Elsevier

European Journal of Pharmacology

Volume 838, 5 November 2018, Pages 69-77
European Journal of Pharmacology

Full length article
Modulation of tetrodotoxin-resistant Na+ channels by amitriptyline in dural afferent neurons

https://doi.org/10.1016/j.ejphar.2018.09.006Get rights and content

Abstract

Migraine is characterized by recurrent and disabling headaches; therefore, several drugs have been widely prescribed to prevent acute migraine attacks. Amitriptyline, a tricyclic antidepressant, is among the most commonly administered. It is poorly known, however, whether amitriptyline modulates the excitability of dural afferent neurons that transmit pain signals from the dura mater. In this study, the effects of amitriptyline on tetrodotoxin-resistant (TTX-R) Na+ channels were examined in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from medium-sized DiI-positive neurons using a whole-cell patch clamp technique. Amitriptyline (3 μM) slightly reduced the peak component of transient INa and induced a marked decrease in the steady-state component of transient TTX-R INa, as well as in the slow ramp-induced TTX-R INa. Our findings suggest that amitriptyline specifically inhibits persistent Na+ currents mediated by TTX-R Na+ channels. While amitriptyline had minor effects on voltage-activation/inactivation, it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Amitriptyline also affected the inactivation kinetics of TTX-R Na+ channels by significantly accelerating the inactivation of TTX-R Na+ channels and slowing the subsequent recovery. Amitriptyline decreased the number of action potentials by increasing the threshold for their generation. In conclusion, the amitriptyline-mediated diverse modulation of TTX-R Na+ channels would be, at least in part, responsible for its prophylactic efficacy for migraine attacks.

Introduction

Migraine is a common recurrent neurological disorder that is typically characterized by disabling headaches and associated symptoms including photophobia, phonophobia, nausea and vomiting (Goadsby, 2007, Diener et al., 2012). As types and symptoms of migraine are diverse and can become severe, a number of drugs have been used for the treatment of migraine; they are generally classified as either abortive or prophylactic (Silberstein and Goadsby, 2002, Silberstein, 2006). Abortive treatment aims to relieve the acute headache and related symptoms. For example, non-steroidal anti-inflammatory drugs are nonspecific agents used to reduce the symptoms of acute migraine, but triptans, selective 5-HT1B/1D receptor agonists (Tepper et al., 2002, Ahn and Basbaum, 2005), are specific drugs used to ameliorate the acute migraine symptoms (Silberstein, 2006, Diener et al., 2012). In contrast, prophylactic treatment is administered to reduce the frequency and duration of migraine attacks, as well as the severity of headaches during the attacks. Anticonvulsants (topiramate, valproate), antidepressants (amitriptyline, fluoxetine), β-adrenoceptor blockers (propranolol, nadolol), and Ca2+ channel antagonists (flunarizine, verapamil) are widely used in this approach (for review, Silberstein and Goadsby, 2002; Silberstein, 2006).

Amitriptyline is a representative tricyclic antidepressant that has been widely used for migraine prophylaxis and the treatment of neuropathic pain (Max et al., 1992, Silberstein, 2006, Zin et al., 2008, Nishishinya et al., 2008). Although amitriptyline has been proven effective preventing migraine attacks (Silberstein, 2006, Xu et al., 2017), the detailed mechanisms underlying its prophylactic efficacy have yet to be fully elucidated. The primary mechanism underlying analgesic efficacy of amitriptyline is the inhibition of serotonin/norepinephrine transporters, and a number of receptors and ion channels are also affected by amitriptyline (Sindrup et al., 2005). For example, amitriptyline has additional pharmacological effects on adrenergic, histaminergic, cholinergic, and serotonergic receptors, to cause its common side effects including dry mouth, dizziness, and weight gain (Sindrup et al., 2005). In addition, amitriptyline has been shown to inhibit voltage-gated Na+ channels, including tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive neurons (Pancrazio et al., 1998, Hur et al., 2008, Liang et al., 2013, Liang et al., 2014). Their inhibition accounts for amitriptyline's antinociceptive efficacy, as these ion channels are directly related to the generation and conduction of action potentials in nociceptive neurons (Elliott and Elliott, 1993, Renganathan et al., 2001). In particular, TTX-R Na+ channels are known to have important roles in the development as well as maintenance of various painful conditions, such as neuropathic pain and inflammatory pain (Akopian et al., 1999, Chahine and O'Leary, 2014, Waxman and Zamponi, 2014).

In the present study, we examined the effect of amitriptyline on TTX-R Na+ channels in dural afferent neurons, which were identified by the retrograde fluorescent tracer DiI. Given that migraine headaches may be transmitted by trigeminovascular neurons (also referred as dural afferent neurons) that innervate blood vessels within the dura mater (Andres et al., 1987, Strassman et al., 2004), the pharmacological experiments using a limited neuronal population would be worth because migraine headaches may be transmitted by these neurons.

Section snippets

Preparation

All experiments complied with the guiding principles for the care and use of animals approved by the Council of Kyungpook National University and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize both the number of animals used and their suffering.

Sensory neurons of the TG innervating the dura were identified after application of the retrograde tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) to

Effects of amitriptyline on TTX-R Na+ channels

The effect of amitriptyline on TTX-R Na+ channels expressed in medium-sized (30–40 µm in a diameter, 61.1 ± 10.9 pF; standard deviation from 152 neurons) DiI-positive neurons was examined using a whole-cell patch clamp technique in the presence of 300 nM TTX and 100 μM Cd2+. Depolarizing step pulses (− 80 to − 10 mV, every 5 s) induced the TTX-R Na+ currents (INa), which were comprised of a rapidly decaying peak component (INaT) and non-inactivating steady-state component (INaS) (Fig. 1B). The

Discussion

Sensory neurons express multiple types of voltage-gated Na+ channels, which are classified as either TTX-S (NaV1.1, NaV1.2, NaV1.6, and NaV1.7) or TTX-R Na+ channels (NaV1.8 and NaV1.9) (Lai et al., 2004, Rush et al., 2007, Gold and Gebhart, 2010). Since TTX-R Na+ channels are expressed exclusively in nociceptive neurons (Akopian et al., 1999, Gold and Gebhart, 2010), the roles of these subtypes in nociceptive transmission have been heavily investigated. TTX-R Na+ channels (NaV1.8) have been

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government, Republic of Korea (2008-0062282).

Conflict of interest

The authors declare no competing financial interests.

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