Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry.
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 ultrastructure 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.
Takizawa
T
T
Department of Anatomy, Jichi Medical School, Tochigi, Japan. ttakizawa@jichi.ac.jp
Takizawa
T
T
Iwasaki
H
H
Saito
T
T
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation; D005615:Freezing; D006651:Histocytochemistry / Q000379:methods; D008297:Male; D010775:Photic Stimulation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology* / Q000648:ultrastructure
D015106:3',5'-Cyclic-GMP Phosphodiesterases