Download Immunohistochemical study of glutathione

The Histochemical Journal 33: 267–272, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Immunohistochemical study of glutathione reductase in rat ocular tissues
at different developmental stages
Tsuneko Fujii1 , Keiji Mori2 , Yoshinori Takahashi3 , Naoyuki Taniguchi1 , Akira Tonosaki2 ,
Hidetoshi Yamashita3 & Junichi Fujii4,∗
1
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871
2
Department of Anatomy, 3 Department of Ophthalmology, 4 Department of Biochemistry, Yamagata University School of
Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan
∗
Author for correspondence
Received 27 February 2001 and in revised form 19 May 2001
Summary
Glutathione, which is found in high levels in eye tissues, is involved in multiple functions, including serving as an antioxidant
and as an electron donor for peroxidases. Although the activities of enzymes related to glutathione metabolism have been
reported in the eye, the issue of which cells produce these proteins, where they are produced and at what levels is an important
one. Glutathione reductase, an enzyme which recycles oxidized glutathione by transferring electrons from NADPH, was
localized immunohistochemically in adult rat eye in this study. The reductase was distributed in the corneal and conjunctival
epithelia, corneal keratocytes and endothelium, iridial and ciliary epithelia, neural retina, and retinal pigment epithelium. In
addition, it was highly expressed in ganglion cells, which are responsible for transmitting photophysiological signals from
the retina to the higher visual centres. To clarify the correlation of glutathione reductase expression and oxidative stress, the
enzymatic activity and the level of protein expression at the pre- and postnatal stages was examined. Expression of the enzyme
was detected first in the ganglion cell layer of a late prenatal stage, and appeared in the inner plexyform layer after birth.
Along with an increasing differentiation between the inner nuclear and outer nuclear layers, glutathione reductase expression
became detectable in the outer plexyform layer. Pigment epithelial cells were positively stained only after birth. Expression
was also detected in the lens epithelium from the prenatal to early postnatal stages although its level was low in the adult
lens. Collectively, these data, except for lens epithelia, suggest the pivotal role of glutathione reductase in recycling oxidized
glutathione for the protection of the tissues against oxidative stress, which is caused by eye opening accompanied by the
initiation of various ocular processes, such as accession of light and transduction of the photochemical signal.
Introduction
Glutathione (GSH) plays multiple roles in living organisms,
such as maintaining intracellular components in a reduced
state, the detoxification of xenobiotics, and as an electron
donor for certain antioxidative enzymes (Meister & Anderson
1983, Arrick 1984). Glutathione reductase (GR) is involved
in converting the oxidized form, GSSG, back to GSH via
an NADPH-dependent reduction reaction and constitutes the
major system in maintaining constant levels of GSH in most
tissues. The recycling of GSSG by the GR system is more
efficient than the de novo synthesis from an energetic point
of view and, hence, is ubiquitous in the body (Carlberg &
Mannervik 1985). Evidence has accumulated to show that
several intracellular events, including signal transduction
and gene expression, are regulated via reduction-oxidation
(redox) reactions (Sen & Packer 1996). In terms of regulation of the redox balance within the cytoplasm, the GSH-GR
system and a thioredoxin-thioredoxin reductase system constitute the two major components.
It is well known that the eye contains high levels of GSH
in conjunction with a large amount of glutathione peroxidase. The enzymatic activities of GR have been investigated
in a variety of tissues, including the eye, under physiological as well as pathological conditions (Black & Wolf 1991,
Zhang & Augusteyn 1994). In order to understand the intrinsic role of GSH and GR in individual organs, which consist of various types of cells, such as the eye, the issue of
which cells produce these proteins, where they are produced,
and at what levels is an important one. Immunohistochemical
studies of the eye aimed at detecting superoxide dismutase
(Ogawa et al. 1997), catalase (Atalla et al. 1987), and glutathione peroxidase (Atalla et al. 1988) have been described.
In addition, only a few reports exist relative to the localization
of GR in some tissues thus far (Ogawa et al. 1993, Knollema
et al. 1996, Gutterer et al. 1999) and no report is available
which specifies the tissues or cells of the eye regardless of its
very high overall content of GSH (Spector 1995). A competent anti-GR does not appear to be available at present
(Yan et al. 1998).
268
To resolve this problem, we recently established a new
antibody against rat GR, using the recombinant GR protein
produced in a baculovirus/insect cell system (Fujii et al. 2000,
2001a). Here, we describe the application of the antibody
to an eye tissue preparation and show that it is abundantly
distributed in the epithelial cells of the cornea, iris, and ciliary
body. In addition, it is present in high levels in ganglion cells
and in the pigment epithelium of the retina. To gain insight
into the role of GR in those cells, its expression in rat eyes at
the pre- and postnatal stages was investigated.
Materials and methods
T. Fujii et al.
(Amersham) under semi-dry conditions with the use of a
Transfer-blot SD Semi-dry transfer cell (Bio-Rad). After
blocking by incubation with 5% skim milk in 20 mM
Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20
(TBST) for 2 h at room temperature, the membranes were
incubated with the rabbit antibody to rat GR (1 : 1000
dilution) (Fujii et al. 2000) for 12 h at 4 ◦ C. After washing
with TBST the membrane was incubated with peroxidaseconjugated goat anti-rabbit IgG (1 : 2000 dilution, Organon
Teknika Corp.) for 1 h. After washing, peroxidase activity
was determined by the chemiluminescence method using an
ECL kit (Amersham).
Materials
Immunohistochemistry
GSSG was obtained from Sigma. NADPH was obtained from
Oriental Yeast Co., LTD. Other reagents were of the highest
grade available. Wistar rats were purchased from Japan SLC,
maintained under 12 h of light and 12 h of darkness schedule
at temperature of 21–23 ◦ C, and fed and given water ad lib.
A streptavidin-biotin method was employed for immunostaining eye tissue sections. After conventional fixation with
Bouin solution and embedding in paraffin wax, 4 µm thick
sections were treated with a goat serum for 10 min to block
non-specific binding, and then reacted with the anti-GR IgG
(Fujii et al. 2000) for 60 min with 1 : 200 dilution. They were
reacted sequentially with biotinylated goat anti-(rabbit IgG),
peroxidase-conjugated streptoavidin, followed by the chromogen 3,3 -diaminobenzidine (DAB) for 3 min. Finally, the
samples were counterstained with Mayer’s haematoxylin for
1 min.
For the analysis of the retina, dissected eye cups were fixed
by immersion with 4% paraformaldehyde in 0.1 M phosphate
buffer for 4–6 h. After rinsing with phosphate-buffered saline
(PBS), the retina was embedded in acrylamide and 8 µm-thick
sections were obtained on a cryostat at −30 ◦ C, followed by
reaction with the anti-GR IgG. A monoclonal antibody H16,
which was raised against lamprey rhodopsin-like protein and
which recognizes rod outer segments in rats, was used to
visualize the outer segment layer (Yoshida et al. 1991). The
sections were examined by a light microscope, AH-2NIC-2
(Olympus), equipped with a Nomarski interference-contrast
device.
Enzyme assay
GR activity was assayed spectrophotometrically by measuring the rate of oxidation of NADPH at 340 nm (Carlberg &
Mannervik 1985). The reaction mixture consisted of 0.1 M
potassium phosphate, (pH 7.0), 1 mM EDTA, 0.1 mM
NADPH, and 1 mM GSSG and the decrease in absorbance
at 340 nm at 30 ◦ C was recorded. One unit of GR activity was
defined as the amount of the enzyme that catalyzes the oxidation of 1 µmol of NADPH per minute. Assays were performed
in duplicate.
Preparation of tissue homogenates and
protein assay
Experiments were performed under the protocol approved
by the Animal Research Committee, Yamagata University
School of Medicine. Rats were sacrificed by decapitation
under anaesthesia with diethyl ether. Dissected eyes were
either fixed immediately in Bouin solution for immunohistochemical analysis or frozen in liquid nitrogen and preserved
at −80 ◦ C until used for enzyme assays. For protein analysis,
tissue samples were homogenized in small volumes of phosphate buffered saline containing 10 µg/ml pepstatin, 10 µg/ml
leupeptin, 100 µM p-amidinophenylmethylsulphonyl fluoride, and 1 mM benzamidine with a polytron homogenizer.
After centrifugation at 10,000 × g for 20 min, the supernatants were collected. Protein concentrations were determined using a BCA kit (Pierce) employing bovine serum
albumin as the standard.
SDS-PAGE and immunoblot analysis
Protein samples were subjected to 10% SDS-PAGE (Laemmli
1970) and then transferred to a Hybond-P membrane
Results
Distribution of GR in the adult rat eye tissues
Figure 1 shows an immunoblot of an extract of a whole eye
from a 21-weeks old rat using the antibody raised against
recombinant rat GR (Fujii et al. 2000). This antibody only
detected the 50 kDa protein corresponding to rat GR on the
blot. Thus the antibody was found to be specific for GR. We
then applied the antibody to immunohistochemical studies of
the adult rat eye tissues. GR was distributed in the cornea and
conjunctival epithelia, corneal keratocytes, corneal endothelium, iridial and ciliary epithelia, neural retina, and retinal
pigment epithelium (RPE) (Figure 2) in comparison with
the negative control (data not shown). A strong staining was
observed in the corneal epithelium but none was observed in
the substantia propia (Figure 2A). Entire regions of the iridial
Glutathione reductase in rat eye
and ciliary tissues were also stained strongly. Of the retina,
the rod outer segments were visualized with the specific
monoclonal antibody H16 (Yoshida et al. 1991) (Figure 2B).
Staining with anti-GR IgG was observed to be strong in the
pigment epithelium (RPE), the subretinal space, and ganglion
cells (GC) and, to a lesser extent, in the inner (IP) and outer
(OP) plexyform layers, but not in the outer (ONL) and inner
nuclear layer (INL) (Figure 2C). Thus the expression of GR
was evident not only in epithelial tissues which are exposed
directly to molecular oxygen, but also in cells which are intercalated in the visual system, which are apparently important
for transmission and integration of visual signals.
269
Developmental changes of the GR activity in
the eye tissues
The levels of GR activities and proteins in the eye tissues
of fetal (E19) and postnatal rats (P0–P16) were examined
to determine whether they become altered in the course of
the development of the eye or with respect to environmental changes. Figure 3 shows changes in the activity of GR
in the soluble fractions of extracts of whole eyes. The enzymatic activity was evaluated to be the highest at the stage
E19 and then gradually decreased thereafter. When examined by immunoblot analysis, the level of GR protein virtually paralleled the activity (Figure 3, inserted picture).
Thus, the contribution of GR protein to whole proteins in
eye tissues decreased continually over the late developmental
stages.
Expression of GR during the development of
the retina
Figure 1. Immunoblot of an adult rat eye with anti-GR IgG. 30 µg of a
homogenate of a whole eye from a 21-weeks old rat was subjected to 10%
SDS-PAGE and then blotted onto the Hybond-P membrane. Western blot
analysis was performed with 1 : 500 dilution of anti-GR IgG. Arrowhead
indicates the position of GR (50 kDa).
Since the contribution of the lens and vitreous body is
increased in volume in developed eyes, a detailed immunochemical analysis is required to evaluate the roles of GR in
pivotal tissues such as the retina. In the embryonic (E19)
to early postnatal (P3) rats, outer and inner nuclear layers were not yet separated. Thereafter, the differentiation
of the inner and outer nuclear layers occurs at a period of
3–6 days, and extends from the central to the peripheral
retina (Weidman & Kuwabara 1968, Obata & Usukura 1992,
Yamashita et al. 1994). The differentiation is accomplished
at about the age of 6 days (P6). The expression of GR was
observed immunohistochemically at E19 to the adult eyes as
Figure 2. Immunohistochemical staining of the adult eye with the anti-GR IgG. Sections of a 16-weeks old rat eye were treated with the IgG specific
for rat GR at a 1 : 200 dilution as the primary antibody (A and C). The retina was reacted with the monoclonal antibody H16 (B) and visualized
with DAB. Regions of the cornea, iris, and ciliary body (A, ×200) and retina (B and C, ×400) are shown. Photographs were obtained by a light
microscope equipped with a Nomarski interference-contrast device. (A) Bar; 50 µm. (B) and (C) Bar; 25 µm.
270
T. Fujii et al.
Figure 3. Changes in GR activities and proteins at the pre- and post-natal
stages. GR activity of the whole eye homogenate was assayed under standard conditions. Means and standard deviations of triplicate experiments
at each time point are shown. An inserted picture shows an immunoblot
of tissue homognate from eyes at indicated stages with anti-GR IgG.
Table 1. GR immunoreactivity in the developing rat eye. Strongly positive (the order is; + < ++ < +++), weakly positive (±), negative (−).
Blank means that the tissue is undifferentiated yet.
Age
Retina
GC
IP
INL
OP
ONL
PL
RPE
Cornea
Lens
Iris
E19
P0
P3
P6
P9
P12
P16
P21
±
+
±
+
+
±
++
±
±
++
+
++
+
−
+
−
+
±
+++
++
++
++
+
−
+
−
+
++
+++
++
++
+++
+
−
+
−
+
++
+++
++
++
+++
++
−
++
−
+
++
+++
++
++
+++
++
−
++
−
+
+++
+++
±
+++
++
±
GC, ganglion cell layer; IP, inner plexyform layer; INL, inner nuclear
layer; OP, outer plexyform layer; ONL, outer nuclear layer; PL, photoreceptor layer; RPE, pigment epithelium; Cornea, corneal epithelium;
Lens, lens epithelium; Iris, iridial epithelium and stroma.
summarized in Table 1. GR expression was first detected in
the ganglion cell layer (GC) at the embryonic stage, and thereafter GR appeared in the IP and the OP at the ages of P0 and
P6, respectively. Figure 4 shows immunohistochemical data
of typical retina at P3 and P21. Along with the differentiation
of the INL and ONL, GR expression became detectable in
the OP at P6. RPE was positively stained after the age of P0.
GR expression was detected in the lens epithelium from E19
through P16, but was considerably decreased in the adult eyes.
Discussion
The lens epithelium contains several antioxidative enzymes,
such as superoxide dismutase, catalase, and glutathione
peroxidase, and is protected from oxidative damage caused
by light and oxygen (Reddy 1990, Spector 1995). Levels of
mRNAs for GR, glutathione peroxidase, catalase, and Cu,
Zn-SOD were examined by Northern blotting and an RNA
protection assay for the case of RNA extracted from a lens
homogenate (Shi & Bekhor 1994). However, in most cases
their levels were evaluated based on individual enzymatic
activity alone. Although the retina is important for receiving
and transducing photochemical stimuli, virtually no attention has been paid to it with respect to GSH and GR. The
new, specific antibody (Fujii et al. 2000, 2001a) enabled us
to investigate GR in discrete eye tissues, including the retina,
for the first time.
Types of cells that exhibited a high level of expression of
GR are multiple. The epithelium of the cornea is directly
exposed to light and molecular oxygen and thus would be
expected to undergo exogenous oxidative damage. Iris is also
directly exposed to light but not ambient oxygen. Ciliary
body is not exposed either, but would be suffered from intracellular oxidative stress due to contraction accompanied by
energy consumption. GR expression in lens epithelium was
low at prenatal stage, increased after birth, and then decreased
after 21 days to adult. GR immunoreactivity in retina was
increasing after P6 (Table 1). GR activity, on the other hand,
decreased at P3 and P6, increased at P9, and decreased again
thereafter. Since specific activity of GR was determined under
the balance of total proteins and GR protein in the tissue, the
increase in the activity at P9 may be due to the increased
expression of GR in retina. Ganglion cells and pigment
epithelial cells also express high levels of GR. It is likely that
a number of additional experiments will be required to complete the causal linkage of GR expression and the impulse
formation of ganglion cells or the phagocytic action of the
pigment epithelium.
A number of reports dealing with the content and role
of GSH and related enzymes in the lens have appeared
(Giblin et al. 1990, Reddy 1990, Spector 1995, Reddan
et al. 1999). The overexpression or knockout of GPX1,
which encodes the cytosolic form of selenium-containing
glutathione peroxidase, effects the response of the lens epithelium to hydrogen peroxide and other oxidative stress to only
a negligible extent (Spector et al. 1996, 1998, Ho et al.
1997, Reddy et al. 1997). While GPX1 accounts for only
about 15% of the detoxification of hydrogen peroxide, other
GSH-dependent systems appear to be involved in about
54%–72% this detoxification, depending on the hydrogen
peroxide concentrations used in the assay (Spector et al.
1997). Although GPX1 was not detected as the result of
an immunogold-marking study in the human iris (Marshall
1997), a high level of expression of GR was found in the
iris of the rat. Recently, a non-selenium-containing GSHdependent peroxidase was identified and purified from the
bovine ciliary body (Shichi 1990, Singh & Shichi 1998). The
enzyme, which is now referred to as peroxiredoxin 6, is able
to reduce alkyl hydroperoxides as well as hydrogen peroxide in a GSH-dependent manner (Fisher et al. 1999, Fujii
et al. 2001b), although Kang et al. (1998) failed to implicate GSH in providing a reducing equivalent to this enzyme.
The localization of GR in the ciliary body matched that of
Glutathione reductase in rat eye
271
Figure 4. Developmental changes of GR expression in the retina. Tissue sections were treated with the IgG reactive to rat GR at 1 : 200 dilution. The
retinas at 3 days (A) and 21 days (B) are shown. Photographs were obtained by a light microscope equipped with a Nomarski interference-contrast
device (×280). Bar; 25 µm.
peroxiredoxin 6. In this context, the possibility that GSH
functions as an electron donor to the peroxidase activity of
peroxiredoxin 6 is supported. Moreover, GSH, which accumulated at a high level in the vitreous body might have been
secreted from surrounding tissues, after reduction by GR.
Low GPX1 and GR activities in adult lens indicate that the
lens is not well protected by these enzymes. However, the lens
is one of the richest tissues in GSH content and, under normal conditions, GSH can protect the tissue nonenzymatically
against oxidative stress.
Since oxidative stress increases markedly after birth and
the opening of the eye, antioxidative enzymes would be
induced during these stages. Of the antioxidant enzymes,
SOD has been the most extensively studied, and an augmented expression of SOD during this process has been
emphasized (Yamashita et al. 1994, Ogawa et al. 1997). The
increase in retinal SOD activity occurs almost simultaneously with the opening of the eye. During postnatal development, GR activities and the corresponding proteins indicated
a less evident expression of GR as compared with other proteins. Such an expression was actually augmented in some
tissues such as the corneal epithelium, retina, and pigment
epithelium. Hence, a compartmentalized expression of this
enzyme in the tissues mentioned above seems to be even
more significant for the function of the eye. Since the inner
and outer segment layers, as well as the pigment epithelium
absorb photoenergy, which triggers the release of reactive
oxygen species, GR would be essential in supplying GSH
for protection against such harmful molecules by both enzymatic and nonenzymatic reactions. In the human, Mn-SOD
activity was higher in the adult than in the fetus. Conceivably this increase is related to a preventive mechanism
against oxidative damage (Yamashita et al. 1994, Ogawa et al.
1997).
High activities at the late prenatal stage suggest an additional function of the GSH/GR system, in addition to a mere
antioxidative role. A putative role of GSH in the redox regulation of the neuronal function has actually been proposed
(Bains & Shaw 1997). The significance of GSH in signal
transduction via the modulation of redox-sensitive receptor
molecules has been postulated in the central nervous system
(Janaky et al. 1999). Thus GR may also participate in maintaining cellular functions and signal transduction in the retina,
in addition to its role in the protection of cells from oxidative stress and harmful cytotoxic compounds. Consequently,
the GSH/GR system, especially in the retina, is likely to be
important both for detoxification reactions of reactive oxygen
species as well as for the redox regulation of a certain cellular
function(s) of the eye.
Acknowledgements
We thank the staff of the Laboratory Animal Center,
Yamagata University School of Medicine, for housing and
caring for the rats. This work was supported, in part, by
a Grant-in-Aid for Scientific Research (C) (No. 13670111)
from the Ministry of Education, Culture, Sports, Science, and
Technology, Japan and by the Nakatomi Foundation, Japan.
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