Amitriptyline modulates calcium currents and intracellular calcium concentration in mouse trigeminal ganglion neurons

Introduction

Amitriptyline (AMT), a tricyclic antidepressant (TCA), has been used for migraine prevention for over three decades. However, the precise mechanisms by which AMT prevents migraines remain incompletely understood. It has been suggested that AMT exerts its prophylactic effects by altering serotonergic and noradrenergic synaptic transmission, similar to its role in treating depression.

Recent theories propose that AMT may act by inhibiting ion channels, particularly voltage-activated sodium (Na⁺) channels. This hypothesis is supported by a substantial body of experimental research. However, evidence regarding AMT’s ability to inhibit calcium (Ca²⁺) channels is more limited. Experimental studies indicate that AMT can block Ca²⁺ channels in cardiomyocytes. Additionally, AMT shares electrophysiological properties with L-type calcium channel antagonists, further suggesting a potential role in calcium channel modulation.

The trigeminovascular system (TGVS), which consists of trigeminal afferents innervating the meningeal blood vessels, plays a crucial role in migraine pathophysiology. Migraine pain is thought to originate from the activation of the TGVS. The neuronal processes underlying this activation involve high-voltage-activated (HVA) Ca²⁺ channels. These channels facilitate calcium ion entry into neurons upon depolarization, thereby influencing neurotransmitter release, cell membrane excitability, intracellular signaling cascades, and gene expression. Given their essential role in both normal sensory function and pathological pain states, the regulation of Ca²⁺ channels is of significant importance in migraine research.

The goal of this study was to investigate the effects of AMT on HVA calcium currents (I_Ca) in trigeminal ganglion (TG) neurons, as well as its impact on intracellular calcium concentration ([Ca²⁺]i) induced by 40 mM KCl. Additionally, we analyzed the involvement of protein kinase C (PKC) and protein kinase A (PKA) in AMT’s action to further understand its mechanism in migraine prophylaxis.

Material and methods

Preparation of TG neurons

All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Trigeminal ganglion (TG) neurons were obtained from male ICR (Institute of Cancer Research) mice weighing 20–25 g, following previously established protocols.

Mice were euthanized by decapitation under ethyl ether anesthesia. A pair of TGs was dissected and collected in Hank’s balanced salt solution (HBSS). The tissue was then incubated in HBSS at 37°C for 15 minutes with 0.5 mg/mL trypsin and 1.5 mg/mL collagenase type I, both obtained from Sigma-Aldrich (USA).

Following enzyme digestion, the tissues were washed in standard extracellular solution and mechanically dissociated using a Pasteur pipette to obtain individual neurons. The dissociated cells were then plated onto 35 mm dishes and maintained for 2–6 hours at 37°C in an incubator with a 95% O₂/5% CO₂ atmosphere.

The composition of HBSS included (in mM): 130 NaCl, 5 KCl, 0.3 KH₂PO₄, 4 NaHCO₃, 0.3 Na₂HPO₄, 5.6 D-glucose, and 10 HEPES, with the pH adjusted to 7.3. The standard extracellular solution contained (in mM): 130 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, and 10 D-glucose, with the pH also adjusted to 7.3.

As previously described, TG neurons were categorized into three groups based on their size: small-sized (0–23 µm), medium-sized (24–37 µm), and large-sized (38–60 µm) neurons.

Patch-clamp recordings

Standard whole-cell patch-clamp recordings were conducted at room temperature using an EPC-9 amplifier and Pulse 8.02 software, both manufactured by HEKA in Germany. Patch pipettes were fabricated from thin-walled borosilicate glass using a two-step vertical puller, specifically the Narishige Scientific Instrument Laboratory model PP-83 from Tokyo, Japan.

These pipettes, when filled with the electrode internal solution, exhibited a resistance ranging from 2 to 5 MΩ. The internal solution comprised the following components in millimolar concentrations: 125 CsCl, 2 MgCl2, 20 HEPES, 11 EGTA, 1 CaCl2, 4.5 Mg-ATP, and 0.3 Li-GTP. The pH of this solution was adjusted to 7.3 using CsOH.

The extracellular solution employed for patch recording consisted of the following in millimolar: 140 tetraethylammonium-Cl (TEA-Cl), 0.8 MgCl2, 5 CaCl2, 10 HEPES, and 11 d-glucose. The pH of this solution was also adjusted to 7.3 using Tris-base. Data were low-pass filtered at 2 kHz, sampled at 10 kHz, and acquired using the Pulse program from HEKA.

Capacity transients were eliminated, and series resistance was compensated by more than 70% using the internal circuitry of the EPC-9 amplifier. Leakage current was digitally subtracted through the application of the p/n protocol. Additionally, the liquid junction potential was corrected throughout all experiments.

Calcium image

Intracellular calcium ion concentration ([Ca2+]i) measurements were performed following previously established methods. Changes in [Ca2+]i within trigeminal ganglion (TG) neurons were detected using confocal laser scanning microscopy. Fluo-4/AM (Invitrogen, USA) was used as the calcium fluorescent indicator, enabling real-time monitoring of [Ca2+]i alterations.

Prior to recording, cells were seeded onto specialized dishes suitable for confocal laser scanning. These cells were then loaded with Fluo-4/AM at a concentration of 2 µM and subsequently incubated at 37 °C for 30 minutes. Following this incubation, the cells were washed three times with standard extracellular solution. One milliliter of this solution, either with or without AMT, was then added.

[Ca2+]i changes were assessed by monitoring alterations in cell fluorescence during the application of KCl or AMT at room temperature. Point scans of selected cells were conducted with a time resolution of 2 seconds and intervals of 2 seconds. The entire drug application process was observed for 5 minutes. To prevent disturbances related to osmotic pressure and pH, all drugs added to the dishes were dissolved in the standard extracellular solution previously described.

A LSM 5 LIVE (Carl Zeiss, Germany) laser scanning confocal system was used, with excitation at 488 nm and emission at 516 nm. Data were collected using LSM 5 LIVE Zen 2007 (Carl Zeiss, Germany). In all instances, fluorescence intensity was normalized to its initial value, recorded before drug application (F0), and expressed as relative fluorescence (F/F0).

Statistical analysis

The patch-clamp recording data from various neuronal treatments were processed using PULSEFIT software developed by Heka Elektronik in Germany. Concentration-response curves were generated and subsequently fitted to the Hill equation, which is expressed as: I/Imax = 1/[1 + (C/IC50)^H].

In this equation, ‘I’ represents the observed blocking percentage of current, while ‘Imax’ denotes the maximum blocking percentage. ‘C’ signifies the drug concentration, and ‘IC50′ corresponds to the concentration that produces a half-maximal current block. Data are presented as means ± standard error of the mean (S.E.M.) across all experiments.

Statistical analysis of the results was conducted using a paired t-test for the effects of AMT on Ca2+ currents and analysis of variance (ANOVA) for the [Ca2+]i changes. All statistical significance tests were two-sided, and a significance level of P < 0.05 was used.

Drugs and chemicals

Amitriptyline, H-89, GÖ-6983, collagenase (type I), trypsin (type I), DMSO, HEPES, EGTA, TEA-Cl, Mg-ATP, KCl, CsOH, and CsCl were all obtained from Sigma Chemical Co. in St. Louis, Missouri, USA. All other chemical reagents utilized were of analytical grade.

AMT was prepared as a 10 mM stock solution using DMSO and then diluted into the extracellular solution to achieve the desired final concentration immediately before each experiment. The concentration of DMSO in the perfusate was maintained below 1% (v/v), which had minimal effects on high-voltage activated calcium currents, exhibiting less than 2% change over 5 minutes.

Drugs were administered directly onto the cells through a tube. The distance between the tube's opening and the cell being examined was approximately 200 µm. The flow rate, set at 1 mL/min, was controlled by gravity to ensure complete replacement of the cell's surrounding environment within less than 1 second.

Results

Effects of AMT on HVA ICa

The present study utilized freshly isolated neurons from mouse trigeminal ganglia (TG), specifically those ranging in size from 20 to 37 µm, which are considered small to medium sized. High-voltage activated calcium currents (HVA ICa) were induced by applying a series of depolarizing pulses. These pulses ranged from -50 mV to +30 mV, with 10 mV increments, each lasting 80 ms and applied every 20 seconds, originating from a holding potential of -60 mV.

The current intensity, calculated as the current amplitude divided by cell membrane capacitance, was then plotted against the depolarizing potential. As observed, the currents were activated at approximately -30 mV and reached their peak at -20 mV. This activation pattern aligns with the characteristic features of HVA ICa.

When amitriptyline (AMT) at a concentration of 5 µM was introduced, it resulted in a reduction of current intensity across voltage potentials higher than -30 mV. Notably, AMT did not alter the activation threshold or the potential at which HVA ICa reached its maximum.
The study then proceeded to examine the effects of amitriptyline (AMT) on the peak amplitude of high-voltage activated calcium currents (HVA ICa). HVA ICa were induced by applying 80 ms depolarizing pulses to -10 mV, originating from a holding potential of -60 mV, with 30-second intervals between pulses.

AMT was found to reduce the peak currents in a concentration-dependent manner. Specifically, AMT reduced the peak currents by 19.0 ± 5.8% (n = 5, P < 0.05) at 1 µM, 42.5 ± 9.9% (n = 5, P < 0.05) at 5 µM, 62.3 ± 4.1% (n = 7, P < 0.05) at 10 µM, 75.4 ± 7.8% (n = 7, P < 0.05) at 50 µM, and 87.3 ± 6.9% (n = 5, P < 0.05) at 100 µM.

The concentration of AMT that resulted in a half-maximal block of the currents (IC50) was determined to be 5.1 µM. The inhibitory effects of AMT at concentrations ranging from 1 to 10 µM began approximately 30 seconds after application, and it took up to 240 seconds to reach a steady state. In contrast, AMT concentrations exceeding 10 µM exhibited more rapid effects.

To investigate the use-dependent block of high-voltage activated calcium currents (HVA ICa) by amitriptyline (AMT), currents were elicited at frequencies of 0.1 Hz, 0.5 Hz, and 1 Hz. This was achieved by applying 10 consecutive 20 ms pulses to -10 mV from a holding potential. The ratio of I10/I1, representing the current amplitude at the 10th pulse divided by the current amplitude at the 1st pulse, was used to quantify the decrease in HVA ICa.

At a frequency of 0.1 Hz, the accumulation of inactivated channels resulted in a reduction in peak current amplitude (I10/I1 = 96.0 ± 3.2%). This reduction was not statistically different from that observed during the application of 5 µM AMT (I10/I1 = 95.1 ± 3.6%, n = 6, P > 0.05).

However, at higher frequencies, a statistically significant reduction in peak current amplitude was observed during drug application. Specifically, I10/I1 was 87.5 ± 4.2% at 0.5 Hz and 82.4 ± 5.3% at 1 Hz, which were significantly different compared to control conditions (I10/I1 = 93.0 ± 2.9% at 0.5 Hz, n = 7, P < 0.05, and 92.3 ± 3.7% at 1 Hz, n = 5, P < 0.05). These results indicate that AMT exhibits a use-dependent block of HVA ICa.

PKC and PKA pathways are not involved in AMT-induced inhibition of HVA Ica

Research has indicated that high-voltage activated calcium (HVA Ca2+) channels are subject to regulation by protein phosphorylation pathways activated by second messengers. To determine whether intracellular signaling cascades play a role in AMT's effects on HVA ICa, protein kinase C (PKC) and protein kinase A (PKA) were inhibited. This was achieved by pre-incubating neurons with GÖ-6983, a selective inhibitor of PKC-α, -β, -γ, and -δ, and H-89, respectively, for 20 minutes before AMT application.

Neither GÖ-6983 (1 µM) nor H-89 (2 µM) exhibited any noticeable effects on HVA ICa when applied alone. As previously established, 5 µM AMT reduced peak currents by 42.5 ± 9.9% (n = 5).

Even with PKC and PKA inhibition, 5 µM AMT continued to suppress HVA ICa, resulting in reductions of 42.7 ± 9.4% (n = 9) and 48.5 ± 4.4% (n = 4), respectively. These values were not statistically different from the 42.5 ± 9.9% reduction observed without kinase inhibition (P = 0.97 and 0.30, respectively). These findings suggest that the inhibitory effect of AMT on HVA ICa is not diminished by PKC or PKA inhibitors.

Discussion

This study successfully illustrated the modulatory effects of amitriptyline (AMT) on high-voltage activated calcium (HVA Ca2+) channels in isolated mouse trigeminal ganglion (TG) neurons. AMT demonstrated a concentration-dependent and use-dependent block of HVA ICa. Notably, protein kinase C (PKC) and protein kinase A (PKA) pathways were not implicated in this process. Furthermore, AMT inhibited KCl-induced increases in intracellular calcium ion concentration ([Ca2+]i), likely due to its direct inhibition of voltage-activated calcium channels.

Migraine is known to be more prevalent in women, potentially due to factors such as a lower potassium concentration threshold for cortical spreading depression in females or the presence of sex steroid receptors, particularly estrogen, within trigeminal circuits. To mitigate the influence of sex steroids, male mice were utilized for tissue collection in this experiment.

Previous research has documented the presence of L-, N-, P/Q-, and R-type HVA Ca2+ channels in TG neurons. The observation that AMT blocked a substantial portion of the HVA currents suggests that it acts as a nonselective blocker, affecting most or all channel isoforms functionally expressed in these cells. In this experiment, the IC50 value for AMT blockade of HVA ICa was determined to be 5.1 µM, which is significantly lower than that observed in cardiomyocytes. Given that AMT's action is voltage-dependent, exhibiting greater apparent potency at more positive holding potentials, the higher IC50 in cardiomyocytes indicates a lower inhibitory potency of AMT in those cells.

Amitriptyline (AMT) demonstrates efficacy in migraine prevention at dosages lower than those employed in depression treatment, where plasma concentrations typically range from 0.36 to 0.90 µM. However, considering the high plasma protein binding of AMT, exceeding 90%, the free drug concentration in plasma is considerably lower. Furthermore, the apparent concentration of AMT in the brain relative to plasma is approximately 20:1. In an in vivo study, intravenous administration of 1.0 to 4.0 mg/kg AMT effectively inhibited activity in the trigeminal nucleus. These findings suggest that the observations from this study may hold clinical significance.

A key kinetic characteristic of AMT is its use-dependent blockade. Nociceptive neurons within the trigeminal ganglion (TG) are typically spontaneously inactive, exhibiting a very low rate of ongoing discharge, ranging from 0.01 to 0.05 Hz. Upon induction of dural mast cell degranulation, the ongoing discharge rate progressively increased by 1.5 ± 0.3 Hz, with a range of 0.4 to 3.6 Hz.

The observed greater blockade when cells were stimulated at 0.5 and 1 Hz, compared to 0.1 Hz, strongly indicates that the open state of calcium channels is more susceptible to AMT-induced inhibition than the closed state. Given that calcium channels in the trigeminovascular system (TGVS) become hyperexcited during migraine, AMT can directly interfere with the enhanced electrophysiological activity of these channels, thereby suppressing TGVS activation.

Protein kinase C (PKC) is known to phosphorylate various cellular components that play crucial regulatory roles in the signal transduction pathways of nociceptor excitation and sensitization. The impact of tricyclic antidepressants (TCAs) on PKC activity has been a subject of debate. For instance, acute amitriptyline (AMT) treatment has been shown to preserve morphine's antinociceptive effect in morphine-tolerant rats by inhibiting phospho-PKA and PKC expression. Conversely, long-term imipramine administration does not appear to alter PKC activity in the rat brain.

In this study, the inhibitory effects of AMT on high-voltage activated calcium currents (HVA ICa) in trigeminal ganglion (TG) neurons were not reversed by pre-incubation with either PKC or PKA inhibitors. This indicates that AMT modulates HVA Ca2+ channels independently of PKC and PKA.

Corroborating the patch-clamp results, which demonstrated AMT's inhibitory effects on HVA Ca2+ channels, this study also found that AMT blocked KCl-induced calcium influx. This inhibition occurred without any noticeable effect on basal calcium levels, suggesting that AMT, at concentrations up to 5 µM, does not interfere with processes involved in intracellular calcium release or sequestration.

The acutely isolated neurons used in this study were not sufficiently viable to effectively pump the elevated intracellular calcium outside the membrane. Consequently, the fluorescent signals did not return to their initial values following KCl stimulation.

Contemporary understanding of migraine, informed by the latest neurobiological and clinical findings, classifies it as a neurovascular disorder. A key aspect of this view is the release of inflammatory neuropeptides from the trigeminal system, which leads to the dilation of meningeal vessels and neurogenic inflammation in the dura.

This inflammation contributes to trigeminal ganglion hyperexcitability, known as peripheral sensitization, and generates abnormal input to second-order trigeminovascular neurons, termed central sensitization. Blocking peripheral L-type, P/Q-type, and especially N-type calcium channels has been shown to prevent subjective pain, hyperalgesia, and allodynia, which are consequences of peripheral and central sensitization, in various experimental and clinical contexts.

The negative modulation of HVA Ca2+ channels by AMT, shown by our results, could be one of the many potential candidates to explain its therapeutic effect of this drug in migraine prevention. In the crisis of migraine, the Ca2+-dependent release of neuropep- tides from trigeminal perivascular axons is considered to be an essential step. By blocking Ca2+ channels and Ca2+ entry, AMT could help reduce the neuronal threshold for excitation and prevent the development of a migraine attack. Go 6983