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Repression of glutaminase I in the rat retina
by administration of sodium-L-glutamate
Jerome K. Freedman and Albert M. Potts
Neioborn albino rats show the same loss of inner retinal layers and of b-wave on treatment
with sodium i,-glutamate as do mice. Values for four enzymes metabolizing glutamate or
glutamine have been determined on normal and glutamate-treated rats. The retinas of normal
rats contain almost twice as much glutaminase I per unit weight as those of glutamate-treated
animals. There is no measurable "glutaminase II" activity in rat retina. There is no difference
between retinas of normal and treated animals in content of glutamosynthetase and glutamotransferase. These findings are consonant with our hypothesis that glutamate action on the
retina of the newborn rat is by repression of synthesis of an enzyme whose reaction product
is glutamate itself. They do not eliminate the possibility that direct competitive inhibition by
glutamate may play a role.
I
n an earlier report1 one of us (A. M. P.)
confirmed the finding of Lucas and Newhouse2 that large doses of glutamate given
to newborn mice resulted in failure to
form or destruction of the ganglion cell
layer and inner nuclear layer of the retina.
Our report further described that such
animals in which retinas consisted chiefly
of receptor cells plus a narrow remnant
of the inner layer showed a characteristically altered electroretinogram. The awave in these animals was intact and the
b-wave was consistently abolished. We
were impressed by the significant toxic
effect of a normal body constituent and
speculated in our previous publication
that feedback repression of enzyme formation might be able to explain the phenomenon.
The present report represents further
investigation of this hypothesis and de-
scribes the activity of a number of glutamate-metabolizing enzymes in treated
and in untreated animals.
Methods
In order to have more retinal material for
chemical study, a brief survey of larger experimental animals was performed with regard to
maturity of the retina at birth and its susceptibility to the glutamate effect. It was found that,
with an identical dose per unit weight, newborn
rats showed the glutamate effect both in the histologic picture and in electrophysiologic behavior.
All the data to be reported here were obtained
with albino rats (Sprague-Dawley strain) which
were treated from the second through the twelfth
day of life with glutamate according to the dose
schedule previously published. Experiments were
performed between the third and fifth weeks of
life, and, in each case, experimental retinas were
compared with those of control animals of
identical age.
Animals were sacrificed by cervical dislocation,
eyes were enucleated, and the retina was quickly
removed and weighed. Retinas were placed in
ice cold distilled water and 5 per cent homogenates were made with the Elvejehm-Potter
homogenizer.
Four enzyme activities were determined: glutaminase I, glutaminase II, glutamosynthetase,
and glutamotransferase.
Glutaminase I. Glutaminase I, a phosphatedependent enzyme with pH optimum between 7.2
From the Section of Ophthalmology, Department
of Surgery, The University of Chicago.
Supported in part by United States Public Health
Service Grants B-2522 and B-2523.
118
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and 7.6, was determined by measuring the amount
of ammonia liberated from a specific quantity of
glutamine, as described by Richterich-van Baerle,
Goldstein, and Dearborn.3 The reaction was run
in 30 c.c. vials incubated in a shaker at 37° C.
At the end of the reaction the ammonia formed
was liberated by K2CO3 and allowed to diffuse to
a drop of ION sulfuric acid on an axial rod
during rotation at room temperature. The collected
ammonia was determined colorimetrically, as
described by Seligson and Seligson,4 and modified
by Richterich-van Baerle and co-workers.3
Glutaminase II. This enzyme system which requires a keto acid for optimal activity and which
is most active at pH 8.6 to pH 8.8 was assayed,
as described by Goldstein, Richterich-van Baerle,
and Dearborn.5 The ammonia formed was determined exactly as described above for glutaminase I.
Glutamosynthetase. Formation of glutamine
from glutamic acid is the function of this enzyme
which requires ATP and Mg++. It can utilize
primary amines as well as ammonia and synthesize
N-y-substituted glutamines. Thus, in the presence
of hydroxylamine, glutamohydroxamic acid is
formed and the color, developed by this product
in the presence of ferric ions, was utilized for its
determination, according to Rudnick
and
Waelsch.0
Glutamotransferase. This enzyme catalyzes the
transfer of the glutamyl radical from glutamine or
glutamine peptides to other amine receptors. Here,
too, hydroxylamine can be used, and the color
of the resultant glutamohydroxamic acid can be
utilized for determination, as described by Rudnick, Mela, and Waelsch.7
It should be noted that, because of the weights
of the retinas obtained, each homogenate was
made with the pooled retinas of 5 to 8 animals.
Results
Glutaminase I. Enzyme activity in liver,
brain, and retina is shown in Table I. The
relatively high level in brain tissue compared to the two other tissues is worth
noting. Krebs'8 figures for higher activity
in retina than brain tissue were obtained
at pH 8.5. The difference between liver in
treated and untreated animals is of questionable significance because of scatter of
results in the two animals in each group. No
such scatter applies to the decrease of
activity in the glutamate-treated retina as
compared to the control. There is nearly
twice as much activity in the control
retinas, and this is a consistent finding in
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Repression of glutaminase I
119
Table I. Glutaminase I activity in rat
tissues (as /xM ammonia liberated/100
mg. wet tissue/30 min. incubation)
Untreated
Treated
Retina
Brain
Liver
20.5
12.3
76.9
79.1
10.3
14.3
Table II. Glutamosynthetase and
glutamotransferase activity of rat retinal
homogenates (as [xM of glutamohydroxamic
acid produced/100 mg. wet tissue/30 min.
incubation)
Glutamosynthetase
Untreated
Treated
7.1
7.0
Glutamotransferase
12.0
12.0
six groups of animals sacrificed at varying
periods after completion of treatment.
Comparison of absolute values for our
tissues with those obtained by others is of
some interest. Although we noted modest
variation in the values found in liver, all
values for the three tissues were within the
same order of magnitude and for any given
tissue results were highly consistent. This
contrasts markedly with glutaminase I
values reported by others. Five different
reports on enzyme activity of normal rat
kidney, when converted to our units, vary
from 0.83 nM to 150 /M per 100 mg. wet
weight per 30 minutes.3'9"12
Glutaminase II. We found no glutaminase activity in the retinas studied that
specifically required pyruvate. Since it has
been shown that glutaminase II activity
represents the action of at least two
enzymes, i.e., a transaminase followed by a
deaminase,5 only one of the two might be
missing. Work in progress shows a very
active transaminase in the retina, so the
likelihood is that the specific deaminase
is lacking.
Glutamosynthetase. Results of this determination (Table II) were consistent and
showed no differences between experimental and control retinas.
Glutamotransferase. Here again there
Investigative Ophthalmology
February 1962
120 Freedman and Potts
was amply measurable transferase activity
but no difference between glutamatetreated and control animals (Table II).
Thus, the only clear-cut effect on the
enzymes studied was the marked decrease
of glutaminase I in the glutamate-treated
animals.
Discussion
We suggested in our previous publication that the mechanism by means of which
the effect of glutamate on the retina can be
explained is that of repression of enzyme
synthesis. Gorini and Maas13 have amply
documented the fact that repression of
enzyme formation by the products of the
specific reaction can occur. Within the last
few years specific instances have been
found among glutamate and glutamine
enzymes in vertebrate tissues. De Mars14
reported on repression of glutamyltransferase formation in cultures of human cells
by glutamine; and, while our own work
was in progress, Goldstein15 reported that
sodium glutamate would suppress the increase of glutaminase I in the rat called
forth by ammonium chloride.
It should be emphasized that our assumption about the effect of glutamate involves one further postulate, namely, that
repression of the specific enzyme in question results in cell death or failure of cell
development. The retinas with which we
are working lack these cells which, according to our supposition, normally contain
glutaminase I and possess only cells (that
is, chiefly receptor cells) in which this
enzyme does not play a major role. It is not
easy to demonstrate that, in fact, the inner
layers of the retina do contain relatively
larger amounts of glutaminase, but experiments on this subject are now in progress.
Determination of glutaminase activity in
retinas where receptor cells have degenerated after iodoacetate treatment, and
histochemical studies of glutaminase in
retinas at various stages of development are
being undertaken with this end in view.
Even if such a demonstration proves
feasible, it does not eliminate the possibility
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that direct competitive inhibition of glutamic acid for glutaminase, as described
by Krebs,s is responsible for our observations. It is not likely that concentrations
capable of competitive inhibition are maintained for a significant period of time by
a single daily dose of glutamate. However,
one cannot rule out the possibility that a
brief daily insult caused by direct inhibition of glutaminase is adequate to explain
our findings. It is hoped that histochemical
studies of glutaminase in developing retinas
at increasing time intervals after the administration of glutamate will throw light
on the matter. These results will be correlated when necessary with retinal content
of radioactively labeled glutamate.
The question has been raised in the past
on just how much injected glutamate
reaches the retina after each dose. We note
the work of Himwich, Petersen, and
Allen,10 who described ready access of
glutamate to the brain at birth and establishment of a barrier to this compound by
the tenth day. This finding makes us confident that glutamate reaches the eye of our
newborn animals in high concentration.
However, we shall again make direct
measurements on the eye with labeled compound to confirm or deny the fact.
Until the studies projected above are
complete, we can only state that the
present results on glutaminase I are not incompatible with our original postulate that
repression of enzyme formation is the
means by which sodium glutamate exerts
its retinotoxic effect.
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