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Journal of Electron Microscopy 47(5): 527-533 (1998)
© 1998 Oxford University Press

Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry

Toshihiro Takizawa1,*, Takami Takizawa1,2, Hideaki Iwasaki1 and Takuma Saito1

1Department of Anatomy, Jichi Medical School 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan
2Cosmetics Laboratory, Kanebo Ltd 5-3-28 Kotobuki-cho, Odawara-shi, Kanagawa 250-0002, Japan

*To whom correspondence should be sent. E-mail: ttakizawa{at}jichi.ac.jp

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultra-structure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light-adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin {alpha} subunit, thereby modulating light adaptation in rod cells.

Keywords     rat retinas, phototransduction, light adaptation, cGMP-phosphodiesterase, transducin GTPase, rapid-freezing enzyme cytochemistry

Received     30 April 1998, accepted 7 July 1998


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