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Introduction |
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9
The Kinetics
of Acetylcholinesterase Inhibition and the Influence
of Fluoride and Fluoride Complexes on the
Permeability of Erythrocyte Membranes - Page 1.
I.
Introduction
The element fluorine was
discovered in ivory at the beginning of the 19th
century by Morodini, a student of Gay-Lussac's, and
first purified in 1886 by Henri Moissan. Fluorine
is, as has only been known for a few years, a so
called essential element, without which at least
vertebrates can not live. Knappwost(1) demonstrated
this in humans after F analysis of urine through the
use of the well known curve for the dependence of
the frequency of caries formation on fluoride
content of the drinking water. McClendon(2) proved
that rats with F-free nutrition do not thrive well.
Hayek et al (3) found that under
physiological conditions traces of F are necessary
to precipitate hydroxyapatite (Abbr. HA).
Research of the effects of small
physiological F-doses was significantly spurred by
the observation that a regular uptake of about 1mg F
per day served as an effective protection against
tooth-decay. (4) This protection is strongest when F
administration is begun before the teeth decay.
This effect is due to an improvement of the
mineralization density of the tooth enamel. An
evenly and densely mineralized enamel offers a
visibly higher protection against the corrosive
influences of cariogenic microorganisms. The
deciding factor is therefore not formation of F-HA
but rather the mineralization density. In vivo the
concentration of F-HA only reaches a level of a few
ppm, at which, according to measurement by Knappwost
and Raju (5), only a few meaningless reductions in
solubility are achieved.
However, a reduction in decay can
even be achieved in teeth that have already begun to
decay, as long as a daily intake of at least 1 mg of
fluoride is maintained. According to Knappwost, the
reduction in decay relies for the most part not on
the presence of fluoride in the saliva, but rather
on an influence on the saliva quality and quantity.
According to McClure(7), the F-content of saliva,
independent of the intake level, should never exceed
0.1 mg/l.
More recent experiments by K.Yao
and P. Groen (8), which were carried out with the
help of an F-specific electrode, confirm these
results. The F-content of the saliva secreted by the
parotid gland, which for test subjects drinking
water with < 0.1ppm of F was 0.007ppm, rose to
0.009ppm upon transition to concentrations of 1ppm
of F in the water.
Knappwost developed a model as
part of his "resistance theory" (9) that describes a
correlation between cariogenic effects and the
viscosity of the saliva. The pH-value of the saliva
also takes on an important role in this model. The
physiologically efficient saliva can thereby be
viewed as a supersaturated solution of HA that has
the task of stabilizing initial corrosive defects on
the enamel surface through remineralization.
The rate of the remineralization
is, at a given supersaturation of HA ions, limited
by the level of diffusion across a boundary layer
attached to the surface of the tooth. The
relationship between the remineralization rate vR
and the viscosity
h
of the saliva can be expressed through the following
equation:
Where n>0 and depends on specific
conditions like, for example, the flow velocity of
the saliva.
The pH-level has an effect on the
solubility of the HA, and therefore also on the
level of supersaturation. A correlation must
therefore exist between oral uptake of fluoride and
the viscosity of the saliva. Experimental findings
support this conclusion (10,11). Of all the factors
that might explain a possible effect of fluoride on
the caries, disregarding the effect on the
mineralization density, Knappwost's theory is least
questionable.
As has already been stated, the
amount of fluoride that is incorporated into the
tooth enamel under physiological conditions is too
small to cause a significant reduction in the
solubility of the HA. An antifermentative effect of
F- on the glycolysis of bacteria in the
mouth only appears at F- concentrations
above 0.5 ppm (12). The concentration of 0.0033 ppm
(8) of F that is reached when the F concentration in
drinking water is 1 mg F/l, a level that was
recognized as sufficient for tooth decay
prophylaxis, however lies well below the
concentration necessary to achieve this effect.
Knappwost's theory assumes an
effect of fluoride on the salivary glands in the
form of an elevated secretion rate, a decrease in
the viscosity, and an increase in the pH. These
symptoms can always be observed with large intakes
of fluoride, both orally as well as parenterally
(11,13). Upon transition to physiological
concentrations the effect is difficult to observe
because of a number of other variables that
influence saliva secretion. In addition, it is
difficult to turn off inductive effects that arise
in conjunction with this outcome. For this reason,
one must attempt to find an influence of the
fluoride on the fundamental biochemical and
biophysical processes involved in saliva secretion.
The salivary glands are
innervated by the autonomic nervous system
(sympathetic and parasympathetic), with the
parasympathetic being of greater importance. The
stimulation occurs as a nervous reflex, the control
center of which lies in the nucleus salivatorius of
the medulla oblangata. The composition of the saliva
depends on the type of stimulus, which creates a
unique stimulatory pattern through the smell and
taste receptors. This stimulatory mechanism allows,
depending on demand, for either a more serous
secretion (stimulation of the parotis) or a more
viscous mucin rich secretion (stimulation of the
sublingual glands) to be created.
For the resistance of the tooth's
surface, however, the so called "resting-saliva" is
of great importance. Like all autonomic organs, the
salivary glands have a basal level of activity,
which in this case serves to moisten the mouth and
throat regions. This moisture is important for
maintenance of muculmembranes and the surfaces of
teeth (by way of remineralization). A general
increase in the tone of the parasympathetic system
has an effect on the composition of the resting
saliva in accordance with Knappwost's resistance
theory, that is to say towards an increased release
of a watery and possibly also more alkaline saliva.
According to recent experiments,
the mechanism of saliva secretion is the following
(14): After stimulation by the neurotransmitter
acetylcholine (ACh), active transport of Cl-
from the interstice into the cell occurs as a result
of hyperpolarization. Passive transport of Na+
follows the Cl- and is in turn followed
by water, which results in an increase in osmotic
pressure within the gland-cell. As a result of the
rising pressure, cellular fluid penetrates the
membrane bordering the lumen. Na+ is
actively reabsorbed at the lumen wall and is
followed by Cl- by way of passive
transport. Water penetrates the lumen wall slowly,
which is the reason for the hypotonia of the saliva.
As a result of the delay in Cl- migration
with respect to that of Na+, the lumen
wall becomes negatively charged on the inside, which
causes a flow of K+ from the inside of
the cell into the lumen. The abnormally high
potassium excess in the saliva results from this
influx. (The K+/Na+ ratio of
the saliva is 1.3, compared to 0.05 in the serum.)
Figure 1
- Schematic Representation of Saliva Secretion
A possible effect of fluoride on
the secretion of saliva could lie either in an
influence on the cholinergic system, or in a direct
influence, perhaps on the membrane permeability for
cations and anions.
1. Fluoride and the
Cholinergic System
Stimulus conduction takes place
by way of the "complete ACh system" at the synapses
of the motor endplate as well as those of the target
organs of the parasympathetic system. The ACh is
synthesized from "activated" acetic acid (in acetyl-CoA,
the acetate is bound to the coenzyme by a high
energy thioester bond) and choline and is collected
in small storage bubbles (vesicles), from which it
is released upon stimulation. The excitation is
passed on by way of depolarizing the bordering cell,
which is the result of a change in Na+
permeability caused by the ACh. The released ACh is
quickly inactivated (saponified) by the enzyme
acetylcholinesterase (AChE). Inactivation is
necessary for the reestablishment of excitability.
Drugs that inhibit AChE (Physostigmine,
Neostigmine, Diisopropylfluorophosphate, E-605,
among others) cause the ACh, which is constantly
released in small amounts, to collect in the
tissues. The build up of ACh leads to the appearance
of parasympathetic stimulation (activation of the
intestinal tract, increased levels of glandular
secretion, decreased blood pressure and heart rate).
In the progressed state a constant depolarization of
the cholinergic membrane, and thereby an
un-excitability, is established. The effected
organism dies as a consequence of this
depolarization.
These symptoms, which are typical
of drugs influencing the parasympathetic nervous
system, are also observed when toxic amounts of
fluoride are administered. MIYAZAKI (15) found
overabundant salivation when toxic amounts of NaF
were given to rats. In 1872 RABOUTEAU (16)
determined that ingesting 0.25g of NaF resulted in
an increase in his salivation. The increase began
after 4.5 hours and lasted 1.5 hours. He could make
the same observation with dogs and rabbits. WEDDEL
(17) induced diarrhea as well as increased
salivation in a dog by administering 0.5g of NaF.
The salivation could not be inhibited by atropine.
This last finding suggests that in this case the
fluoride must be influencing the salivary glands
directly. Otherwise, atropine would have inhibited
the salivation by displacing the ACh, collecting due
to AChE, from its receptors. An anti-cholinesterase
effect of fluoride at lower concentrations than
those applied here is thereby however not out of the
question.
Inhibition of AChE by fluoride
has been described often. E. HEILBRONN (18) and R.M.
KRUPKA (19) completed detailed studies. The authors
describe the inhibition of AChE by NaF, as well as
the pH dependence of the inhibition. This dependence
is, however, not traced back to the un-dissociated
HF molecule, in contrast to which we will, over the
course of this report, show that the inhibition of
AChE by fluoride occurs in proportion to the
concentration of HF.
It follows from Heilbronn and
Krupka's experiments that an inhibition of AChE by
fluoride only arises at concentrations that are
acutely toxic and even lethal in vivo. If one shifts
to physiological concentrations (0.1-1 ppm) the
inhibitions become so small that they lie below the
threshold for accurate measurement.
The inhibition of AChE by
fluoride can be drawn into the discussion of a
vagotonic fluoride influence if effects are found
that, in vivo, can lead to an increase of fluoride's
normal inhibitory influence. The inhibitory effect
of fluoride was assigned to F- in all
previous investigations of the inhibition of AChE by
fluoride. In our opinion, the inhibition does not
necessarily appear only in this form in the
organism. For example, if one dissolves magnesium
hexafluorosilicate (MgSiF6) or cryolite
(Na3AlF6) in a buffer at pH of
7.4, which corresponds to that of human blood, only
partial hydrolysis occurs, as we will show over the
course of this work. The residual complexes, at
least in the case of (SiF6)2-,
inhibit AChE more strongly than fluoride. Therefore,
if one postulates the existence of such complexes in
the organism, the range in which inhibition still
appears shifts towards physiological F
concentrations.
Due to the constant contact of
natural waters with silicates as well as Fe and Al
compounds, one must expect that these compounds and
silicates will form complexes with the fluoride
contained in the water. These complexes can then, by
way of drinking water, enter the body, where they
persist and carry out their influences. New, and as
of yet unreleased, experiments by Knappwost and
Rastaedter, suggest that fluoride is present in
several mineral springs as a Si complex. Taking such
compounds into account one can easily imagine that
fluoride causes an AChE inhibition in vivo, which
makes itself noticeable as a slight vagotonia.
2. Effect of Fluoride on
Membrane Permeability
As Weddel describes (17), a
strong saliva flow developed at high fluoride
concentrations. Since this flow could not be
inhibited by atropine we assume a direct effect of
fluoride on the salivary glands in this case. In
studying the influence of toxic F doses on the
nervous system and muscles, TAPPEINER (13) found
that depression of the central nervous system and
stimulation of the motor endplate appeared
initially. Uncontrolled fibrillary twitches, which
were removed by Curare, arose as well. It is true
that these observations suggest a cholinergic effect
of F, since Curare blocks ACh from binding the
receptor of the effected membrane. This does not,
however, necessarily contradict Weddel's
observations of the salivary glands, since the
stimulatory processes of gland cells differ from
those of the skeletal musculature. (Stimulation at
the motor endplate is preceded by a depolarization;
at the gland cells a hyperpolarization precedes
stimulation).
After long term exposure, high
doses of F eventually led to a blockage of all
stimulus conduction, which suggests a constant
depolarization of nerve and muscle cells.It has long
been known that fluoride can affect (Na+-K+)
distribution at cell membranes. (20) This influence
was first observed in red blood cells, which have a
high intra-cellular K+ concentration
together with a low Na+
concentration. In serum, on the other hand, the
relationship is reversed.
The unequal distribution of these
ions, which is found in all bodily cells, can only
be maintained with constant energy use, as expressed
by the "Gibbs-Helmholtz" equation:
Since the membrane is permeable
to the cations, the T•D
S term is positive when the distribution is uneven.
In the case of a quasi stationary equilibrium the
change in free energy (D
G) must equal 0. This means that T•D
S must be counteracted by an equal, but opposite,
D
H, in this case the enthalpy change (D
H = -7 Cal./Mol.) that results from the splitting of
ATP in the membrane.
Fluoride can affect the (Na+-K+)
distribution at cell membranes and, thereby for
nerve cells, also the resting potential, in three
ways:
-
By inhibiting the enzymatic
degradation of glucose, and therefore also ATP
synthesis.
-
By suppressing the splitting of
ATP at the membrane, which normally provides the
energy for the active transport of cations, by
inhibiting the membrane bound (Na+- K+) activated
ATPase.
-
By fluoride directly affecting
the permeability of the membrane for the
aforementioned ions. This effect perhaps involves
a reciprocal action by fluoride with the membrane
proteins by changing their spatial conformation.
There is evidence to support all
of these possibilities. O. WARBURG (21) has already
reported on the inhibition of glycolysis by F-.
He suggests that fluoride's effect is caused by an
inhibition of enolase by a Mg-fluorophosphate
complex. We discovered a decline in ATP formation in
our own experiments at F- concentrations
> 10-3 M.
L.J. OPIT (22) reports of an
inhibition of the (Na+-K+)
activated ATPase of kidney cells (guinea pig).
According to OPIT, 4 x 10-3 M NaF
inhibits the enzyme up to 50%. S. LEPKE and H.
PASSOW (23) could determine a direct effect on the
membrane in that they discovered K+
efflux in so called "Erythrocyte Ghosts" after
action of 4 x 10-2 M NaF.
Which of these effects dominates
in vivo has not been determined. The concentrations
used here all lie above the physiological
concentration. We must still investigate if similar
effects can be observed at smaller F concentrations.
We must also determine which effects become
effective at which concentrations, when F-
acts on the entire system. The possibilities for a
fluoride effect, possibly also a selective effect on
the salivary glands, are very complex. The
possibilities can be divided into:
-
an effect on the cholinergic system, that
is, on synthesis, storage, release, and
inactivation of ACh.
-
an effect on the (Na+-K+)
distribution and thereby on the resting potential
of nerve cells, whereby the parasympathetically
stimulated cells should be most sensitive.
-
a direct effect on the
processes at the salivary gland, perhaps through
activation of the active ion transport, or through
independent enlargement of the hyperpolarization
during the stimulatory phase.
II.
Presentation of the Problem
According to a theory of
KNAPPWOST's (9), watery vagotonic saliva causes an
increase in the rate of the natural processes that
maintain the surface of teeth, known as
remineralization. The saliva functions as a
supersaturated solution of HA in this process. The
level of supersaturation rises with the pH level, so
that a watery and slightly alkaline saliva possesses
the best reparative properties.
Numerous findings show that
vagotonic symptoms can be observed after
administration of fluoride. (10,11,13,15,17) We
undertook the task of looking for a possible
influence of fluoride ions on the tone of the vagus
nerve by measuring the inhibition of AChE,
and at the same time of studying the kinetics of
this inhibition. We also felt it necessary to study
the effect of fluoride complexes on the ACh-AChE
system, due to their frequent occurrence in nature.
Since it could be assumed that
fluoride can also trigger vagotonic effects
indirectly, we were also interested in the effect of
fluoride on the transport of ions and molecules
through the cell membrane. Furthermore, radioactive
tracing methods, with the help of which biochemical
reactions and even entire chain reactions can be
studied in a single procedure, were to be applied
for this experiment. The pathway of ions and
molecules in the body and at cell membranes can also
be followed using this technique.
Introduction |
Contents |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
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