TECHNICAL NOTE |
Correspondence to: Toshihiro Takizawa, Dept. of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: ttakizawa@jichi.ac.jp.
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Summary |
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This report describes the subcellular distribution of 5'-nucleotidase (5'-NT) in rat photoreceptor cells and pigment epithelial cells processed by rapid-freeze enzyme cytochemistry. There was a striking difference in the ultrastructural localization of 5'-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme cytochemistry, 5'-NT activity was localized in the extradiscal space of intact nonvacuolated discs, whereas by conventional cytochemistry it was shown in the intradiscal space of artifactual vacuolated discs. In the freeze-substituted retinal cells, an appreciable difference in functional 5'-NT molecules was also found. The soluble 5'-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active in the pigment epithelial cells. Rapid-freezing enzyme cytochemistry should be worth employing as a method to reveal the fine localization of enzyme activity at the level of cell ultrastructures, which are poorly preserved by conventional fixation, and should provide information approximate to that in living cells. (J Histochem Cytochem 46:10911095, 1998)
Key Words: rapid freezing, freeze-substitution fixation, enzyme cytochemistry, 5'-nucleotidase, cGMP-related enzymes, rat retinas, photoreceptor cells, pigment epithelial cells, electron microscopy
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Introduction |
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Electron microscopic enzyme cytochemistry is a morphological technique for analyzing topology and dynamics of enzymes in situ. Although chemical fixation is always essential in this technique, immobilization of cell ultrastructures containing enzyme molecules by conventional fixation often reveals contradictory preservation of enzyme activity (i.e., inactivation of enzyme activity vs artifactual diffusion of enzyme activity). Freeze-substitution fixation is a technique that overcomes the obstacles of conventional fixation. Rapid-freezing enzyme cytochemistry, which combines rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry, enables us to reveal the precise localization of enzymes on well-preserved cell structures (
In retinal photoreceptor cells, the enzyme 5'-nucleotidase (5'-NT) is one of the enzymes in the cGMP metabolism cascade of the vertebrate visual transduction system. Although some researchers have studied the enzyme cytochemical localization of 5'-NT in retinal rod cells processed by conventional fixation (
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Materials and Methods |
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Reagents
The following chemicals were purchased from Sigma (St Louis, MO): adenosine 5'-monophosphate (5'-AMP), guanosine 5'-monophosphate (5'-GMP), levamisole, and adenosine 5'-[,ß-methylene]diphosphate (AOPCP). Glutaraldehyde (25% and 70% aqueous) and lead nitrate were obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals were of the highest grade available.
Rapid-freezing Enzyme Cytochemistry
Enzyme cytochemical studies of 5'-NT in the retina were performed primarily on SpragueDawley rats. Additional experiments performed on the frog (Xenopus laevis) retina gave basically similar results. This report therefore focuses on results obtained in the rat.
Thirty-six adult male SpragueDawley rats (200300 g) were used. The eye of the rat was removed under sodium pertobarbital anesthesia and then dissected along the equator in Hanks' solution. Subsequently, the neural retina, which is composed of the layers from the rod outer segment to the optic nerve fibers, was gently separated from the pigment epithelium. Within a minute after removal from the animal, the neural retina or the pigment epithelium was rapidly frozen on a liquid nitrogen-cooled copper block in a Polaron E7200 Slammer quick-freezing apparatus. After rapid-freezing, the frozen samples were freeze-substituted as described previously (
After freeze-substitution, the samples were rehydrated by gradually adding 0.1 M cacodylate buffer, pH 7.2, with 6% sucrose and then rinsed three times in the same buffer. Forty-µm sections were made with a Dosaka EM DTK-3000 Microslicer (Osaka, Japan) or a Komatsu Electronics MB201 freezing microtome (Tokyo, Japan). The sections were then incubated in a lead-containing reaction medium for detection of 5'-NT activity, a modification of the procedure of
Conventional Enzyme Cytochemistry
To compare the effect of freeze-substitution fixation on localization of 5'-NT with that of conventional chemical fixation, immersion fixation was carried out as a parallel experiment. Some samples were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) with 6% sucrose for 30 min at 4C before the cytochemical reaction. In this case, the medium and the incubation parameters (time, temperature, mixing conditions) were identical to those mentioned above.
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Results |
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In the samples processed by immersion fixation, 5'-NT activity was detected both in the rod outer segment of the photoreceptor cells and on the apical processes (microvilli and other protrusions) of the pigment epithelial cells (Figure 1C and Figure 1G). The reaction product of 5'-NT activity was localized in the intradiscal space of the vacuolated discs of the rod cells (Figure 1C) and on the outer surface of the apical processes of the pigment epithelial cells (Figure 1G). Thus, the reaction product indicating 5'-NT activity was associated primarily with the exoplasmic side of the cell membranes of both cells.
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In the samples processed by rapid-freezing and subsequent freeze-substitution fixation, cell ultrastructure, especially the rod outer segment, was well preserved. Many discs were compactly packed and uniformly flattened (Figure 1A and Figure 1B). It is noteworthy that the intradiscal space was hardly recognizable in the freeze-substituted samples (Figure 1B), whereas it was easily identified in the conventionally fixed retina (Figure 1C). These observations are consistent with earlier morphological findings suggesting that an intact disc is flat (
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Discussion |
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5'-NT is widely distributed in a number of cells and tissues. Animal 5'-NT has been classified into two major forms according to its biochemical properties: a membrane-bound form and a soluble form (for review see
A striking difference in the subcellular localization of 5'-NT between the rod outer segment processed by freeze-substitution fixation and that fixed conventionally by immersion is shown in this study (see Figure 1B and Figure 1C). The reason could be that detection of 5'-NT activity and morphological alterations are affected by conventional fixation. The results showing that 5'-NT activity was present on the cytoplasmic side of the freeze-substituted disc membrane and not on the exoplasmic side can be explained by assuming that the cytochemically visible 5'-NT activity represents more functional activity of the soluble 5'-NT. In addition, the membrane-bound 5'-NT activity was detected not in the discs but on the apical processes of pigment epithelial cells (see Figure 1B and Figure 1F). The membrane-bound 5'-NT activity in discs may be inactive in situ and different from that on the apical processes of pigment epithelial cells because each disc membrane is not connected directly to the plasma membrane and the lumen of the disc was almost obliterated (
The question of cytochemical heterogeneity of 5'-NT in the other portions of photoreceptor cells remains to be answered (
This study presents the localization of 5'-NT activity in the outer segment of the rod cells and in the apical portion of the pigment epithelial cells processed by rapid-freezing enzyme cytochemistry. In the rod outer segments, the soluble 5'-NT on the cytoplasmic side of the disk membrane was vital, whereas in the pigment epithelial cells the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active. Rapid-freezing enzyme cytochemistry is a recommended approach for detection of other enzymes associated with subcellular structures that are not well preserved by conventional fixation, and can provide information about cells approximating, as closely as possible, the conditions in living cells.
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Acknowledgments |
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Supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan.
I am grateful to Dr Takuma Saito of Jichi Medical School for critical comments on this manuscript. I wish to thank Ms Kiyomi Inose, Ms Chiaki Ishijima, and Ms Megumi Yatabe for excellent technical assistance.
Received for publication April 15, 1998; accepted May 5, 1998.
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