A Pulsed DC Electric Field Affects P2-Purinergic Receptor Functions by Altering the ATP Levels in Vitro and in Vivo Systems
J. C. Seegers,1 M.-L. Lottering,1 A. M. Joubert,1 F. Joubert,2 A. Koorts,1C. A. Engelbrecht,3 D. H. van Papendorp1
Departments of 1
Physiology and 2
Biochemistry, University of Pretoria, Pretoria, South Africa; 3
Department of Physics, Rand Afrikaans University,
Johannesburg, South Africa
Summary Recently it was shown that extracellular ATP, acting through purinergic receptors, has many
physiological functions, including opening of Ca2 -ion channels, activation and mediation of signal tranduction
mechanisms as well as activation of the pain sensation. Since electrical stimulation is also known to affect many
signal transduction processes as well as the alleviation of pain, we hypothesized that electric stimulation may affect
the extracellular release of ATP. We investigated the effects of a small DC electric field (101
±102 V mÿ1 range and
with frequencies below 150 Hz) on the release of ATP in vitro (HeLa cells), and on the levels of ATP in vivo (the plasma of
healthy volunteers). In HeLa cells ATP release was increased 50 fold, while the total amount of ATP in the cells was
increased by 163%. In the plasma a significant decrease (P < 0.05) in ATP concentration was seen after electrical
stimulation, in all the volunteers. The small DC electric field also affected the cAMP signal transduction system
in vitro (HeLa cells and human lymphocytes) and in vivo (human plasma). Decreased levels of cAMP (P < 0.05) were
seen in HeLa cells and increased levels of cAMP (P < 0.05) in isolated human lymphocytes. The cAMP levels in the
plasma of the electrically treated volunteers were lower than control values. These results show that the frequency,
waveform and signal strength of the applied electric field are suitable for effecting measurable changes on signal
transduction in vitro and in vivo. & 2002 Harcourt Publishers Ltd
INTRODUCTION
Extensive research has shown that, apart from its well
known intracellular effects, ATP also has many extracellular physiological functions (1). Extracellular ATP has
been identified as a ligand, as a transmitter and also as a
co-transmitter that affects numerous cellular functions by
activating P2-purinergic receptors (see 1 for review). There
are different types of P2-receptors, the two major classes
being
increased, which will then activate many calciumdependent cellular activities (3). The P2Y11 receptor
specifically activates adenylyl cyclase, causing increased
cAMP production (4,5). In addition, phospholipase A2 is
also activated by extracellular ATP, which can lead to
increased levels of arachidonic acid in the stimulated cells
(6). Apart from the functions mentioned above, a possible
role of extracellular ATP as an instigator and mediator of
the pain sensation has also been described by several
groups (2,7±10).
There are many reports of effective treatment of pain
with exogenously applied small electric fields overthe area
of treatment (11). The cellular mechanisms involved in the
successful treatment of pain with electric devices remain
unclear. Mechanisms that may possibly be affected after
exposure to an electric field include the following: (a)
enhanced release of dynorphins and enkephalins, especially b-endorphins (12,13); (b) internalization of substance
P receptors (14); (c) the gate control theory of pain (15); (d)
activation of different opioid receptors (16). We suggest
that electric stimulation may affect the release of ATP
which may also influence aspects of the pain sensation. It
has been shown that electric stimulation enhances the
release of ATP in in vitro studies (17), but less is known of
the release in in vivo systems. In a previous paper we
hypothesized that most of the therapeutic effects attributed to electric treatment can be explained by effects on
cell membrane-linked signal transduction mechanisms
(11). Similar to extracellular ATP (2,7±10), electric stimulation of cells also affects signal transduction mechanisms by increasing intracellular calcium, arachidonic acid
and cAMP levels in a variety of cells (18±20). The positive
effects of electric stimulation on bone fracture healing and
tissue regeneration have been attributed to activation of
some of these signal transduction mechanisms (20±22).
Since it has been shown that ATP release is affected in
vitro after exposure to a small electric field (17), we suggest
that electric field-induced extracellular alterations in ATP
levels may affect the pain sensation via P2-purinergic
receptors. Because it is difficult to determine ATP release
in vivo in an electrically treated area, we tested our theory
on ATP release by determining ATP levels in the plasma of
healthy volunteers exposed to a small applied electric
field, which was in the form of periodic DC pulses with
peak strength in the 101
±102 V mÿ1 range, and with frequencies below 150 Hz. We used HeLa cells to study ATP
release in vitro.
Since both extracellular ATP and electric stimulation
affect cAMP levels, and therefore signal transduction
(4,5,18), the effects of the application of the small electric
field on cAMP levels in HeLa cells, human plasma and isolated human lymphocytes were investigated to furthertest
our hypothesis that signal tranduction mechanisms are
affected when cells are exposed to a small DC electric field.
APS Technologies (Tech Pulse, Pretoria, South Africa)
supplied two devices delivering a periodic, direct current,
pulsed electric field. The pulse waveform was a brief
monophasic square pulse (duration 0.8 ms) followed by
exponential decay to base level. The median of the applied
current strength was set to 600 mA for all the experiments,
which corresponds to a peak voltage of approximately
20 V for the in vivo experiments and a peak voltage of
approximately 10 V for the in vitro experiments. The
applied signal translates into peak field strengths of
approximately 101
±102 V mÿ1 in the samples. The pulse
frequency used was 150 Hz.
ATP levels in vitro
A marked, highly significant (P < 0.05) increase in the
release of ATP into the medium in electrically stimulated
HeLa cells was seen (Fig. 1), which corroborates our
assumption that electric stimulation may enhance ATP
Fig. 1 The levels of ATP in HeLa cells before and after electric
treatment. The release of ATP after exposure was significantly higher
than in the controls (P < 0.05). The procedures followed to obtain
these values were as follows: Equal numbers of HeLa cells
( 5105 cells/well) were seeded into two six-well culture plates
(9.4 cm2 surface area/well). The cell cultures were incubated for
24 h in MEM containing 10% heat inactivated fetal calf serum. The cell
cultures were incubated at 37 C in a humidified atmosphere
containing 5% CO2. After 24 h near confluent layers of HeLa cells
were washed three times with PBS (37 C). Two ml of warm
(37 C) PBS were placed in each of the wells. The wells were fitted
with electrodes embedded in resin. Electrodes in each well, attached
to the two APS devices, were separated by 3.4 cm, and were in
electrical contact with the PBS. In each plate two of the wells were left
untreated and four subjected to electrical stimulation (pulsed DC
current with median strength set to 600 mA), for 8 minutes. All the
experiments were performed in an incubator at 37 C. After electrical
stimulation of HeLa cells, triplicate PBS samples were taken from
each well for the measurement of secreted ATP. To measure
intracellular ATP, the remaining PBS was discarded and the HeLa
cells were lysed with the provided cell lysis reagent for 5 min at
room temperature. Luciferase reagent (100 ml) was added to 100 ml
of either PBS sample or cell lysate and ATP content was measured
according to protocol described in the pack insert of Roche’s
Bioluminescence assay kit. A BioOrbit 1251 Luminometer (O.E.N.
Enterprises, SA) was used. (Data are presented as follows
in Figures 1±5: Means are presented in bar charts, with T-bars
referring to standard deviations (SD). P values were obtained with
Student’s t-test).
172 Seegers et al.
Medical Hypotheses (2002) 58(2), 171±176 & 2002 Harcourt Publishers Ltd
release. The cell membrane is not usually permeable to
ATP, but it is a general finding that ATP is released into the
media of cultured cells when there is a mechanical disturbance present, including the changing of medium (1).
Therefore, as expected, a small amount of ATP was present
in the media of control HeLa cells (Fig. 1). Electric stimulation, however, increased extracellular ATP more than
50-fold (Fig. 1), indicating that an ATP-releasing mechanism in the membranes of the HeLa cells might have
been activated.
To verify that the ATP release was not caused by cell
membrane damage, electrically treated HeLa cells were
immediately stained with trypan blue (1%) after treatment, to establish cell viability and membrane integrity.
No increase in trypan blue uptake and therefore no toxic
effect were seen. Cells were also stained with haematoxylin and eosin using standard procedures previously
described (23), to detect morphological changes. Again
there were no detectable effects on cytoplasmic, nuclear
and mitotic morphology (morphological studies are not
shown).
In contrast to the extracellular effects, intracellular ATP
levels were significantly decreased in the HeLa cells after
stimulation (Fig. 1). The total concentration of ATP in each
well (the combined value of ATP in the medium and in
the intracellular fraction) was increased (163%) compared to the untreated control (Fig. 1), indicating that ATP
synthesis was increased by electric stimulation.
ATP levels in vivo
ATP concentrations in the plasma of all nine volunteers
were, however, significantly lower than their untreated
control samples (Fig. 2). This was an interesting finding
emphasizing that in vitro results cannot be extrapolated to
in vivo results.
cAMP levels in vitro and in vivo
Total cAMP levels were determined in HeLa cells and
lymphocyte experiments. In the HeLa cells cAMP production was inhibited by electric stimulation (Fig. 3)
whereas the cAMP production was stimulated significantly in the lymphocytes (Fig. 4). These results suggest that, because of their specific receptor populations,
the two cell types are differently affected by electric
stimulation.
In all nine plasma samples, cAMP levels were decreased
after exposure to the applied electric field (Fig. 5),
although only three of the values were statistically significant. Although it is difficult to explain the various
results on cAMP levels we can conclude that the cAMP
concentration and therefore second messenger functions
are affected by exposure to a small electric field.
Fig. 2 ATP levels after treatment were all significantly (P < 0.05)
lower than a 100% control before exposure to the electric field.
To obtain these values the following procedure was followed.
Electrodes (separated by approximately 30 cm) linked to
an APS device were attached to healthy volunteers. Two negative
electrodes were placed on the dorsum of the left hand, and the two
positive electrodes on the medial aspect of the left arm above the
elbow joint. Nine healthy volunteers were used in this study.
Blood was taken from the left brachial vein of the volunteers,
prior to electric stimulation and again 7 minutes after start of treatment
(just before the electrodes were removed after 8 min of electric
treatment). Blood was collected into tubes containing
7.5 mM EDTA and centrifuged at 3000 rpm for 10 minutes to
remove cells. Aliquots (0.5 ml) of plasma were stored at
ÿ70 C prior to analysis. The procedure provided by Roche’s
Bioluminescence assay kit was followed to determine ATP
values. See legend to Fig. 1.
Fig. 3 In HeLa cells cAMP concentration was significantly lower
(P < 0.05) after exposure to a small DC electric field (procedure
described in Fig. 1). Equal numbers of HeLa cells ( 5105 cells/well)
were seeded into two six-well culture plates. After 24 h cells
in four wells were exposed to a small electric field as described
in Fig. 1. Intracellular as well as secreted cAMP levels were
determined in the untreated and electrically treated cells.
Tenconcentrated lysis reagent (200 ml/well) was added to the
wells containing HeLa cells and 2 ml PBS cell suspension.
The cells were agitated for 10 min at room temperature to facilitate
cell lysis. Cell lysis was monitored by microscopic evaluation with
Trypan blue. Quadruplicate aliquots of cell lysate (100 ml) were
transferred to a donkey anti-rabbit Ig coated plate for total cAMP
measurement according to the non-acetylation EIA procedure as
described in Amersham’s Biotrak kit pack insert. For total cAMP
the two sets of values were added.
Pulsed DC electric field affects P2-purinergic receptor functions 173
& 2002 Harcourt Publishers Ltd Medical Hypotheses (2002) 58(2), 171±176
DISCUSSION
Numerous membrane signal transduction mechanisms
are activated, inhibited or mediated by extracellular ATP,
since there are so many different purinergic receptors (1).
The very high levels of electric field-stimulated ATP
released in the treated HeLa cells, may therefore influence
many cellular processes in the cells in an auto- or paracrine fashion. The cellular responses will depend on the
type of purinergic receptors, PX2- (ion-channel linked)
or PY2 (G-protein linked), present in the HeLa cell
membranes. Apart from the effects on the purinergic
receptors, it has also been shown that extracellular ATP
acts as a substrate for ectokinases (protein kinases that are
active on the surface of cell membranes) in HeLa cells (24)
and may therefore influence the phosphorylation of
extracellular membrane proteins.
The increase in the total amount of ATP (Fig. 1) in stimulated HeLa cells, indicates that the cytoplasmic ATP
production must have been increased by electric stimulation. It is known that electric stimulation will enhance
Ca2 inflow in exposed cells, through the activation of
ligand-gated calcium channels (18). This influx of Ca2
enhances the Crabtree (anaerobic ATP production) effect,
which enhances glycolysis in the cytoplasm but inhibits
ATP-synthase in the mitochondria (25). Thus, the increased ATP may be attributed to the anaerobic breakdown
of glucose in the HeLa cells.
ATP can be released from cells when damage occurs in
the cell membrane (1). There are also physiologically important transport mechanisms of ATP across cell
membranes, which include the release of cytosolic ATP
through secretory vesicles and through specific ATPtransporting systems (26±28). From this study it is unclear
how the ATP was released to such a large extent into
the medium. Although we did not see enhanced Trypan
blue uptake after the HeLa cells were exposed to the
DC electric field, there might have been a transient
electro-poretic effect of very short duration. The reason
for this statement is that Trypan blue does not indicate
enhanced membrane permeability (rather loss of membrane integrity), which could not be identified with the
Trypan blue studies. The morphological study showed no
cytotoxic effects on the HeLa cells. We therefore conclude
that a low-amplitude, periodic, pulsed DC electric field will
enhance ATP production and release in HeLa cells.
The decrease in ATP levels in the plasma obtained from
blood drawn from the area where the electric field was
applied, was an unexpected finding. We do not know how
to explain this effect. It is known that red blood cells (RBC),
not only take up adenosine in the plasma but that ATP is
also removed by RBC (29). Therefore one possibility is that
the uptake of ATP is enhanced by RBC in an applied DC
electric field which may in part explain the lower levels of
ATP in the plasma after electric treatment. Extracellular
ATP is also rapidly metabolized by extracellular ATPases
and nucleotidases. Activation of these enzymes in the DC
electric field can also lead to lower ATP levels in the
plasma (1). Adenosine, the final metabolite of ATP, is a
known inhibitor of peripheral pain signals (30). Therefore
it is important to know whether enhanced ATP metabolism could lead to an increased adenosine concentration
in the plasma. If ATP metabolism is enhanced by the
electric field leading to the production of high levels of
adenosine, this may also explain the alleviation of pain
Fig. 4 In isolated lymphocytes cAMP concentration was
significantly higher (P < 0.05) after exposure to a small electric
field. Human lymphocytes were isolated from heparinized blood using
the protocol provided by the Histopaque-1077 kit. Two ml of blood
yielded between 3106 and 5.4106 lymphocytes. The isolated
lymphocytes were suspended in PBS and 2 ml of this suspension
containing 1.2106 cells were seeded into each well of the
six-well plates. The cells in four of the wells were used as controls
and the cells in eight of the wells were electrically stimulated as
described above. CAMP levels were determined as described
in Fig. 3.
Fig. 5 Compared to their 100% controls, cAMP levels in all
nine of the plasma samples obtained after electric treatment were
decreased (for procedure see Fig. 2). The decreased cAMP
concentration of only three of the samples were significant at the
P < 0.05 level (*). The following procedure was followed to
determine cAMP in the plasma (samples were stored at ÿ70 C,
see Fig. 2): the plasma samples were thawed and diluted 1 : 100
with assay buffer. cAMP levels were measured following the
acetylation EIA procedure as described in the Biotrak kit pack
insert.
174 Seegers et al.
Medical Hypotheses (2002) 58(2), 171±176 & 2002 Harcourt Publishers Ltd
observed after electric stimulation. The levels of adenosine are usually very low in the blood, since adenosine
is rapidly taken up by the erythrocytes in vivo (31).
The red blood cells (RBC) have to be treated with erythro9-(2-hydroxy-3-nonyl)-adenine (EHNA), a compound
rendering used to stabilize the RBC membranes in
heparinized blood, high enough adenosine levels are
present that can be detected using HPLC (32). We could
not determine adenosine levels in the plasma of the
volunteers since we were unsuccessful in obtaining
EHNA from the suppliers. The levels of adenosine in our
samples were therefore too low to be detected. Although
there may be several explanations for the low ATP levels,
what is interesting however, is that the decrease in
plasma ATP may reflect a decrease of ATP concentration
in the extracellular fluid. Since it is known that extracellular ATP contributes to the pain sensation (2,7±10), the
decrease seen after electric stimulation may be linked to
the alleviation of pain experienced in patients exposed
to pulsed DC electric treatment (33).
A DC electric field affected cAMP levels differently in
the two in vitro systems (Figs 3 and 4). It is evident that
different cell types with different receptor populations will
not react similarly to treatment that affects their signal
transduction mechanisms. The HeLa cells are furthermore
transformed whereas the lymphocytes are normal cells, a
factor that may also contribute to the different effect
on the cAMP concentration. According to Lader et al. (27),
increased levels of cAMP are necessary to release ATP
from cardiac myocytes by activating the ATP-permeable
pathway in these cells. Such an ATP cAMP-dependent
transport pathway is apparently not applicable in the
HeLa cells because of lower than control cAMP levels.
Walleczek (34), in an extensive review paper, showed that
the effects of a small electric field on DNA synthesis and
lymphocyte growth can be attributed to the inflow of
Ca2 which activates calcium-dependent signaling. We
can now add increased cAMP production acting as an
additional signaling system affected in lymphocytes by a
small DC electric field. How this will affect lymphocyte
functioning and subsequently the immune system, is
unknown. It will be interesting to determine the effects of
the electric treatment on lymphocytes exposed in vivo.
The significance of the lower than control levels in the
plasma (Fig. 5) is at present also obscure.
As far as a physical interpretation of the effect of a
periodic, pulsed applied electric field is concerned, the
driven oscillator model (11 and references therein) is
addressed as follows by the results of this set of experiments: The clear effects on ATP and cAMP levels seen in
these experiments indicate that the frequency, waveform
and signal strength of the applied field have appropriate
values for effecting measurable change in the human
body. The different effects observed on HeLa cells and
lymphocytes (both in vitro) respectively, support the proposal that the applied field interacts with specific membrane receptors individually. Consequently, an applied
field with a specific form, strength, and periodicity will
selectively enhance the functioning of particular receptors, while affecting other receptors less effectively.
However, the differences in the nature of the effects seen
in the in vivo and in vitro samples indicate that the effects
are of a complex nature. Comparative studies of a range of
applied signals, at a high-frequency resolution, might
shed new light on the finer details of cellular membrane
response to artificially applied electric fields.
Exciting results have been obtained by comparing the
effect of a standardized applied field to two different products of membrane function: ATP and cAMP concentration inside and outside the cell. These results call for
studies of the effect of standardized fields on a broad
range of the products of membrane function, in the
hope that specifically targeted effects could in future
be achieved by an informed specification of an applied
electrical signal.
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