THE ACTION OF ELECTROMAGNETIC FIELDS ON REGULATION OF PARAMECIUM MOTOR FUNCTIONS

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THE ACTION OF ELECTROMAGNETIC FIELDS ON REGULATION OF Paramecium MOTOR
FUNCTIONS
STAT
Moscow TRUDY MOSKOVSKOGO OBSHCHESTVA ISPYTATELEY PRIRODY in Russian Vol 28,
1968 pp 130-136
[Text] Research on the sensitivity of Paramecium motor functions to the
action of electromagnetic fields (EMF) is one aspect of research on EMF
action at the cellular level, on the effect of EMF on selfregulating systems
and, finally, on the participation of such fields in the very processes of
vital cellular regulation.
A great deal of experimental information has been accumulated to date on
the action of EMF on protozoan motor functions. The subjects of such re-
search have been both orientation effects arising in response to EMF (3,5,
18,19) and the action of these fields on the body's controlling systems
(9,10,12-14).
Naturally the question arises as to whether there is similarity between re-
actions of Faramecium to EMF and changes in movements of these animals under
natural conditions. In other words, to what degree are EMF adequate stimuli
for Paramecium locomotor fanction?
A sequence (in order of increasing voltage) of over 10 different types of
movements have been noted in Paramecium in response to prolonged stimulation
by alternating current from 50 to 50,000 Hz -- slight rotation with short,
straight movements, oscillation relative to the center of the animal's long
body axis, and so on (13,25). These same types of movements have been des-
cribed for Paramecium under natural conditions (24). In this case cessation
of movement in Infusoria, which is similar to electroshock reaction (ESR)
described by A. S. Presman (9), is caused by reversal of cilia on half of
its body (5).
In addition a bioelectric difference in potentials has been discovered in
the cilia of Infusoria between the internal and external surfaces of the
body. This difference changes rhythmically, synchronously with movement
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of the cilia (21). A comparison of these data permits us to consider that the
EMF we employed is to some extent an adequate natural bioelectric stimulus
of Paramecium locomotive functions.
What are the mechanisms underlying EMF action on excitable structures of
Paramecium?
According to the enzymochemical theory, a reaction of acetylcholine with a
receptor protein, which increases the permeability and serves as the starting
mechanism of rapid movement of sodium and calcium ions, is the initiator of
the excitation process (7). The receptor-acetylcholine complex is in dynamic
equilibrium with free acetylcholine and receptor protein. The free ester is
attacked by acetylcholinesterase and is quickly inactivated as a result of
hydrolysis, after which the receptor (and the permeability) return to the
initial rest state.
Let us examine the possible action of microwaves on the acetylcholine-cholin-
esterase system. The presence of active cholinesterase in the pellicle of
ciliated Infusoria (27) and experimental data on participation of the ace-
tylcholine-cholinesterase system in stimulation of Paramecium (6) indicate
that such an approach .s valid.
The procedure for studying Paramecium excitability has been described in
detail in previous publications (9,13). Series of alternating current pulses
with a pulse repetition frequency of 600 Hz and a series duration of 50 usec
(produced by GS-100I generator followed by electronic modulation) were used
as the stimulating pulses.
Irradiation by microwaves was conducted in a special chamber, the layout of
which had been described earlier in detail (14). A VNIIM i 0 pulse generator
was used as the microwave source. Microwave irradiation was conducted in
the pulse-pulse mode, the wavelength was 10 cm, the pulse repetition frequency
was 700 pulses/sec, the duration of internal pulses was 1 usec, the pulse
series duration was 50 cosec, and the irradiation power was 10 percent lower
than that necessary for direct stimulation. The reagent solutions were pre-
pared out of the medium in which Paramecium was cultured. An MBS-2 micro-
scope was used to observe the behavior of Paramecium. An aluminum hood
lined within by absorbent KhV-l0 plates was used to protect the experimentor's
eyes from microwave side effects. There was an opening in the hood for the
microscope objective.
Irradiation of Paramecium by microwaves in this mode reduced the excitation
threshold by 55 percent as compared to controls. The maximum effect was
manifested 5-10 minutes after the start of irradiation (Figure 2 [sic]).
We hypothesized that by blocking cholinesterase we could disturb the dynamic
equilibrium between free acetylcholine and acetylcholine bound to the re-
ceptor. In this case we suggested that the disturbance would depend on the
concentration of inhibitory substance, and that it would be manifested either
in an increase in the excitation threshold or in its decrease. Such a depen-
dence on inhibitor concentration stems from the fact that when the concentration
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of acetylcholine is increased, the cholinesterase activity also grows in
parallel to a particular level. After this,activity begins to increase, and
a high acetylcholine concentration severely inhibits enzyme action (17).
0
~ 50
t
(1) g is 15 2p 25 30 35 40 Y5 5~^ d0
6nu..&iue (2 ) ff r ( nuN
100
00
%
J I
40
j _j7 15 20 25 30 35 40 45 50 60
0J,^ eo+ue (2) ( roux
Figure 1. Change in ESR Thresholds of Parmenium Upon Stimulation
by Alternating Current Pulses: a -- upon one-time micro-
wave irradiation in continuous mode; hatched area --
variation in the threshold voltage of alternating current
pulses (frequency 12 kHz, duration 50 msec) without micro-
wave irradiation; b -- on the background of 'the action of
urea in a 1-percent concentration, and upon combined action
of 1-percent urea and microwave irradiation; 1 -- effect
of the action of 1-percent urea; 2 -- effect of the com-
bined action of 1-percent urea and microwave irradiation
in the continuous mode.
Key:
1. Threshold voltage (percent 2. Irradiation
of control) 3. Minutes
We used proserine -- a reversible inhibitor of cholinesterase activity at
a concentration of 1.5- 10-3 percent as the substance ')locking the action of
cholinesterase. At this concentration it causes reduction of Paramecium
excitability. The ESR threshold increases in this case by 60 percent as
compared to controls (see Figure 2).
When the action of proserine (at a concentration of 1.5 ? 10-3 percent) is
combined with microwave action, in a sense these effects are summated, as a
result of which the excitation threshold is restored to its initial level
during 15 minutes of irradiation (see Figure 2).
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Figure 2. Change in ESR Thresholds of Paramecium Upon Stimulation
by Alternating Current Pulses and Simultaneous Micro-
wave Irradiation, Proserine and the Combined Action of
Microwave and Proserine: .1 -- effect of irradiation by
series of microwave pulses at a frequency of 700 pulses/
sec and a duration of 50 msec; 2 -- effect of proserine
at a concentration of 1.5- 10-3 percent; 3 -- effect of
the combined action of proseri.:ie (1.5 ? 10-3percent) and
microwave irradiation in the pulse-pulse mode. Hatched
area -- variation of the threshold voltage of alternating
current pulses (frequency 600 Hz, duration 50 msec) with-
out microwave irradiation. [Key: See key, Figure 1]
Thus microwave irradiation acting on a background of reduced Paramecium ex-
citability in a sense cancels out the effect of excitability reduction, nor-
malizing the ESR thresholds, probably due to restoration of the disturbed
dynamic equilibrium between the acetylcholine-receptor complex and free ace-
tylcholine, or between free acetylcholine and cholinesterase. To verify the
action that microwave irradiation would have on the second component of the
starting reaction --- the protein, we made use of the possibilities for actively
changing the excitability level of Paramecium using SH-group donors and ac-
ceptors (4,8,15).
The research procedures were similar to those described above with the single
difference that microwave irradiation was conducted in continuous mode,
using a wavelength of 16 cm (produced by a GCh-8 generator), specific power
of 5 mw/cm3, and an irradiation time of 20 minutes.
Microwaves irradiation in this mode reduces the excitation threshold by 30
percent, the maximum effect being manifested in the last 5 minutes of ir-
radiation during the aftereffect period (Figure 1).
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A thiolic toxin -- mercuric chloride -- at a concentration of 1L5 ? 10-5 was
used to bind the S11-groups of the proteins. In response to this reagent the
Paramecium excitation threshold doubled the control threshold in 45 minutes.
When Paramecium is subj(cted to simultaneous action of microwave radiation
and mercuric chloride in the concentration indicated above, a 10-15 percent
reduction is observed in the excitation threshold as compared to the excita-
tion threshold at the same moments in time in the presence of mercuric chloride
alone. In this case this effect persists for 20 minutes after irradiation
;Figure 3a). Thus microwave irradiation partially cancels out the effect of
mercuric chloride -- that is, in this case it normalizes the excitation thres-
hold to a certain degree.
Inasmuch as thiolic toxins can, in addition to binding with SH-groups, have
an inhibitory action on various enzyme systems, we used an oxidation reaction
to inhibit SH-groups. For this ouriose we employed oxidized cysteine, which
is one of the metabolites formed during normal vital activity.
T T -J5
:r.
!0 r5 20 30 35 40 45 50 10 15 20 25 30 .:5 40 4; SO
OJnywem ie (2) ~ 06np'eHue 1 (2) (3) xua,
Figure 3. Change in ESR Threshold of Paramecium Upon Stimulation
by Alternating Current Pulses and Simultaneous Micro-
wave Irradiation: a -- in the presence of mercuric
chloride and combined action of microwaves and mercuric
chloride: 1 ?-- effect of mercuric chloride at d concen-
trat.ion of 1.5 ? 10-; percent; 2 -- effect of irradiation
by continuous m.i:.?rowaves; 3 -- effect of the combined
action of mercuric chloride (1.5. 10-5 percent) and
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microwave irradiation in continous mode; hatched area --
variation in threshold voltage of alternating current pulses
(frequency 12 kHz, duration 50 msec) without microwave ir-
radiation. b -- in the presence of cysteine and combined
action of cysteine and microwave irradiation; 4 -- effect
of cysteine in a 1-percent concentration; 5 -- effect of irra-
diation by continuous microwaves; 6 -- effect of the com-
bined action of cysteine (1 percent) and microwave irra-
diation in the continuous mode; hatched area -- variation
in threshold voltage of alternating current pulses (fre-
quency 12 kHz, duration 50 msec) without microwave irradia-
tion.
Key:
(See Key, Figure 1]
When cysteine at concentrations of 1-1.5 ? 10-5 percent is added to the Para-
mecium medium, an excess of mercaptide RS- ions, which oxidize more readily
than do undissociated SH-groups, arises within it. Since the oxidation op-
timum of cysteine lies in the alkaline pH (16) and the Parmneeium medium has
a weakly alkaline reaction (pH 7.5-8.1), and the concentration of mercaptide
ions is rather large in the medium, cysteine oxidation would occur in the
following way:
RS- -> F-S. + e-
RS. + RS. -} RS-RS;
I
II
In this case the rate of the oxidation process under these conditions would
be defined by reaction II. The disulfide compounds that are formed may in
turn cause oxidation of the SH-groups of Paramecium proteins -- that is, they
may promote configurational changes in the proteins.
In the indicated concentrations, cysteine evokes a rise in the Paramecium
excitation thresholds, the degree to which this effect is pronounced depend-
inq on the cysteine concentration: When cysteine is at a concentration of
1.5 ? 10-5 percent the ESR threshold increases by 45 percent, while when the
concentration is 1 percent the threshold increases by 250 percent (Figure 3b).
When Parameciumn is subjected to microwave irradiation on a background of cys-
teine action at concentrations from 1.5- 10-3 to 1.5. 10-5 percent, the ex-
citation threshold becomes fully restored, rising in responce to cysteine.
When higher concentrations of cysteine are employed (1-1.5. 10-4 percent) in
combination with microwave irradiation the excitation threshold drops by a
factor of two during irradiation as compared to the size of the excitacior
threshold at the same moments in time in the presence of cysteine alone
(Figure 3b).
Thus depending on cvsteine concentration, microwave irradiation partially
or completely cancels out the effect of cysteine -- that is, it normalizes
excitability, apparently hindering oxidation of protein SH-groups.
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GOVERNMENT USE ONLY
Data on increasing resistance of animals to the action of ionizing radiation as
a result of preliminary microwave irradiation (11,12) can serve as indirect evi-
dence for such a hypothesis. The effect stems from the evoked action of micro-
waves, reduction of the oxygen partial pressure in arterial blood, and inhi-
bition of electron excitation in protein molecules. As a consequence of these
processes the formation of free radicals in response to ionizing radiaticn is
prevented or reduced (22). We can suggest on the basis of these experiments
that microwaves affect SH-groups. To test these hypotheses we attempted to
increase the number of reactive SH-groups in protein of Paramecium itself.
We used urea as the agent freeing SH-groups o:F native proteins. As in the
case of just microwave action alone (Figure la), when a 4-percent solution of
urea is introduced into the Paramecium medium the excitation threshold of the
Infusoria drops. Urea action is manifested in the very first minutes, reach-
ing a maximum at 35 minutes (the ESR threshold drops by 40 percent). At 60
minutes the excitation threshold reaches its initial level (Figure ih).
When Paramecium is subjected to simultaneov.s action of urea and microwave
irradiation the excitation threshold, which is altered by urea, is restored
to its initial level after 20 minutes of irradiation. Although as had been
described above (Figure la), microwave irradiation in this mode can itself
raise Paramecium excitability, when these two factors are combined their uni-
directional effects are totally canceled out.
Thus normalization of the excitability level is a general effect of micro-
waves, irrespective of whether the particular agent reduces (proserine, mer-
curic:: chloride, cysteine) or raises (urea) Paramecium excitability.
Although the regulatirg effect of electromagnetic fields is obvious in this
case, we can discuss its mechanism only hypothetically.
Biochemical research has shown that protein molecules in protozoan cilia and
flagella exist simultaneously in a and S-configurations (2), and that any de-
structive changes (for example, the action of urea) disturb the initial ratio
between these configurations.
It would be natural to hypothesize that by canceling the effect of urea, micro-
wave irradiation can restore this equilibrium. Data on change in the ratio
of the two possible states or configurations of macromolecular systems in re-
sponse to high-energy electric fields (20) argue in favor of this hypothesis.
An electric field shifts the equilibrium between the two states in the direc-
tion of greatest polarization and changes the elasticity of protein chains.
The latter, in particular, may be at the basis of muscular contraction. Con-
sequently it is fully probable that a-configuration proteins transform into
the stabler a-confiquration in response to EMF.
At the same time when protein solutions are irradiated by microwaves all
polarized side chains of such molecules would become oriented along the
field lines, exhibiting a tendency for breakage of hydrogen bonds and chang-
ing the hydration zone (26). This can apparently only partially explain the
external analogy between the effects of microwaves and urea, but not their
joint action.
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Thus we can say on the basis of the obtained data that in all probability
electromagnetic fields act on both components examined by us of the acetyl-
choline-receptor protein starting reaction.
Conclusions
1. Electromagnetic fields, including microwaves, are active and apparently
adequate stimuli for Paramecium.
2. The action of electromagnetic fields is based on the association between
locomotor functions and electromagnetic regulation.
3. Obviously the action of EMF in the microwave range is manifested at the
level of enzyme processes in the pellicle of Paramecium (the acetylcholine-
cholinesterase system), and in the form of configurational changes in pro-
teins of the acetylcholine-receptor protein complex.
In conclusion I express my gratefulness to Candidate of Biological Sciences
A. S.. Presman for his constant attention and assistance in completion of
this project.
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23. Parducz, B., "Reizphysiologische Untersuchunaen an Zili.aten. I. Uber das
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