Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina

Abstract

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.