Saurabh Gupta

Mechanisms of Pain Modulation by Sex Hormones in Migraine

(Headache 2011;51:905‐922)

Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery.

Objective: We pharmacologically characterized pituitary adenylate cyclase–activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC1, VPAC2 and PAC1 receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries. Methods: Concentration response curves to PACAP38, PACAP27, VIP and the VPAC1 receptor agonist ([Lys15,Arg16,Leu27]-VIP[1-7]-GRF[8-27]) were constructed in the absence or presence of the PAC1 receptor antagonist PACAP6-38 or the VPAC1 receptor antagonist, PG97269. mRNA expression was measured using qPCR. Results: PACAP38 was less potent than VIP in both arteries. Both peptides had lower potency and efficacy in meningeal than in coronary arteries, while mRNA expression of VPAC1 receptor was more pronounced in meningeal arteries. PACAP6-38 reduced the Emax of PACAP27, while PG97269 right-shifted the VIP-induced relaxation curve only in the coronary arteries. Conclusion: The direct vasodilatory effect of VIP and PACAP might be less relevant than the central effect of this compound in migraine pathogenesis.

A naturalistic glyceryl trinitrate infusion migraine model in the rat

Background: and aim Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose. Methods: Awake rats were infused with GTN (4u2009µg/kg/min, for 20u2009min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively. Results: A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30u2009min and 2u2009h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2u2009h and 4u2009h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4u2009h, demonstrating the specificity of this model for migraine. Conclusion: We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1xa0μM), the neurokinin NK1 receptor antagonist L-733060 (0.5xa0μM), the voltage-sensitive calcium channel blocker ruthenium red (100xa0μM), the TRPV1 receptor antagonist capsazepine (5xa0μM), the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl (l-NAME; 100xa0μM), the gap junction blocker 18α-glycyrrhetinic acid (10xa0μM), as well as the RhoA kinase inhibitor Y-27632 (1xa0μM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1xa0mM), charybdotoxin (0.5xa0μM)u2009+u2009apamin (0.1xa0μM) and iberiotoxin (0.5xa0μM)u2009+u2009apamin (0.1xa0μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

Current and prospective pharmacological targets in relation to antimigraine action.

Migraine is a recurrent incapacitating neurovascular disorder characterized by unilateral and throbbing headaches associated with photophobia, phonophobia, nausea, and vomiting. Current specific drugs used in the acute treatment of migraine interact with vascular receptors, a fact that has raised concerns about their cardiovascular safety. In the past, α-adrenoceptor agonists (ergotamine, dihydroergotamine, isometheptene) were used. The last two decades have witnessed the advent of 5-HT1B/1D receptor agonists (sumatriptan and second-generation triptans), which have a well-established efficacy in the acute treatment of migraine. Moreover, current prophylactic treatments of migraine include 5-HT2 receptor antagonists, Ca2+ channel blockers, and β-adrenoceptor antagonists. Despite the progress in migraine research and in view of its complex etiology, this disease still remains underdiagnosed, and available therapies are underused. In this review, we have discussed pharmacological targets in migraine, with special emphasis on compounds acting on 5-HT (5-HT1–7), adrenergic (α1, α2, and β), calcitonin gene-related peptide (CGRP1 and CGRP2), adenosine (A1, A2, and A3), glutamate (NMDA, AMPA, kainate, and metabotropic), dopamine, endothelin, and female hormone (estrogen and progesterone) receptors. In addition, we have considered some other targets, including gamma-aminobutyric acid, angiotensin, bradykinin, histamine, and ionotropic receptors, in relation to antimigraine therapy. Finally, the cardiovascular safety of current and prospective antimigraine therapies is touched upon.

Female sex hormones and rat dural vasodilatation to CGRP, periarterial electrical stimulation and capsaicin

Background.—The prevalence of migraine is 2 to 3‐fold higher in females than in males, and it is intricately related to the levels of female sex hormones. These hormones may regulate the synthesis and receptor expression of calcitonin gene‐related peptide (CGRP), which mediates neurogenic dural vasodilatation and is implicated in migraine pathogenesis.

Functional reactivity of 5-ht receptors in human umbilical cord and maternal subcutaneous fat arteries after normotensive or pre-eclamptic pregnancy

Objective To investigate the functional reactivity of 5-hydroxytryptamine (serotonin; 5-HT) receptors in foetal umbilical cord arteries (UCA) and maternal subcutaneous fat resistance arteries (SFA) in normotensive and pre-eclamptic pregnancy. Design Study groups were divided based on the presence or absence of pre-eclampsia and the duration of gestation. Methods Segments of UCA and SFA were mounted in tissue baths and concentration–response curves to 5-HT and sumatriptan (5-HT1B/1D receptor agonist) were constructed in the absence or presence of ketanserin (5-HT2A receptor antagonist) or GR125743 (5-HT1B/1D receptor antagonist). Results Both 5-HT and sumatriptan contracted all UCA segments studied. The responses to 5-HT and the potency of ketanserin in UCA were not different between the study groups, indicating a similar profile of the 5-HT2A receptor. In contrast, the potencies of sumatriptan and GR125743 were significantly higher in normotensive full-term pregnancies than in normotensive pre-term pregnancies in UCA. The response to sumatriptan in UCA arteries was not significantly different between pre-eclamptic and normotensive pregnancies. However, the potency of both sumatriptan and GR125743 was positively correlated to the gestational age in the normotensive group, whereas this relationship was absent in the pre-eclamptic group. In SFA, responses to 5-HT and sumatriptan were not different between the pre-eclamptic patients and normotensive controls. Conclusions In both UCA and SFA, 5-HT1B/1D and 5-HT2A receptors mediate vasoconstriction. The sensitivity of 5-HT1B/1D receptors increases in the last trimester in the UCA in normal pregnancies, which seems to be expedited in pre-eclamptic patients. Further studies on 5-HT1B/ID receptors will thus give new insights into the foetal development and pathophysiology of pre-eclampsia.

Effects of current and prospective antimigraine drugs on the porcine isolated meningeal artery

Vasoconstriction to agonists at serotonin (5-hydroxytryptamine; 5-HT) receptors and α-adrenoceptors, as well as vasodilatation induced by α-CGRP, have been well described in the porcine carotid circulation in vivo. The present study sets out to investigate the effects of current and prospective antimigraine drugs on porcine meningeal artery segments in vitro. Sumatriptan, ergotamine, dihydroergotamine, isometheptene and clonidine failed to contract the meningeal artery, but 5-HT, noradrenaline and phenylephrine induced concentration-dependent contractions. The contractions to 5-HT were competitively antagonized by the 5-HT2A receptor antagonist ketanserin, whilst those to noradrenaline were antagonized by α1-(prazosin), α2-(rauwolscine and yohimbine) and α2C/2B-(OPC-28326) adrenoceptor antagonists. Whilst dobutamine and salbutamol were ineffective, α-CGRP produced concentration-dependent relaxations that were antagonized by the CGRP1 receptor antagonist olcegepant. In agreement with their lack of contractile effect, sumatriptan and ergotamine failed to influence forskolin-stimulated cyclic AMP accumulation in the porcine meningeal artery; in contrast, both compounds decreased forskolin-stimulated cyclic AMP accumulation in the human isolated saphenous vein, where they induced contractions. Finally, using RT-PCR, we could demonstrate the presence of mRNAs encoding for several 5-HT receptors (5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A and 5-HT7) and adrenoceptors (α1A, α1B, α1D, α2A, α2B, α2C, β1 and β2), as well as that for the calcitonin receptor like receptor, a component of the CGRP1 receptor. These results suggest that: (i) the porcine meningeal artery may not be involved in the vasoconstriction of the carotid vascular bed elicited by antimigraine drugs in anaesthetized pigs, and (ii) the mismatch between the presence of receptor mRNA and the lack of response to sumatriptan, dobutamine and salbutamol implies that mRNAs for the 5-HT1B receptor and β1- and β2-adrenoceptors are probably unstable, or that their density is too low for being translated as receptor protein in sufficient quantities.

KATP channel openers in the trigeminovascular system

Background: The ATP-sensitive K+ (KATP) channel openers levcromakalim and pinacidil are vasodilators that induce headache in healthy people. The neuropeptide calcitonin gene-related peptide (CGRP) induces headache in healthy people and migraine in migraineurs, potentially through a mechanism that involves opening of vascular or neuronal KATP channels and mast cell degranulation. Using rat as a model, we studied the molecular presence of KATP channels in the trigeminovascular system. Furthermore, we examined whether KATP channel openers stimulate the in vitro release of CGRP and whether they degranulate dural mast cells. Methods: mRNA and protein expression of KATP channel subunits were studied in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) by qPCR and western blotting. In vitro CGRP release was studied after application of levcromakalim (1u2009µM) and diazoxide (10u2009µM) to freshly isolated rat dura mater, TG and TNC. Rat dural mast cells were challenged in situ with levcromakalim (10−5u2009M) to study its potential degranulation effect. Results: mRNA and protein of KATP channel subunits Kir6.1, Kir6.2, SUR1 and SUR2B were identified in the TG and TNC. KATP channel openers did not release or inhibit capsaicin-induced CGRP release from dura mater, TG or TNC. They did also not induce dural mast cell degranulation. Conclusions: KATP channel openers do not interact with CGRP release or mast cell degranulation. Activation of these channels in the CNS is antinociceptive and therefore cannot explain the headache induced by KATP channel openers. Thus, they are likely to induce headache by interaction with extracerebral KATP channels, probably the SUR2B isoforms.

Rat Carotid Artery Responses to α-Adrenergic Receptor Agonists and 5-HT After Ovariectomy and Hormone Replacement

Objective.—To compare the contractile responses to α‐adrenergic receptor agonists and 5‐HT in the rat carotid artery after ovariectomy and subsequent hormone replacement with 17β‐estradiol, progesterone, or the combination of 17β‐estradiol and progesterone.