全 文 ：© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
OCCURRENCE OF INDUCIBLE NITRIC
OXIDE SYNTHASE IMMUNOREACTIVE
MATERIAL IN THE RETINA OF THE
CLAWED FROG, Xenopus laevis
Huang Shile1 　Hubert H. Kerschbaum2 　Chen Zuyi1 　Anton Hermann2
( College of Science , Nanjing A gricult ural U niversity , Nanjing 210095 , PR China1)
( Instit ute of Zoology , U niversity of S alzburg , Hellbrunnerst r. 34 , A25020 S alzburg , A ust ria2)
此文于 1997 年 5 月 27 日收到。
Supported by Austrian FWF ( P9247 to A. Hermann) , Austrian National Bank ( P5389 to A. Hermann) and the Medical Re2
search Coordination Center , University of Salzburg , Austria ( P95/ 992101 to S. Huang) .
　　NOS immunocytochemistry , using specif ic antibodies to NOS Ⅱand Ⅲ, was used to
local ize NOS2containing structures in the retina of adult Xenopus laevis . Photoreceptor
ell ipsoids, occasional somata of gangl ion and amacrine cells contained NOS Ⅱ im2
munoreactive material . Müller cells exihibited intense NOS Ⅱ immunoreactivity. The
neurites in the outer plexiform layer were also intensely immunolabeled for NOSⅡ. No
NOSⅢ immunoreactivity was found in the cellular structures of Xenopus retina. The
results suggest that NO , generated by inducible NOS Ⅱmay be involved in the media2
tion of visual transduction.
Key words : Nitric oxide synthase , retina neuron , immunocytochemistry , isoform ,
localization
Introduction
Nitric oxide (NO) , a labile , diffusible and free radical gas and generated by NO synthase
(NOS) , acts as a signaling molecule in nervous , vascular and immune system[5 ] . Biochemical and
molecular biological evidence reveals that there are at least two types and three isoforms of NOS :
one is constitutive (cNOS) , calcium/ calmodulin dependent and contains brain ( bNOS) or neu2
ronal (nNOS or NOS I) and endothelial (eNOS or NOS Ⅲ) isoforms mainly from neuronal and
endothelial tissues respectively , and another one is inducible (iNOS) , calcium/ calmodulin inde2
pendent and primarily f rom macrophages (mac NOS or NOS Ⅱ) [5 ] .
No modulates visual t ransduction in vertebrates[1 ,11 ] . Using immunocytochemistry for NOS
or histochemistry for NADPH2diaphorase , a marker enzyme of NOS , NOS2containing cells have
been found in the retina of different vertebrates[2 ,9 ,10 ,12 ,13 ] . The majority of studies have focused
on the cellular localization of NOS I in the retina[2 ,12 ] , which have demonstrated that NADPH2di2
403　 核 农 学 报　1998 ,12 (5) :304～308Acta A gricult urae N ucleatae Sinica
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
aphorase histochemical staining colocalizes with NOS I immunoreactive material in the retina.
However , occurrence of other isoforms of NOS can not be excluded , since NADPH2dpaphorase
histochemistry can not distinguish NOS I from NOS Ⅱ or Ⅲ, in the different cells or tis2
sues
[2 ,6 ,15 ]
. Recently , NOS Ⅱ isoform has been found in hippocampal pyramidal cells in the
mouse hippocampus[4 ] . Moreover , normal human retina expresses both constitutive and inducible
isoforms of NOS mRNA[14 ] . Therefore , in this study we check whether there is NOS Ⅱand Ⅲ
in the normal retina of the clawed frog , Xenopus laevis .
Materials and Methods
Materials 　Adult Xenopus laevis were kept in tapwater at room temperature , and fed with
liver and heart once or twice a week. Adult animals were used several months after metamorpho2
sis. Their length was about 6 cm snout2vent . All chemicals used were perchased from Sigma un2
less otherwise stated.
Tissue preparation 　Adult Xenopus laevis were anaesthetized with MS2222 and decapitated.
Eyes were at once removed , fixed in Bouin’s solution for 2 h at 4 ℃, then washed in 011 mol/ L
phosphatebuffered saline ( PBS p H 714) , dehydrated in a graded series of ethanol and embedded in
paraffin using xylene as an intermedium. Serial sections (7μm thick) were cut and mounted on
poly2D2lysine2coated slides.
Immunocytochemistry 　NOS immunoreactive material was localized using the peroxidase an2
tiperoxidase method as previously described[8 ] . Briefly , the paraffin sections were dewaxed in xy2
lene and rehydrated in a descending series of ethanol. The rehydrated paraffin sections were rinsed
3 ×5 min in 011 mol/ L PBS (p H714) and incubated in normal goat serum (1∶30 , Dakopatts ,
Denmark) for 30 min at room temperature , followed by incubation with the primary antibodies ,
i. e. affinity purified rabbit polyclonal antibodies to human NOS Ⅲ (1∶250) and mouse NOS Ⅱ
(1∶125) ( Transduction Laboratories , Kentucky , USA) overnight at 4 ℃. Thereafter , sections
were rinsed 3 ×5 min with 011 mol/ L PBS (p H714) and incubated in goat anti2rabbit antiserum
(1∶100 , BioMaker , Rehovot , Israel) for 30 min at room temperature , rinsed 3 ×5 min in 011
mol/ L PBS (p H714) and incubated in rabbit horseradish peroxidase2conjugated antibody (1∶100 ,
Dakopatts) for 30 min at room temperature. Peroxidase was visualized with 3 , 3’2diaminobenzi2
dine2HCL (015 mg/ ml , Aldrich) and 0103 % H2O2 (v/ v) in 011 mol/ L PBS (p H714) . After
washing in PBS , the sections were dehydrated and mounted. For controls , sections were incubat2
ed with normal rabbit serum in place of antibodies against isoforms of NOS. Replacement of rabbit
anti2NOS antibodies by normal rabbit serum failed to stain the cells.
Results
In the retina of adult Xenopus laevis existed srong NOS Ⅱimmunoreactivity , whereas NOS
Ⅲimmunoreactivity did not occur. The intensity of NOS Ⅱ immunoreactivity varied widely a2
503　5 期 爪蛙视网膜中诱导型一氧化氮合成酶免疫反应物质的存在 (英文)
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
mong the subpopulation of retinal cells (plate Ⅰ) . In the outer nuclear layer (ONL) , weak2to2
moderate mNOS immunoreactivity was found in the rod and cone photoreceptor ellipsoids (plate
Ⅰ21) . In the outer plexiform layer (OPL) , neurites were intensely stained. In the inner nuclear
layer ( INL) , the somata of presumptive amacrine cells were occasionally immunolabelled. As de2
scribed in rabbits[13 ] , two types of amacrine cells were present . One was large and intensely stained ,
Plate Ⅰ
Horizontal sections show NOS Ⅱimmunoreactivit y in the retina of the clawed frog , Xenopus laevis . Both rod (small ar2
rowhead) and cone (large arrowhead) photoreceptor ellipsoids are immunolabelled (plate Ⅰ21) . In the inner nuclear layer exist
two types of amacrine cells : the large intensely stained cell (large arrowhead) and the smaller weakly stained cell (small arrow2
head) (plate Ⅰ22) . A cell body (arrowhead) in the ganglion cell layer is also immunostained (plate Ⅰ23) . Müller cells (ar2
rows) and the processes in the outer plexiform layer contain intense NOS Ⅱimmunoreactivity (plate Ⅰ21 and 4) . Scale bars : .
1～4 , 10μm.
whereas the other type smaller and weakly stained (plate Ⅰ22) . The ganglion cell layer ( GCL )
603 核　农　学　报 12 卷
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
showed also NOS Ⅱimmunoreactive putative displaced amacrine cells or ganglion cells (plate Ⅰ2
3) . The cell bodies in the GCL were of diverse size and showed different intensities of immunore2
activity. Müller cells , glia spanning in the retina , posessed intense immunolabeled radial processes
f rom the OPL to the INL , occasionally even through the IPL to the GCL (plate Ⅰ21 and 4) .
The dist ribution of NOS Ⅱimmunoreactivity was not always uniform. Within the amacrine cells
and ganglion cells only the cell body was labeled while the neurites were not immunoreactive.
Discussion
In this study , we localize NOS Ⅱimmunoreactivity in retinal neuronal cells , including pho2
toreceptor cells , amacrine and ganglion cells , and retinal glial cells (Müller cells) in the retina of
Xenopus laevis , which is in agreement with the molecular biological results in human retina[14 ] .
Using reverse transcription polymerase chain reaction ( RT2PCR) , Park and coworkers[14 ]
demontrated that human retina contained brain type , rbNOS and inducible type , riNOS , which
had almost the same cDNA sequences as human/ rat brain NOS and human chondrocyte iNOS , re2
spectively. In this study , we did not found NOS Ⅲimmunoreactivity in the Xenopus retina , al2
though the ocular blood vessels of Xenopus expressed intense immunoreactivity for NOS Ⅲ (data
not shown) . Taken together , in the vertebrate retina the constitutive NOS may only be att ribut2
ed to NOS I isoform , which has been identified in different vertebrates[2 ,12 ] .
Evidence shows that NO may be a signal messenger in visual t ransduction[1 ,11 ] . It is general2
ly considered that the role of NO in the nervous system is due to NOS Ⅰisoform , and that iNOS
is responsible for the synthesis of NO , mediating cytotoxic effects[5 ] . Experimentally , iNOS , in2
deed , was expressed by treatment of cytokine or endotoxin in retinal Müller cells[7 ] . Clinically , in
the retina of AIDS patients high levels of NO production has been observed , particularly in Müller
cells[3 ] . However , in the previous research[14 ] and this study , iNOS isoform was found in the
retina , without t reatment of cytokines or endotoxin , or other pathological stimuli. Moreover , we
demonstrated NOS Ⅱimmunoreactivity not only in Müller cells but also in subpopulation of reti2
nal neurons , implying that NO produced by iNOS in the retina might , besides as one of cytotoxic
factors , play a modulatory role in visual t ransduction as well.
References
1 　Ahmad Ⅰ, Leinders2Zufall T , Kocsis J , Shepherd GM , Zufall F , Barnstable CJ . Retinal ganglion cells express a c GMP2gated
cation conductance activatable by nitric oxide donors. Neuron , 1994 , 12 :155～165
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10 　Kurenni DE , Thurlow GA , Turner RW , Moroz LL , Sharkey KA , Barnes S. Nitric oxide synthase in tiger salamander retina.
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13 　Osborne NN , Barnett NL , Herrera AJ . NADPH diaphorase localization and nitric oxide synthase activity in the retina and an2
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14 　Park C2S , Pardhasaradhi K , Gianotti C , Villegas E , Krishna G. Human retina expresses both constitutive and inducible iso2
forms of nitric oxide synthase mRNA. Biochem Biophys Res Conmun , 1994 ,205 :85～91
15 　Tracey WR , Nakane M , Pollock J S and FÊrstermann U. Nitric oxide synthases in neuronal cells , macrophages and endotheli2
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爪蛙视网膜中诱导型一氧化氮合成酶免疫反应物质的存在
黄世乐1 　H. H. kerschbaum2 　陈祖义1 　A. Hermann2
(南京农业大学理学院 ,南京 2100951
奥地利萨尔茨堡大学动物研究所 ,萨尔茨堡 ,奥地利2)
借助 Ⅱ和 Ⅲ型一氧化氮合成酶 (NOS)特异性抗体对成年爪蛙 ( Xenopus laevis ) 视网膜中含 NOS 的结构
进行了免疫细胞化学定位研究。发现视杆和视锥细胞的视网膜杆、少数神经节细胞和无足细胞的细胞体均含
有 NOS Ⅱ免疫反应物质 ,缪勒细胞呈现强的 NOS Ⅱ免疫反应性 ,外神经丛层的神经突起亦含强的 NOS Ⅱ免疫
反应性。在视网膜的细胞结构中 ,未见 NOS Ⅲ免疫反应性。该结果暗示由 NOS Ⅱ产生的一氧化氮可能参与
视觉传导的调节。
关键词 :一氧化氮合成酶 　视网膜 　神经元 　免疫细胞化学 　异型 　定位
803 Acta A gricult urae N ucleatae Sinica
1998 ,12 (5) :304～308