Topology of the Outer Segment Membranes of Retinal Rods and Cones Revealed by a Fluorescent Probe

Abstract

When N,N'-didansyl cystine binds to the cell membranes of vertebrate rods and cones its fluorescence efficiency increases about 20-fold. The entire outer segments of living cones become brilliantly fluorescent. Stained live rods, as well as most freshly detached outer segments, are only weakly fluorescent, but they become brightly fluorescent within a few seconds if their plasma membranes are osmotically ruptured. The difference in staining of rod and cones suggests that disk membranes of rods are not continuous with the plasma membranes are osmotically ruptured. The difference in staining of rod and cones plasma membrane on outer segments of photoreceptors in electrophysiological and biochemical experiments, and to study the infolding pattern of rod and cone disks.

Rhodopsin-to-metarhodopsin II transition triggers amplified changes in cytosol ATP and ADP in intact retinal rod outer segments.

“…Bullfrogs (Rana catesbiana) were dark-adapted for [12][13][14][15][16][17][18][19][20][21][22][23][24] hr, and the retinas were dissected free from the eyecup and pigment epithelium in aerated Ringer solution under infrared illumination, with the aid of image converters. ROS were harvested by gentle shaking and were used as soon after isolation as possible (=1-2 min).…”

“…It is believed that the combination of immediate use of ROS after isolation and the elimination of washing and purification procedures help to make processes studied in the isolated intact ROS relevant to the biochemistry of ROS in functional intact rod photoreceptors. Typically 80-90% of ROS isolated in this manner were judged to be osmotically intact by using the didansylcysteine fluorescent staining procedure (13), and inner segment saccules numbered <10% of outer segments present as judged by Janus Green B staining (14). Aliquots (200 1LI) of Ringer solution (100 mM NaCl/5 mM NaHCO3/3.5 mM KCl/ 0.18 mM CaCl2/2 mM MgCl2/20 mM glucose/20 mM Tris, pH 7.7) containing =104 ROS, exposed to light or kept in darkness, were quenched by rapid injection into 2 ml of boiling Tris buffer (20 mM) for 2 min.…”

Fundamental Studies in the Molecular Basis of Laser Induced Retinal Damage

“…Phallicidin can be introduced into live animal cells by permeabilizing the plasma membrane with lysolecithin (14) or by pinocytosis (13). Since fluorescent dye can be introduced into rod cells by vesicle fusion (20), it is possible that actin can be fluorescently labeled by these techniques in intact functioning photoreceptors.…”

“…Fluoride ions (13)(14)(15)(16)(17)(18) vanadate (19) and molybdate (20,21) with brief centrifugation times to avoid increasing the dark PDE activity (2). Isolation procedures were carried out at 4 0 C or with reagents on ice using infrared illumination (.>750 nm) and an image convertor (NI-Tec, Inc., Niles, IL).…”

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors.

“…The pellet was resuspended in pseudo-intracellular medium, and the cell suspension incubated for 20 rain at room temperature in total darkness to permit the loss of > 95% of the endogenous nucleotides. The efficacy of electropermeabilization was ascertained by epifluorescent light microscopy of EP-ROS cells incubated with the membrane-impermeant dye, didansylcysteine (Yoshikami, Robinson, and Hagins, 1974); in all experiments, > 80% of the total EP-ROS population were made permeable to the fluorescent dye by the electropermeabilization conditions cited above. The morphology of EP-ROS, as judged by phase contrast microscopy, was indistinguishable from cell suspensions which had not undergone the electropermeabilization procedure.…”