Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark...

Frog rod outer segments swell slowly after being shaken from an excised retina into a modified Ringer's solution. The swelling has the following characteristics: (a) It is suppressed by illumination which bleaches only 500 rhodopsin molecules per outer segment per second. This is approximately the level required to saturate the in vivo receptor potential. (b) Light suppression is seen in NaCl but not in KCl solutions. (c) Dark swelling is labile and is enhanced by calf serum, low calcium concentrations, dithiothreitol, and cyclic nucleotide phosphodiesterase inhibitors. (d) Lowering the pH to 5.5 or removing magnesium reversibly reduces dark swelling to the same extent as illumination. (e) The amount of light required for maximal suppression of dark-swelling increases approximately 10-fold if the calcium concentrations is lowered by EGTA addition. (f) The effect of illumination is irreversibly abolished by antimycin and other inhibitors of mitochondrial electron transport. (g) A process analogous to dark adaptation in vivo can be observed: If 10-50% of the rhodopsin present is bleached and the outer segments are then kept dark, rapid dark swelling returns after a period of 15-45 min. This swelling is again sensitive to light. We tentatively ascribe the light suppression of swelling to the same decrease in sodium permeability which is observed on illuminating living receptor cells. The experiments suggest that outer segments retain their competence to perform both transduction and dark adaptation after their separation from the retina.