Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects

Abstract

The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca(2+) /calmodulin stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein kinase G pathway stimulation. A well-studied example of such an apparent therapeutic response, EMF stimulation of bone growth, appears to work along this pathway. However, pathophysiological responses to EMFs may be as a result of nitric oxide-peroxynitrite-oxidative stress pathway of action. A single such well-documented example, EMF induction of DNA single-strand breaks in cells, as measured by alkaline comet assays, is reviewed here. Such single-strand breaks are known to be produced through the action of this pathway. Data on the mechanism of EMF induction of such breaks are limited; what data are available support this proposed mechanism. Other Ca(2+) -mediated regulatory changes, independent of nitric oxide, may also have roles. This article reviews, then, a substantially supported set of targets, VGCCs, whose stimulation produces non-thermal EMF responses by humans/higher animals with downstream effects involving Ca(2+) /calmodulin-dependent nitric oxide increases, which may explain therapeutic and pathophysiological effects.

Full text

Electromagnetic fields act via activation of voltage-gated
calcium channels to produce beneficial or adverse effects
Martin L. Pall *
Professor Emeritus of Biochemistry and Basic Medical Sciences, Washington State University, Portland, OR, USA
Received: January 8, 2013; Accepted: May 20, 2013
●Introduction
●Possible modes of action following
voltage-gated calcium channel stimulation
●Therapeutic bone-growth stimulation
via Ca
2+
/nitric oxide/cGMP/protein kinase G
●Ca
2+
/nitric oxide/peroxynitrite and
pathophysiological
responses to EMF exposures: the example of
single-strand DNA breaks
●Discussion and conclusions
Abstract
The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not
been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three
studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC
blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically
plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca
2+
/calmodulin
stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein
kinase G pathway stimulation. A well-studied example of such an apparent therapeutic response, EMF stimulation of bone growth, appears to
work along this pathway. However, pathophysiological responses to EMFs may be as a result of nitric oxide-peroxynitrite-oxidative stress path-
way of action. A single such well-documented example, EMF induction of DNA single-strand breaks in cells, as measured by alkaline comet
assays, is reviewed here. Such single-strand breaks are known to be produced through the action of this pathway. Data on the mechanism of
EMF induction of such breaks are limited; what data are available support this proposed mechanism. Other Ca
2+
-mediated regulatory changes,
independent of nitric oxide, may also have roles. This article reviews, then, a substantially supported set of targets, VGCCs, whose stimulation
produces non-thermal EMF responses by humans/higher animals with downstream effects involving Ca
2+
/calmodulin-dependent nitric oxide
increases, which may explain therapeutic and pathophysiological effects.
Keywords: intracellular Ca
2+

voltage-gated calcium channels

low frequency electromagnetic field exposure

nitric
oxide

oxidative stress

calcium channel blockers
Introduction
An understanding of the complex biology of the effects of electromag-
netic fields (EMFs) on human/higher animal biology inevitably must
be derived from an understanding of the target or targets of such
fields in the impacted cells and tissues. Despite this, no understand-
ing has been forthcoming on what those targets are and how they
may lead to the complex biological responses to EMFs composed of
low-energy photons. The great puzzle, here, is that these EMFs are
comprised of low-energy photons, those with insufficient energy to
individually influence the chemistry of the cell, raising the question of
how non-thermal effects of such EMFs can possibly occur. The author
*Correspondence to: Martin L. PALL, Ph.D.,
Professor Emeritus of Biochemistry and Basic Medical
Sciences, Washington State University, 638 NE 41st Ave.,
Portland, OR 97232 USA
Tel: +01-503-232-3883
E-mail: martin_pall@wsu.edu
ª2013 The Author.
Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
doi: 10.1111/jcmm.12088
J. Cell. Mol. Med. Vol 17, No 8, 2013 pp. 958-965

has found that there is a substantial literature possibly pointing to the
direct targets of such EMFs and it is the goal of this study to review
that evidence as well as review how those targets may lead to the
complex biology of EMF exposure.
The role of increased intracellular Ca
2+
following EMF exposure
was already well documented more than 20 years ago, when Wallec-
zek [1] reviewed the role of changes in calcium signalling that were
produced in response EMF exposures. Other, more recent studies
have confirmed the role of increased intracellular Ca
2+
following EMF
exposure, a few of which are discussed below. His review [1]
included two studies [2, 3] that showed that the L-type voltage-gated
channel blocker, verapamil could lower or block changes in response
to EMFs. The properties of voltage-gated calcium channels (VGCCs)
have been reviewed elsewhere [4]. Subsequently, extensive evidence
has been published clearly showing that the EMF exposure can act to
produce excessive activity of the VGCCs in many cell types [5–26]
suggesting that these may be direct targets of EMF exposure. Many
of these studies implicate specifically the L-type VGCCs such that var-
ious L-type calcium channel blockers can block responses to EMF
exposure (Table 1). However, other studies have shown lowered
responses produced by other types of calcium channel blockers
including N-type, P/Q-type, and T-type blockers (Table 1), showing
that other VGCCs may have important roles. Diverse responses to
EMFs are reported to be blocked by such calcium channel blockers
(Table 1), suggesting that most if not all EMF-mediated responses
may be produced through VGCC stimulation. Voltage-gated calcium
channels are essential to the responses produced by extremely low
frequency (including 50/60 Hz) EMFs and also to microwave fre-
quency range EMFs, nanosecond EMF pulses, and static electrical
and magnetic fields (Table 1).
In a recent study, Pilla [27] showed that an increase in intracellu-
lar Ca
2+
must have occurred almost immediately after EMF exposure,
producing a Ca
2+
/calmodulin-dependent increase in nitric oxide
occurring in less than 5 sec. Although Pilla [27] did not test whether
VGCC stimulation was involved in his study, there are few alternatives
that can produce such a rapid Ca
2+
response, none of which has been
implicated in EMF responses. Other studies, each involving VGCCs,
summarized in Table 1, also showed rapid Ca
2+
increases following
EMF exposure [8, 16, 17, 19, 21]. The rapidity of these responses rule
out many types of regulatory interactions as being involved in produc-
ing the increased VGCC activity following EMF exposure and sug-
gests, therefore, that VGCC stimulation in the plasma membrane is
directly produced by EMF exposure.
Possible modes of action following
VGCC stimulation
The increased intracellular Ca
2+
produced by such VGCC activation
may lead to multiple regulatory responses, including the increased
nitric oxide levels produced through the action of the two Ca
2+
/cal-
modulin-dependent nitric oxide synthases, nNOS and eNOS.
Increased nitric oxide levels typically act in a physiological context
through increased synthesis of cGMP and subsequent activation of
protein kinase G [28, 29]. In contrast, in most pathophysiological
contexts, nitric oxide reacts with superoxide to form peroxynitrite, a
potent non-radical oxidant [30, 31], which can produce radical prod-
ucts, including hydroxyl radical and NO
2
radical [32].
Therapeutic bone-growth stimulation
via Ca
2+
/nitric oxide/cGMP/protein
kinase G
An example of a therapeutic effect for bone repair of EMF exposure in
various medical situations includes increasing osteoblast differentia-
tion and maturation and has been reviewed repeatedly [33–44]. The
effects of EMF exposure on bone cannot be challenged, although
there is still considerable question about the best ways to apply this
clinically [33–44]. Our focus, here, is to consider possible mecha-
nisms of action. Multiple studies have implicated increased Ca
2+
and
nitric oxide in the EMF stimulation of bone growth [44–49]; three
have also implicated increased cGMP and protein kinase G activity
[46, 48, 49]. In addition, studies on other regulatory stimuli leading to
increased bone growth have also implicated increased cGMP levels
and protein kinase G in this response [50–56]. In summary, then, it
can be seen from the above that there is a very well-documented
action of EMFs in stimulating osteoblasts and bone growth. The avail-
able data, although limited, support the action of the main pathway
involved in physiological responses to Ca
2+
and nitric oxide, namely
Ca
2+
/nitric oxide/cGMP/protein kinase G in producing such
stimulation.
Ca
2+
/nitric oxide/peroxynitrite and
pathophysiological responses to EMF
exposures: the example of single-
strand DNA breaks
As was noted above, most of the pathophysiological effects of nitric
oxide are mediated through peroxynitrite elevation and consequent
oxidative stress. There are many reviews and other studies, implicat-
ing oxidative stress in generating pathophysiological effects of EMF
exposure [see for example 57–64]. In some of these studies, the rise
in oxidative stress markers parallels the rise in nitric oxide, suggest-
ing a peroxynitrite-mediated mechanism [64–67].
Peroxynitrite elevation is usually measured through a marker of
peroxynitrite-mediated protein nitration, 3-nitrotyrosine (3-NT). There
are four studies where 3-NT levels were measured before and after
EMF exposure [66, 68–70]. Each of these studies provides some evi-
dence supporting the view that EMF exposure increases levels of per-
oxynitrite and therefore 3-NT levels [66, 68–70]. Although these
cannot be taken as definitive, when considered along with the evi-
dence on oxidative stress and elevated nitric oxide production in
response to EMF exposure, they strongly suggest a peroxynitrite-
mediated mechanism of oxidative stress in response to EMFs.
ª2013 The Author.
Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
959
J. Cell. Mol. Med. Vol 17, No 8, 2013

Table 1 EMF responses blocked or lowered by calcium channel blockers
Ref. no. EMF type Calcium channel Cell type or organism Response measured
2 Pulsed magnetic
fields
L-type Human lymphocytes Cell proliferation; cytokine
production
3 Static magnetic
field (0.1 T)
L-type Human polymorphonuclear
leucocytes
Cell migration; degranulation
5 ELF L-type Rat chromaffin cells Differentiation; catecholamine release
6 Electric field L-type Rat and mouse bone cells Increased Ca
2+
, phospholipase A2, PGE2
7 50 Hz L-type Mytilus (mussel) immunocytes Reduced shape change, cytotoxicity
8 50 Hz L-type AtT20 D16V, mouse pituitary
corticotrope-derived
Ca
2+
increase; cell morphology,
premature differentiation
9 50 Hz L-type Neural stem/progenitor cells In vitro differentiation, neurogenesis
10 Static magnetic
field
L-type Rat Reduction in oedema formation
11 NMR L-type Tumour cells Synergistic effect of EMF on anti-tumour
drug toxicity
12 Static magnetic field L-type Myelomonocytic U937 cells Ca
2+
influx into cells and anti-apoptotic
effects
13 60 Hz L-type Mouse Hyperalgesic response to exposure
14 Single nanosecond
electric pulse
L-type Bovine chromaffin cells Very rapid increase in intracellular Ca
2+
15 Biphasic electric current L-type Human mesenchymal stromal cells Osteoblast differentiation and cytokine
production
16 DC & AC magnetic
fields
L-type b-cells of pancreas, patch clamped Ca
2+
flux into cells
17 50 Hz L-type Rat pituitary cells Ca
2+
flux into cells
18 50 Hz L-type, N-type Human neuroblastoma IMR32 and
rat pituitary GH3 cells
Anti-apoptotic activity
19 Nanosecond pulse L-type, N-type,
P/Q-type
Bovine chromaffin cells Ca
2+
dynamics of cells
20 50 Hz Not determined Rat dorsal root ganglion cells Firing frequency of cells
21 700–1100 MHz N-type Stem cell–derived neuronal cells Ca
2+
dynamics of cells
22 Very weak electrical
fields
T-type Sharks Detection of very weak magnetic fields
in the ocean
23 Short electric pulses L-type Human eye Effect on electro-oculogram
24 Weak static magnetic
field
L-type Rabbit Baroreflex sensitivity
25 Weak electric fields T-type Neutrophils Electrical and ion dynamics
26 Static electric fields,
‘capacitive’
L-type Bovine articular chondrocytes Agrican & type II collagen expression;
calcineurin and other Ca
2+
/calmodulin
responses
EMF: electromagnetic field; ELF: extremely low frequency.
960 ª2013 The Author.
Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Such a peroxynitrite-mediated mechanism may explain the many
studies showing the single-stranded breaks in DNA, as shown by
alkaline comet assays or the similar microgel electrophoresis assay,
following EMF exposures in most such studies [71–89], but not in all
[90–97]. Some of the factors that are reported to influence whether
such DNA single-strand breaks are detected after EMF exposure
include the type of cell studied [79, 86], dosage of EMF exposure
[78] and the type of EMF exposure studied [73, 77]. Oxidative
stress and free radicals have roles, both because there is a con-
comitant increase in oxidative stress and because antioxidants
have been shown to greatly lower the generation of DNA single-
strand breaks following EMF exposure [72, 75, 81, 82] as has
also been shown for peroxynitrite-mediated DNA breaks produced
under other conditions. It has also been shown that one can block
the generation of DNA single-strand breaks with a nitric oxide
synthase inhibitors [82].
Peroxynitrite has been shown to produce single-strand DNA
breaks [98–100], a process that is inhibited by many but not all an-
tioxidants [99, 100]. It can be seen from this that the data on genera-
tion of single-strand DNA breaks, although quite limited, support a
mechanism involving nitric oxide/peroxynitrite/free radical (oxidative
stress). Although the data on the possible role of peroxynitrite in
EMF-induced DNA single-strand breaks are limited, what data are
available supports such a peroxynitrite role.
Discussion and conclusions
How do EMFs composed of low-energy photons produce non-thermal
biological changes, both pathophysiological and, in some cases,
potentially therapeutic, in humans and higher animals? It may be sur-
prising that the answer to this question has been hiding in plain sight
in the scientific literature. However, in this era of highly focused and
highly specialized science, few of us have the time to read the relevant
literature, let alone organize the information found within it in useful
and critical ways.
This study shows that:
1Twenty-three different studies have found that such EMF
exposures act via activation of VGCCs, such that VGCC channel
blockers can prevent responses to such exposures (Table 1).
Most of the studies implicate L-type VGCCs in these responses,
but there are also other studies implicating three other classes
of VGCCs.
2Both extremely low frequency fields, including 50/60 cycle
exposures, and microwave EMF range exposures act via activa-
tion of VGCCs. So do static electric fields, static magnetic fields
and nanosecond pulses.
3Voltage-gated calcium channel stimulation leads to
increased intracellular Ca
2+
, which can act in turn to stimulate
the two calcium/calmodulin-dependent nitric oxide synthases
and increase nitric oxide. It is suggested here that nitric oxide
may act in therapeutic/potentially therapeutic EMF responses
via its main physiological pathway, stimulating cGMP and pro-
tein kinase G. It is also suggested that nitric oxide may act in
pathophysiological responses to EMF exposure, by acting as a
precursor of peroxynitrite, producing both oxidative stress and
free radical breakdown products.
4The interpretation in three above is supported by two spe-
cific well-documented examples of EMF effects. Electromagnetic
fields stimulation of bone growth, modulated through EMF
stimulation of osteoblasts, appears to involve an elevation/nitric
oxide/protein kinase G pathway. In contrast to that, it seems
likely that the EMF induction of single-stranded DNA breaks
involves a Ca
2+
/elevation/nitric oxide/peroxynitrite/free radical
(oxidative stress) pathway.
It may be asked why we have evidence for involvement of VGCCs
in response to EMF exposure, but no similar evidence for involvement
of voltage-gated sodium channels? Perhaps, the reason is that there
are many important biological effects produced in increased intracel-
lular Ca
2+
, including but not limited to nitric oxide elevation, but much
fewer are produced by elevated Na
+
.
The possible role of peroxynitrite as opposed to protein kinase G
in producing pathophysiological responses to EMF exposure raises
the question of whether there are practical approaches to avoiding
such responses? Typically peroxynitrite levels can be highly elevated
when both of its precursors, nitric oxide and superoxide, are high.
Consequently, agents that lower nitric oxide synthase activity and
agents that raise superoxide dismutases (SODs, the enzymes that
degrade superoxide) such as phenolics and other Nrf2 activators that
induce SOD activity [101], as well as calcium channel blockers may
be useful. Having said that, this is a complex area, where other
approaches should be considered, as well.
Although the various EMF exposures as well as static electrical
field exposures can act to change the electrical voltage-gradient
across the plasma membrane and may, therefore, be expected to
stimulate VGCCs through their voltage-gated properties, it may be
surprising that static magnetic fields also act to activate VGCCs
because static magnetic fields do not induce electrical changes on
static objects. However, cells are far from static. Such phenomena as
cell ruffling [102,103] may be relevant, where thin cytoplasmic sheets
bounded on both sides by plasma membrane move rapidly. Such
rapid movement of the electrically conducting cytoplasm, may be
expected to influence the electrical charge across the plasma mem-
brane, thus potentially stimulating the VGCCs.
Earlier modelling of electrical effects across plasma membranes
of EMF exposures suggested that such electrical effects were likely to
be too small to explain EMF effects at levels reported to produce bio-
logical changes (see, for example [22]). However, more recent and
presumably more biologically plausible modelling have suggested
that such electrical effects may be much more substantial [104–109]
and may, therefore, act to directly stimulate VGCCs.
Direct stimulation of VGCCs by partial depolarization across the
plasma membrane is suggested by the following observations dis-
cussed in this review:
1The very rapid, almost instantaneous increase in intracellular
Ca
2+
found in some studies following EMF exposure [8, 16, 17,
19, 21, 27]. The rapidity here means that most, if not all indi-
rect, regulatory effects can be ruled out.
2The fact that not just L-type, but three additional classes of
VGCCs are implicated in generating biological responses to EMF
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J. Cell. Mol. Med. Vol 17, No 8, 2013

exposure (Table 1), suggesting that their voltage-gated proper-
ties may be a key feature in their ability to respond to EMFs.
3Most, if not all, EMF effects are blocked by VGCC channel
blockers (Table 1).
4Modelling of EMF effects on living cells suggests that plasma
membrane voltage changes may have key roles in such effects
[104–109]. Saunders and Jefferys stated [110] that ‘It is well
established that electric fields …or exposure to low frequency
magnetic fields, will, if of sufficient magnitude, excite nerve tissue
through their interactions with …voltage gated ion channels’.
They further state [110] that this is achieved by direct effects on
the electric dipole voltage sensor within the ion channel.
One question that is not answered by any of the available data is
whether what is known as ‘dirty electricity’ [111–113], generated by
rapid, in many cases, square wave transients in EMF exposure, also
acts by stimulating VGCCs. Such dirty electricity is inherent in any
digital technology because digital technology is based on the use of
such square wave transients and it may, therefore, be of special con-
cern in this digital era, but there have been no tests of such dirty elec-
tricity that determine whether VGCCs have roles in response to such
fields, to my knowledge. The nanosecond pulses, which are essen-
tially very brief, but high-intensity dirty electricity do act, at least in
part, via VGCC stimulation (Table 1), suggesting that dirty electricity
may do likewise. Clearly, we need direct study of this question.
The only detailed alternative to the mechanism of non-thermal
EMF effects discussed here, to my knowledge, is the hypothesis of
Friedman et al. [114] and supported by Desai et al. [115] where the
apparent initial response to EMF exposure was proposed to be NADH
oxidase activation, leading to oxidative stress and downstream regu-
latory effects. Although they provide some correlative evidence for a
possible role of NADH oxidase [114], the only causal evidence is
based on a presumed specific inhibitor of NADH oxidase, diphenyle-
neiodonium (DPI). However, DPI has been shown to be a non-specific
cation channel blocker [116], clearly showing a lack of such specific-
ity and suggesting that it may act, in part, as a VGCC blocker. Conse-
quently, a causal role for NADH oxidase in responses to EMF
exposure must be considered to be undocumented.
In summary, the non-thermal actions of EMFs composed of low-
energy photons have been a great puzzle, because such photons are
insufficiently energetic to directly influence the chemistry of cells. The
current review provides support for a pathway of the biological action
of ultralow frequency and microwave EMFs, nanosecond pulses and
static electrical or magnetic fields: EMF activation of VGCCs leads to
rapid elevation of intracellular Ca
2+
, nitric oxide and in some cases at
least, peroxynitrite. Potentially therapeutic effects may be mediated
through the Ca
2+
/nitric oxide/cGMP/protein kinase G pathway. Patho-
physiological effects may be mediated through the Ca
2+
/nitric oxide/
peroxynitrite pathway. Other Ca
2+
-mediated effects may have roles as
well, as suggested by Xu et al. [26].
Conflicts of interest
The author confirms that there are no conflicts of interest.
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