HT1D ligand, raises questions about their possible correlation with the 5-HT1D receptor subtype. Since a number of drugs display high affinity for the 5-HT1D (GR127935) and 5-HT1F (e.g. methysergide and oxymetazoline) receptor subtypes, in this study we have used these drugs to determine whether the above vasoconstrictor 5-HT1-like receptors correlate with the 5-HT1D and/or 5-HT1F receptor subtypes.One-minute intracarotid infusions of 5-HT (0.3–30 μg/min), sumatriptan (1–30 μg/min), oxymetazoline (0.03–3 μg/min) and noradrenaline (0.3–3 μg/min) resulted in dose-dependent decreases in external carotid blood flow without changes in arterial blood pressure or heart rate. These vasoconstrictor responses remained unaltered after i.v. administration of physiological saline (0.015, 0.05 and 0.15 ml/kg; n = 4) or ritanserin (1 mg/kg; n = 5). In contrast, GR127935 (1, 3 and 10 μg/kg, n = 6) potently blocked the responses to 5-HT (unmasking a dose-dependent vasodilator component) and sumatriptan without affecting those to oxymetazoline or noradrenaline. Interestingly, methysergide (10, 30 and 100 μg/kg, n = 5) also blocked the vasoconstrictor responses to 5-HT and sumatriptan, but unlike GR127935, did not revert the vasoconstrictor response to 5-HT; the responses to oxymetazoline remained unaffected, but those to noradrenaline were apparently attenuated by the highest dose.Taken together, the above findings suggest that the sumatriptan-sensitive 5-HT1-like receptors mediating canine external carotid vasoconstriction resemble 5-HT1D receptors, probably of the 5-HT1Dβ subtype on the basis of the resistance to blockade by ritanserin. The pharmacological profile of these receptors could be similar (bovine and human cerebral arteries, porcine carotid arteriovenous anastomoses and human coronary arteries) to other putative 5-HT1D receptors mediating vascular responses.

5-Hydroxytryptamine inhibits the tachycardia induced by selective preganglionic sympathetic stimulation in pithed rats.

It has been shown in several species that serotonin (5-hydroxytryptamine; 5-HT) is able to inhibit the responses produced by sympathetic stimulation in a wide variety of blood vessels and other organs, including the heart. However, in pithed rats, the analysis of potential sympatho-inhibitory actions of 5-HT is hampered by the fact that 5-HT (given as i.v. bolus injections) produces tachycardia per se. Moreover, most studies have investigated 5-HT-induced sympatho-inhibition at only one frequency of stimulation. Thus, the present study set out to find the experimental conditions to overcome these problems. In this regard, we analyzed the potential ability of 5-HT, administered as i.v. continuous infusions, to inhibit the tachycardia caused by stimulation of the preganglionic (C7-T1) sympathetic outflow in pithed rats. Sympathetic cardiostimulation (0.01-3 Hz) resulted in frequency-dependent increases in heart rate; these responses were potentiated after desipramine (50 microg/kg, i.v.). During continuous infusions of 5-HT (3.1-10 microg/kg.min, i.v.), but not saline, the sympathetically-induced tachycardia was dose-dependently inhibited in both control and desipramine-pretreated rats. This inhibitory effect of 5-HT was significantly more pronounced at lower frequencies of stimulation. In contrast, the above infusions of 5-HT did not inhibit the tachycardia induced by i.v. bolus injections of noradrenaline in both control and desipramine-pretreated rats. Taken together, the above findings confirm that 5-HT induces inhibition of the sympathetic chronotropic outflow in the rat by acting at receptors located prejunctionally, without evoking tachycardia, over a wide range of stimulation frequencies.

Effects of sumatriptan on capsaicin-induced carotid haemodynamic changes and CGRP release in anaesthetized pigs

It is suggested that during a migraine attack capsaicin-sensitive trigeminal sensory nerves release calcitonin gene related peptide (CGRP), resulting in cranial vasodilatation and central nociception. Hence, inhibition of trigeminal CGRP release may prevent the above vasodilatation and, accordingly, abort migraine headache. Therefore, this study investigated the effects of sumatriptan (100 and 300 μg/kg, i.v.) on capsaicin-induced carotid haemodynamic changes and on CGRP release. Intracarotid (i.c.) infusions of capsaicin (10 μg/kg/min, i.c.) increased total carotid, arteriovenous anastomotic and capillary conductances as well as carotid pulsations, but decreased the difference between arterial and jugular venous oxygen saturations. Except for some attenuation of arteriovenous anastomotic changes, the capsaicin-induced responses were not affected by sumatriptan. Moreover, i.c. infusions of capsaicin (0.3, 1, 3 and 10 μg/kg/min, i.c.) dose-dependently increased the jugular venous plasma concentrations of CGRP, which also remained unaffected by sumatriptan. The above results support the contention that the therapeutic action of sumatriptan is mainly due to cranial vasoconstriction rather than trigeminal (CGRP release) inhibition.

Donitriptan, but not sumatriptan, inhibits capsaicin-induced canine external carotid vasodilatation via 5-HT1B rather than 5-HT1D receptors.

It has been suggested that during a migraine attack capsaicin‐sensitive trigeminal sensory nerves release calcitonin gene‐related peptide (CGRP), resulting in cranial vasodilatation and central nociception; hence, trigeminal inhibition may prevent this vasodilatation and abort migraine headache. This study investigated the effects of the agonists sumatriptan (5‐HT1B/1D water‐soluble), donitriptan (5‐HT1B/1D lipid‐soluble), PNU‐142633 (5‐HT1D water‐soluble) and PNU‐109291 (5‐HT1D lipid‐soluble) on vasodilator responses to capsaicin, α‐CGRP and acetylcholine in dog external carotid artery.

Evidence that some imidazoline derivatives inhibit peripherally the vasopressor sympathetic outflow in pithed rats

Imidazoline derivatives (e.g. clonidine and moxonidine) and alpha(2)-adrenoceptor agonists (e.g. B-HT 933) have been shown to inhibit sympathetically-induced [(3)H]noradrenaline release in several isolated blood vessels. The present study has compared the potential capability of agonists at imidazoline I(1/2) receptors and/or alpha(1/2)-adrenoceptors to inhibit the sympathetically-induced vasopressor responses in pithed rats. For this purpose, male Wistar rats were pithed and prepared for measurement of diastolic blood pressure and heart rate. Then, the vasopressor responses induced by either selective electrical stimulation (2 ms, 60 V; 0.03, 0.1, 0.3, 1 and 3 Hz) of the vascular sympathetic outflow (T(7)-T(9)) or i.v. bolus injections of exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 microg/kg) were determined before and during i.v. continuous infusions of the agonists B-HT 933 (alpha(2)), clonidine (alpha(2), I(1)), moxonidine (alpha(2), I(1)), cirazoline (alpha(1), I(2)), agmatine (putative endogenous ligand of imidazoline receptors) and methoxamine (alpha(1)), or equivalent volumes of physiological saline. Electrical sympathetic stimulation elicited frequency-dependent vasopressor responses which were significantly inhibited during the continuous infusions of B-HT 933, clonidine, moxonidine, cirazoline and agmatine, but not of physiological saline. Interestingly, the vasopressor responses to exogenous noradrenaline, which remained unaffected during the infusions of physiological saline, B-HT 933, moxonidine, cirazoline and agmatine, were significantly blocked during the infusions of clonidine or methoxamine. These results suggest that B-HT 933, moxonidine, cirazoline and agmatine induced a prejunctional inhibition of the vasopressor sympathetic outflow in pithed rats, whilst clonidine inhibited the vasopressor sympathetic outflow by both prejunctional and postjunctional mechanisms.

Evidence for 5‐HT1B/1D and 5‐HT2A receptors mediating constriction of the canine internal carotid circulation

The present study has investigated the preliminary pharmacological profile of the receptors mediating vasoconstriction to 5‐hydroxytryptamine (5‐HT) in the internal carotid bed of vagosympathectomised dogs. One minute intracarotid infusions of the agonists 5‐HT (0.1–10 μg min−1), sumatriptan (0.3–10 μg min−1; 5‐HT1B/1D), 5‐methoxytryptamine (1–100 μg min−1; 5‐HT1, 5‐HT2, 5‐HT4, 5‐ht6 and 5‐HT7) or DOI (0.31–10 μg min−1; 5‐HT2), but not 5‐carboxamidotryptamine (0.01–0.3 μg min−1; 5‐HT1, 5‐ht5A and 5‐HT7), 1‐(m‐chlorophenyl)‐biguanide (mCPBG; 1–1000 μg min−1; 5‐HT3) or cisapride (1–1000 μg min−1; 5‐HT4), resulted in dose‐dependent decreases in internal carotid blood flow, without changing blood pressure or heart rate. The vasoconstrictor responses to 5‐HT, which remained unaffected after saline, were resistant to blockade by i.v. administration of the antagonists ritanserin (100 μg kg−1; 5‐HT2A/2B/2C) in combination with tropisetron (3000 μg kg−1; 5‐HT3/4) or the cyclo‐oxygenase inhibitor, indomethacin (5000 μg kg−1), but were abolished by the 5‐HT1B/1D receptor antagonist, GR127935 (30 μg kg−1). Interestingly, after administration of GR127935, the subsequent administration of ritanserin unmasked a dose‐dependent vasodilator component. GR127935 or saline did not practically modify the vasoconstrictor effects of 5‐MeO‐T. In animals receiving GR127935, the subsequent administration of ritanserin abolished the vasoconstrictor responses to 5‐MeO‐T unmasking a dose‐dependent vasodilator component. The vasoconstriction induced by sumatriptan was antagonized by GR127935, but not by ritanserin. Furthermore, ritanserin (100 μg kg−1) or ketanserin (100 μg kg−1; 5‐HT2A), but not GR127935, abolished DOI‐induced vasoconstrictor responses. The above results suggest that 5‐HT‐induced internal carotid vasoconstriction is predominantly mediated by 5‐HT1B/1D and 5‐HT2A receptors.