Improved cytochemical method for cyclic 3',5'-nucleotide phosphodiesterase in rat rod outer segments.

Tajima, Toshihiro; Saito, Takuma

doi:10.1267/ahc.25.697

Cited by 1 publication

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“…The membrane-bound 5 Ј-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 (Yamada 1982). The results are in good agreement with the biochemical analyses mentioned above and our previous observations that the localization of other cGMP-related enzymes (i.e., guanylate cyclase and cGMP phosphodiesterase) for the phototransduction system is in the extradiscal space (Saito and Takizawa 1991;Takizawa and Saito 1992).…”

Section: Discussionsupporting

confidence: 89%

5′-Nucleotidase in Rat Photoreceptor Cells and Pigment Epithelial Cells Processed by Rapid-freezing Enzyme Cytochemistry

Tajima

1998

J Histochem Cytochem.

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SUMMARYThis report describes the subcellular distribution of 5 Ј-nucleotidase (5 Ј-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 Ј-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme cytochemistry, 5 Ј-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 Ј-NT molecules was also found. The soluble 5 Ј-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5 Ј-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. 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 (Klaushofer and von Mayersbach 1979; Saito 1989,1992;Robinson and Karnovsky 1991;Saito and Takizawa 1991).In retinal photoreceptor cells, the enzyme 5 Ј-nucleotidase (5 Ј-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 Ј-NT in retinal rod cells processed by conventional fixation (Kreutzberg and Hussain 1984;Hussain and Baydoun 1985;Saito 1991), they have not been in good agreement on its localization. One of the reasons is that rod cells are one of the most delicate cells to treat by conventional fixation and morphological modifications during fixation are often encountered. Yamada (1982) showed the fine structure of intact discs in the rod outer segment processed by rapid-freezing and subsequent freezesubstitution fixation. Therefore, it would be interesting to detect 5 Ј-NT activity in freeze-substituted photoreceptor cells. This article presents the ultrastructural localization of 5 Ј-NT in rat photoreceptor cells an...

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Section: Discussionsupporting

confidence: 89%