Medical/biological study (experimental study)

Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na(+) currents through intracellular Ca(2+) release med./bio.

By: Liu DD, Ren Z, Yang G, Zhao QR, Mei YA

Published in: J Cell Mol Med 2014; 18 (6): 1060-1070

Aim of study (acc. to author)

To investigate the effects of melatonin on extremely low frequency magnetic field-induced activation of voltage-gated sodium channels and the related pathways in rat cerebellar granule cells.

Background/further details

In a previous study (He et al. 2013), the authors showed that extremely low frequency magnetic fields significantly activated the voltage-gated sodium channels of cerebellar granule cells. Melatonin is known to modulate the delay in outward rectifying K⁺ channels, resulting amongst others in a protection against apoptosis. However, only few studies examined the effect of melatonin on Na⁺ channels, especially in combination with extremely low frequency magnetic fields.
Cell cultures were examined in 5 groups: 1.) sham exposure, 2.) magnetic field exposure, 3.) magnetic field exposure + 1 µM melatonin, 4.) magnetic field exposure + 5 µM melatonin and 5.) sham exposure + 5 µM melatonin. To verify the results, a melatonin receptor agonist (IIK-7) or an antagonist (4-P-PDOT) were partially added as well as an calmodulin inhibitor (KN93) and a ryanodine-sensitive receptor blocker (inhibits the release of Ca²⁺).

Endpoint

molecular biosynthesis: protein expression of phosphorylated protein kinase A
cell function: sodium current; intracellular Ca²⁺ level

Exposure

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: continuous for 60 minutes	magnetic flux density: 1 mT

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 60 minutes

Exposure setup

Exposure source	Helmholtz coils
Setup	magnetic field was generated by a pair of Helmholtz coils placed in opposition to each other, coils were powered by a generator system that produced sinusoidal input voltage, device was powered by an AC power generator, magnetic field frequency and density were monitored by a sensor that was connected to a digital multimeter, geometry of the system assured a uniform field for the exposed cultured cells, surfaces of the culture plates were parallel to the force lines of the alternating magnetic field in the solenoid, maximum temperature increase recorded in the exposed cultures was 0.4 ± 0.1°C
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1 mT	-	measured	-	-

Reference articles

Ongaro A et al. (2012): Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts
Varani K et al. (2012): Effect of pulsed electromagnetic field exposure on adenosine receptors in rat brain

Exposed system:

intact cell/cell culture
rat cerebellar granule cells

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: protein expression of phosphorylated protein kinase A (Western blot)
cell function: sodium current (patch-clamp technique); steady state activation of sodium current after depolarization in 5 mV steps from -70 to 20 mV with 5 s intervals; patch-clamp technique); intracellular Ca²⁺ level (fluorescence intensity, inverted phase contrast microscopy)

Investigated system:

intact cell/cell culture
supernatant of cell lysates

Time of investigation:

after exposure

Main outcome of study (acc. to author)

In magnetic field exposed cells, the sodium current in voltage-gated sodium channels was significantly increased compared to the control group. An addition of 5 µM melatonin inhibited the exposure-induced effect. This inhibitory effect of melatonin was mimicked by a melatonin receptor agonist and was diminished by an antagonist. The voltage-gated sodium channel steady-state activation curve was significantly shifted towards hyperpolarization by the magnetic field exposure, but this effect was diminished by an addition of melatonin.
The protein expression level of phosphorylated protein kinase A was significantly elevated in exposed cell cultures (also with the addition of melatonin or a melatonin receptor agonist) in comparison to the unexposed control group.
The intracellular Ca²⁺ level was significantly increased by melatonin and by melatonin in addition with magnetic field exposure compared to the control group, but not by magnetic field exposure alone and not in the presence of a ryanodine-sensitive receptor blocker. The inhibitory effect of melatonin on the magnetic field exposure-induced activation of voltage-gated sodium channels was diminished by a calmodulin inhibitor.
The authors conclude that melatonin could protect against the magnetic field-induced activation of voltage-gated sodium channels in cerebellar granule cells of rats through an elevated Ca²⁺ release.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

National Basic Research Program (Program 973), China
National Science Fund for Talent Training in Basic Science, China
Shanghai Leading Academic Discipline Project, China

Sun ZC et al. (2016): Extremely Low Frequency Electromagnetic Fields Facilitate Vesicle Endocytosis by Increasing Presynaptic Calcium Channel Expression at a Central Synapse
Yang G et al. (2015): Exposure to 50 Hz magnetic field modulates GABA currents in cerebellar granule neurons through an EP receptor-mediated PKC pathway
Luo FL et al. (2014): Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons
He YL et al. (2013): Exposure to Extremely Low-Frequency Electromagnetic Fields Modulates Na(+) Currents in Rat Cerebellar Granule Cells through Increase of AA/PGE(2) and EP Receptor-Mediated cAMP/PKA Pathway
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