P.R. Saxena

Interactions of GR127935, a 5-HT1B/D receptor ligand, with functional 5-HT receptors

GR127935 (N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) [1, 1-biphenyl]-4-carboxamide hydrochloride) has been recently introduced as an experimental tool to antagonize 5-HT1B/D receptor-mediated functional responses. The compound indeed exhibits a very high affinity and selectivity for 5-HT1B/D binding sites and it antagonizes a number of 5-HT1B/D receptor-mediated responses. The present experiments were performed to investigate the selectivity of GR127935 against functional responses mediated by 5-HT1-like, ‘orphan’ 5-HT1-like (5-ht7?), 5-HT2, 5-HT3 or 5-HT4 receptors in several invivo preparations. Intravenous (i.v.) treatment with GR127935 (300μg˘kg-1) potently antagonized decreases in total carotid blood flow as well as hypotensive responses induced by the 5-HT1-like receptor agonist sumatriptan in rabbits. I.v. bolus injections of GR127935 (up to 500 and/or 1500μg˘kg-1) did not significantly modify 5-HT-induced: (i) tachycardia in the pig (5-HT4 receptor-mediated) and cat (‘orphan’ 5-HT1-like or, perhaps, 5-ht7 receptor-mediated); (ii) depressor effects in the rat and cat (‘orphan’ 5-HT1-like or 5-ht7 receptor-mediated); (iii) vonBezold-Jarisch reflex in the rat or the early phase of the urinary bladder contraction in the cat (both 5-HT3 receptor-mediated). In contrast, high doses (500-1500μg˘kg-1) of GR127935 suppressed 5-HT-induced pressor responses in the rat and cat and urinary bladder contractions (secondary phase) in the cat as well as the DOI ((±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride)-induced pressor responses in the rat, which are all mediated by 5-HT2A receptors. In conclusion, the present study demonstrates that GR127935 is a selective 5-HT1B/D receptor antagonist devoid of interactions at ‘orphan’ 5-HT1-like (5-ht7?), 5-HT3 and 5-HT4 receptors. However, GR127935 possesses a moderate 5-HT2A receptor blocking property, which is consistent with its binding profile (pKi: 7.4). Lastly, in view of the potent antagonist action of GR127935, the sumatriptan-induced hypotension in rabbits seems to be mediated by 5-HT1B/D receptors.

Characterization of sumatriptan‐induced contractions in human isolated blood vessels using selective 5‐HT1B and 5‐HT1D receptor antagonists and in situ hybridization

The 5-HT1B/1D receptor agonist sumatriptan is effective in aborting acute attacks of migraine and is known to cause constriction of cranial arteries as well as some peripheral blood vessels. The present study set out to investigate whether 5-HT1B and/or 5-HT1D receptors mediate contractions of the human isolated middle meningeal and temporal arteries (models for anti-migraine efficacy) and coronary artery and saphenous vein (models for side-effect potential). Concentration-response curves were made with sumatriptan (1 nM-100 μM) in blood vessels in the absence or presence of selective antagonists at 5-HT1B (SB224289) and 5-HT1D (BRL15572) receptors. SB224289 antagonized sumatriptan-induced contractions in all blood vessels, although the antagonism profile was different amongst these blood vessels. In the temporal artery, SB224289 abolished contraction to sumatriptan, whereas in the middle meningeal artery and saphenous vein sumatriptan-induced contractions were blocked in an insurmountable fashion. Moreover, SB224289 acted as a weak surmountable antagonist in the coronary artery (pKB: 6.4 ± 0.2). In contrast, BRL15572 had little or no effect on sumatriptan-induced contractions in the four blood vessels investigated. In situ hybridization revealed the expression of 5-HT1B receptor mRNA in the smooth muscle as well as endothelial cells of the blood vessels, whereas the mRNA for the 5-HT1D receptor was only very weakly expressed. These results show that the 5-HT1B receptor is primarily involved in sumatriptan-induced contractions of human cranial as well as peripheral blood vessels.

Comparison of the cardiovascular effects of the 5-HT1A receptor agonist flesinoxan with that of 8-OH-DPAT in the rat

The cardiovascular response to flesinoxan and 8-OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin), 5-HT1A receptor agonists, has been investigated in anaesthetized Wistar rats and spontaneously hypertensive rats (SHR) and in conscious SHR. Flesinoxan and 8-OH-DPAT potently lowered blood pressure and heart rate in these models. In conscious SHR, atropine reversed the bradycardia induced by flesinoxan partially and that induced by 8-OH-DPAT completely. In pithed rats with vasopressin-raised blood pressure, neither flesinoxan nor 8-OH-DPAT lowered blood pressure or heart rate. Intracisternal administration of either flesinoxan or 8-OH-DPAT was less efficacious than intravenous administration. The cardiovascular responses to flesinoxan and 8-OH-DPAT in the anaesthetized Wistar were inhibited by the putative 5-HT1A antagonists methiothepin, buspirone, spiroxatrine and 8-MeO-C1EPAT (8-methoxy-2-(N-2-cholroethyl-N-n-propylamino)tetralin). 8-MeO-C1EPAT appeared to be the most suitable antagonist in this model. The 5-HT1C, antagonist ritanserin or the 5-HT3 antagonist GR 38032F had no effect on the responses to flesinoxan or 8-OH-DPAT. In conscious SHR however, 8-MeO-C1EPAT did not antagonize these cardiovascular responses. This study confirms the involvement of central 5-HT1A receptors in the cardiovascular effects of flesinoxan and 8-OH-DPAT.

Intravital microscopy on a closed cranial window in mice: a model to study trigeminovascular mechanisms involved in migraine.

The purpose of the study was to develop a mouse model to study trigeminovascular mechanisms using intravital microscopy on a closed cranial window. In addition, we studied exogenous and endogenous calcitonin gene-related peptide (CGRP)-mediated vasodilation in dural arteries. Arteries in C57BL/6Jico mice were constricted with endothelin-1, which reduced the baseline diameter by 65-75%. Subsequently, vasodilation was induced by α-CGRP, capsaicin or transcranial electrical stimulation of perivascular trigeminal nerves in the absence or presence of different concentrations of BIBN4096BS or sumatriptan. Both α-CGRP and capsaicin induced vasodilation in preconstricted arteries. Transcranial electrical stimulation also induced current-dependent relaxation of dural arteries with 100 μA producing maximal dilation in the control group. BIBN4096BS blocked the responses evoked by ä-CGRP and capsaicin, as well as electrical stimulation, whereas sumatriptan attenuated only vasodilation induced by electrical stimulation. This model is likely to prove useful in dissecting elements of the trigeminovascular system and for exploring pathophysiological aspects of migraine, especially in future studies using transgenic mice with mutations relevant to those observed in patients with migraine.

No vasoactive role of the angiotensin II type 2 receptor in normotensive Wistar rats.

OBJECTIVE To investigate the vasoactive consequences of angiotensin II type 2 receptor stimulation in vivo. DESIGN AND METHODS Three consecutive 10 min intravenous infusions of angiotensin (Ang) II (100, 300 and 1000 ng/kg per min) were given to 20 pentobarbitone-anaesthetized normotensive Wistar rats (weight 330+/-6 g, mean +/- SEM). The rats had been pretreated with saline (n = 8), the angiotensin II type 1 receptor antagonist, irbesartan (100 microg/kg per min for 30 min, n = 6), or the angiotensin II type 2 receptor antagonist, PD123319 (20 microg/kg per min for 30 min followed by continuous infusion throughout the entire experiment, n = 6). Regional haemodynamic effects of Ang II were studied using the radioactive microsphere method. RESULTS Ang II increased mean arterial blood pressure (MAP) and heart rate by, maximally, 44+/-9 and 26+/-6%, respectively (P < 0.05 compared with baseline), and decreased cardiac output and systemic vascular conductance (cardiac output/MAP) by, maximally, 24+/-8 and 47+/-4%, respectively (P < 0.05 compared with baseline). The Ang II-induced decrease in systemic vascular conductance was caused by decreases in vascular conductances (regional flow/MAP) of the gastrointestinal tract (52+/-4%), kidney (63+/-3%), skeletal muscle (39+/-8%), skin (63+/-4%), mesentery + pancreas (32+/-11%), adrenal (27+/-11%) and spleen (57+/-6%) (all P < 0.05 compared with baseline). Irbesartan increased baseline vascular conductances in adrenal, brain and kidney, and inhibited all haemodynamic responses induced by Ang II. PD123319 affected neither baseline values nor the Ang II-induced haemodynamic responses. CONCLUSIONS Ang II-induced systemic and regional haemodynamic effects in normotensive Wistar rats are mediated exclusively via angiotensin II type 1 receptors. No evidence for angiotensin II type 2 receptor-mediated vasoactive responses was obtained.

Porcine carotid vascular effects of eletriptan (UK-116,044): a new 5-HT1B/1D receptor agonist with anti-migraine activity

It has been suggested that opening of cephalic arteriovenous anastomoses may be involved in the headache phase of migraine. Indeed, a number of acutely acting anti-migraine drugs, including the ergot alkaloids and sumatriptan, constrict porcine carotid arteriovenous anastomoses. In this study, using pentobarbital anaesthetised pigs, we investigated the effects of eletriptan, a close structural analogue of sumatriptan, on the distribution of common carotid artery blood flow into arteriovenous anastomotic and nutrient (capillary) fractions. Eletriptan (10, 30, 100, 300 and 1000 µg kg–1, i.v.) decreased the total carotid blood flow, exclusively by decreasing cephalic arteriovenous anastomotic blood flow; nutrient blood flow, particularly to the ear, skin and fat, was significantly increased. The doses of eletriptan needed to reduce arteriovenous anastomotic blood flow and conductance by 50% (ED50) were, respectively, 117±21 µg kg–1 (251±45 nmol kg–1) and 184±42 µg kg–1 (396±91 nmol kg–1); the highest dose caused reductions of 84±3% and 77±4%, respectively. The eletriptan-induced changes in carotid haemodynamics were clearly attenuated by pretreating the pigs with the selective 5-HT1B/1D receptor antagonist GR127935 (0.5 mg kg–1). On the basis of these results, we conclude that (1) the eletriptan-induced constriction of cephalic arteriovenous anastomoses as well as the arteriolar dilatation in head tissues is predominantly mediated by 5-HT1B/1D receptors, and (2) eletriptan should be effective in aborting migraine headache. Clinical studies have already demonstrated its therapeutic action in migraine patients.

Effects of donitriptan on carotid haemodynamics and cardiac output distribution in anaesthetized pigs.

We investigated the effects of donitriptan, which possesses a uniquely high affinity and efficacy at 5-HT1B/1D receptors, on carotid and systemic haemodynamics in anaesthetized pigs. Donitriptan (0.16-100 μg kg-1, i.v.) dose-dependently decreased total carotid blood flow and vascular conductance (maximum response: -25 ± 3%). This effect was entirely due to a selective reduction in the cephalic arteriovenous anastomotic fraction (maximum response: -63 ± 3%; ED50%: 92 ± 31 nmol/kg); the nutrient vascular conductance increased. Donitriptan did not decrease vascular conductances in or blood flow to a number of organs, including the heart and kidneys; in fact, vascular conductances in the skin, brain and skeletal muscles increased. Cardiac output was slightly decreased by donitriptan, but this effect was confined to peripheral arteriovenous anastomoses. The haemodynamic effects of donitriptan were substantially reduced by the 5-HT1B/1D receptor antagonist GR127935. These results show that donitriptan selectively constricts arteriovenous anastomoses via 5-HT1B receptor activation. The drug should be able to abort migraine headaches and it is unlikely to compromize blood flow to vital organs.

Assessment of anti‐migraine potential of a novel α‐adrenoceptor agonist S19014: effects on porcine carotid and regional haemodynamics and human coronary artery

Taking into account the drawbacks associated with the use of triptans, attempts are being made to explore other avenues for the treatment of migraine. Recently, it has been shown that both α1- and α2-adrenoceptors mediate the constriction of porcine carotid arteriovenous anastomoses, which has effectively served as an experimental model predictive of anti-migraine activity. The present study investigated the effects of a novel α-adrenoceptor agonist S19014 {spiro[(1,3- diazacyclopent-1-ene)-5 : 2′-(4′,5′-dimethylindane)] fumarate} on carotid and systemic haemodynamics in anaesthetized pigs, and on human isolated coronary arteries. Increasing doses of S19014 (1-30 μg/kg, i.v.) produced a dosedependent initial short-lasting vasopressor response and a decrease of total carotid blood flow and conductance. The carotid blood flow and conductance changes were exclusively due to constriction of carotid arteriovenous anastomoses (capillary blood flow increased) and were accompanied by an increase in arterio-jugular venous oxygen saturation difference. Whereas prazosin (100 μg/kg, i.v.) was ineffective, rauwolscine (300 μg/kg, i.v.) attenuated the responses to S19014. The compound did not much affect the distribution of cardiac output to peripheral organs when compared with the vehicle group. Furthermore, S19014 only slightly contracted the human isolated coronary artery and its contractions, contrary to those of sumatriptan, were not increased in blood vessels precontracted with U46619. These results suggest that (i) the systemic and carotid vascular effects of S19014 are mainly mediated by α2-adrenoceptors, and (ii) S19014 could be effective in the treatment of migraine with an improved cardiovascular tolerance.

α1-adrenoceptor subtypes mediating vasoconstriction in the carotid circulation of anaesthetized pigs: possible avenues for antimigraine drug development

It has recently been shown that the α-adrenoceptors mediating vasoconstriction of porcine carotid arteriovenous anastomoses resemble both α1- and α2-adrenoceptors, but no attempt was made to identify the specific subtypes (α1A, α1B and α1D) involved. Therefore, the present study was designed to elucidate the specific subtype(s) of α1-adrenoceptors involved in the above response, using the α1-adrenoceptor agonist phenylephrine and α1-adrenoceptor antagonists 5-methylurapidil (α1A), L-765 314 (α1B) and BMY 7378 (α1D). Ten-minute intracarotid infusions of phenylephrine (1, 3 and 10 μgkg−1.min−1) induced a dose-dependent decrease in total carotid and arteriovenous anastomotic conductance, accompanied by a small tachycardia. These carotid vascular effects were abolished by L-765 314 (1000 μgkg−1; i.v.), while these responses were only attenuated by 5-methylurapidil (1000 μgkg−1; i.v.), and BMY 7378 (1000 μgkg−1; i.v.). Furthermore, intravenous bolus injections of phenylephrine (3 and 10 μgkg−1) produced a dose-dependent vasopressor response, which was only affected by 1000 μgkg−1 of 5-methylurapidil, while the other antagonists were ineffective. These results, coupled to the binding affinities of the above antagonists at the different α1-adrenoceptors, suggest that both α1A- and α1B-adrenoceptors mediate constriction of carotid arteriovenous anastomoses in anaesthetized pigs. In view of the less ubiquitous nature of α1B- compared to α1A-adrenoceptors, the development of potent and selective α1B-adrenoceptor agonists may prove to be important for the treatment of migraine.

The Lack of Vasoconstrictor Effect of the Pineal Hormone Melatonin in an Animal Model Predictive of Antimigraine Activity

The pineal hormone, melatonin, has been implicated in the pathophysiology of migraine and several studies have demonstrated its vasoconstrictor properties. In the present study, systemic and carotid haemodynamic effects of melatonin, administered directly into the carotid artery, were investigated in anaesthetized pigs. Ten-minute intracarotid infusions of melatonin (1, 10 and 100 μg kg−1 min−1) produced slight decreases in blood pressure and total carotid and arteriovenous anastomotic blood flows, but nutrient blood flow was not affected. The decrease in carotid blood flow was entirely caused by the hypotension, since no changes in vascular conductance values were observed. It is concluded that melatonin itself is not capable of producing vasoconstriction in the cranial circulation of anaesthetized pigs. Thus, it appears that melatonin has no anti-migraine potential via a vasoconstrictor mechanism.