KINETICS OF ROD OUTER SEGMENT RENEWAL IN THE DEVELOPING MOUSE RETINA

The kinetics of rod outer segment renewal in the developing retina have been investigated in C57BL/6J mice. Litters of mice were injected with [³H]amino acids at various ages and killed at progressively later time intervals. Plastic 1.5 µm sections of retina were studied by light microscope autoradiography. The rate of outer segment disk synthesis, as judged by labeled disk displacement away from the site of synthesis, is slightly greater than the adult level at 11–13 days of age; it rises to more than 1.6 times the adult rate between days 13 and 17, after which it falls to the adult level at 21–25 days. The rate of disk disposal, as measured by labeled disk movement toward the site of disposal, is less than 15% of the adult level at 11–13 days of age; it rises sharply to almost 70% of the adult level by days 13–15 and then more gradually approaches the adult rate. The net difference in rates of synthesis and disposal accounts for the rapid elongation of rod outer segments in the mouse between days 11 and 17 and the subsequent, more gradual elongation to the adult equilibrium length reached between days 19 and 25. The changing rate of outer segment disk synthesis characterizes the late stages of cytodifferentiation of the rod photoreceptor cells.     

THE RENEWAL OF PHOTORECEPTOR CELL OUTER SEGMENTS

The utilization of methionine-³H by retinal photoreceptor cells has been studied by radioautographic technique in the rat, mouse, and frog. In all three species, the labeled amino acid is concentrated initially in the inner segment of the cell. Within 24 hr, the radioactive material is displaced to the base of the outer segment, where it accumulates as a distinct reaction band. The reaction band then gradually moves along the outer segment and ultimately disappears at the apex of the cell, which is in contact with the retinal pigment epithelium. These findings are interpreted to indicate that the photoreceptor cell outer segment is continually renewed, by the repeated lamellar apposition of material (membranous discs) at the base of the outer segment, in conjunction with a balanced removal of material at its apex. The outer segment renewal rate is accelerated in frogs when ambient temperature is raised, and is elevated in both frogs and rats when the intensity of retinal illumination is increased.     

THE ISOLATION OF RETINAL OUTER SEGMENT FRAGMENTS

Bovine retinal outer segment fragments were isolated by density gradient centrifugation in a high centrifugal field. Assays of the final preparation for enzymes of the mitochondrial respiratory chain indicated mitochondrial contamination not in excess of 1 per cent. Glucose-6-phosphatase and TPNH-cytochrome c reductase activities, presumably diagnostic for microsomes, were also absent. Electron micrographs did not disclose the presence of significant numbers of particles other than fragments of the outer segment discs. The red fragments were characterized by an ascorbate-oxidizing system and a high lipid content.     

THE OSMOTIC BEHAVIOR OF CRENATED RED CELLS

The anomalously small swelling which the red cells of human oxalated blood undergo in hypotonic plasma is related to the extent to which the cells are crenated. Reasons are given for regarding crenation as corresponding to gelation, and the bulk modulus for crenated cells, calculated from the measurements of swelling in hypotonic plasma, is shown to be of the same order as that for gelatin gels.     

THE RENEWAL OF ROD AND CONE OUTER SEGMENTS IN THE RHESUS MONKEY

The renewal of retinal rod and cone outer segments has been studied by radioautography in rhesus monkeys examined 2 and 4 days after injection of leucine-³H. The cell outer segment consists of a stack of photosensitive, membranous discs. In both rods and cones some of the newly formed (radioactive) protein became distributed throughout the outer segment. Furthermore, in rods (but not in cones), there was a transverse band of concentrated radioactive protein slightly above the outer segment base 2 days after injection. This was due to the formation of new discs, into which labeled protein had been incorporated. At 4 days, these radioactive discs were located farther from the outer segment base. Repeated assembly of new discs had displaced them away from the basal assembly site and along the outer segment. Measurements of the displacement rate indicated that each retinal rod produces 80–90 discs per day, and that the entire complement of outer segment discs is replaced every 9–13 days. To compensate for the continual formation of new discs, groups of old discs are intermittently shed from the apical end of the cell and phagocytized by the pigment epithelium. Each pigment epithelial cell engulfs and destroys about 2000–4000 rod outer segment discs daily. The similarity between visual cells in the rhesus monkey and those in man suggests that the same renewal processes occur in the human retina.     

ELECTRON MICROSCOPE OBSERVATIONS ON FORM CHANGES IN PHOTORECEPTOR OUTER SEGMENTS AND THEIR SACCULES IN RESPONSE TO OSMOTIC STRESS

Isolated retinas of rats or frogs were incubated in various salt or sucrose solutions over a wide range of osmolarities and then fixed in 1% osmium tetroxide solutions of matching osmolarities. Light and electron microscope observations were concentrated on the outer segments of the intact photoreceptors. These became globular and of increasing size with increasing hypoosmolarity and irregularly linear and condensed in hyperosmotic solutions. Isoosmotic incubations of rat retinas in solutions containing potassium as the only cation also produced swelling of the outer segments when chloride or acetate was present; but swelling was less when the cation was sodium and it was not seen with either cation when the anion was methylsulfate. The effects of various metabolic and membrane poisons are also reported. The behavior of the saccules within the outer segments was equivocal. While there was a tendency toward more saccules with wider lumina with hypoosmolarity, most of the saccules were not swollen. Surprisingly, intrasaccular space was consistently enlarged in rat retinas exposed to hyperosmotic sucrose but not to salt. The saccules or their derivatives within swollen outer segments tend to maintain their intersaccule spacing and approximation to the cell membrane. It was also noted that the ciliary connectives resist swelling and that retinal Müller cells swell readily.     

NEW EVIDENCE SUPPORTING THE LINKAGE TO EXTRACELLULAR SPACE OF OUTER SEGMENT SACCULES OF FROG CONES BUT NOT RODS

Previous electron microscopic examinations of outer segments of photoreceptors suggest that many flattened saccules of cones are continuous with the cell membrane and that their lumina connect with the extracellular compartment but that most saccules in rods appear to lack these connections. The saccules probably contain photolabile pigment, and certain potentials appear to result from dipole formation during pigment bleaching. The detection of dipoles from rod saccules may require that the lumina of rod saccules connect with extracellular space, and questions have been raised whether the interpretation of micrographs is correct or the isolation of rod saccules is the result of artifact. Accordingly, lanthanum and barium precipitates were produced near fixed and unfixed frog photoreceptors. Lanthanum precipitates appeared to infiltrate the saccules of fixed cones and the few surviving cones exposed prior to fixation, but no rod saccules were infiltrated except occasional, most basal saccules or saccules within narrow zones of probable damage. Barium precipitates did not infiltrate saccules of either variety of unfixed photoreceptor, but they did occasionally infiltrate around the saccules at points of damage in rod outer segments. The results thus support the view of the patency of saccules of frog cones and are consistent with, but do not prove, the isolation of saccules of frog rods.     

OSMOTIC EFFECT ON THE PHOTOSHRINKAGE OF CHLOROPLASTS

Shrinkage of spinach chloroplasts by illumination and that byexposure to a high tonicity raised by addition of sucrose wereinvestigated by means of the rapid measurement of chloroplastvolume with a COULTER counter. The osmotic shock in the darkinduced two steps of volume change; almost instantaneous shrinkagefor the osmotic shock and follow-up gradual swelling. The finalvolume attained after equilibration was smaller than the originalvolume below 0.6 M, and greater above this concentration. Whenchloroplasts under osmotic shock were illuminated, the photoshrinkagecompeted with the swelling induced by the osmotic shock, andthese reverse effects were balanced at a certain volume. Photoshrinkageactivity measured after equilibration decreased with increasingconcentrations, and the activity curve plotted against sucroseconcentration showed a stationary level of 50% of the originalactivity between 0.2 and 0.6 M, indicating the resistance ofa structure in chloroplasts to the denaturation by the osmoticeffect of sucrose. The osmotic effect in the dark as well asin the light was completely reversible below 0.2 M and was partiallyreversible or irreversible above this concentration. Glucoseshowed qualitatively the same osmotic effect as sucrose. ¹ Present address: Laboratory of Chemistry of Natural Products,Tokyo Institute of Technology, Meguroku, Tokyo.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

SOME PROPERTIES OF THE PROTEIN FORMING THE OUTER FIBERS OF CILIA

Cilia were isolated from Tetrahymena pyriformis by an ethanol-calcium procedure. Solutions of outer-fiber protein were obtained either by aqueous extraction of an acetone powder of whole cilia, or by dissolving the isolated outer-fibers in 0.6 M KCl. In aqueous solution, the outer-fiber protein has a sedimentation coefficient of 6.0S and a molecular weight of 104,000 ± 14,000. In 5 M guanidine hydrochloride solution the molecular weight falls to 55,000 ± 5,000. After reduction and alkylation in 8 M urea, about 95% of the protein migrates as a single band on electrophoresis in polyacrylamide gel at pH 8.9; the migration velocity is identical with that of reduced and alkylated actin. Freshly prepared outer-fiber protein contains about 7.5 sulfhydryl groups per 55,000 g of protein. The amino acid composition of outer-fiber protein resembles that of actin, with such differences as occur being of the same order as those between actins from different species of animal.     

THE KINETICS OF OSMOTIC SWELLING IN LIVING CELLS

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V _eq., V ₀, and V_t stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q ₁₀ = 2 to 3, or µ = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15° in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.     

ENDOGENOUS LEVELS OF NEUROTRANSMITTER CANDIDATES IN PHOTORECEPTOR CELLS OF THE TURTLE RETINA

The neurotransmitter used by vertebrate photoreceptors has not been identified, although aspartic acid, glutamic acid and acetylcholine have all been suggested as possible candidates. In the present study, we have measured the endogenous levels of these substances in isolated photoreceptors of the turtle retina. Fractions containing over 80% of identified photoreceptors were obtained by enzymatic dissociation of the retina followed by velocity sedimentation at unit gravity. The endogenous levels of free amino acids in these fractions were measured by amino acid analysis and the ACh content was measured by a radiochemical assay. In addition, identified photoreceptors were drawn into a micropipet individually under visual observation and their ACh content was determined. Our results show that while the concentrations of free aspartic acid and glutamic acid in isolated photoreceptors are similar to those found in the retina, the concentration of ACh in cone photoreceptors (0.1–0.2 mM) is 2- to 3-fold higher than that in the turtle retina. This result combined with the findings of others suggests that turtle cone photoreceptors may be cholinergic