Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.     

Some properties of old and new rhodopsin in single Bufo rods

Rod photoreceptors renew the membranous disks of the outer segments (ROS). New disks are assembled at the proximal base and old disks are shed at the distal tip. Rhodopsin, the major protein of the disk, remains with the disk into which it was inserted. Thus, it is true that the oldest rhodopsin is at the tip and the newest at the base. A microspectrophotometer is used to examine the properties of rhodopsin in the two ends of the toad ROS. No differences between the two are found in absorption spectrum, concentration, dichroism, photoconversion rates, or lateral diffusion rates. Regeneration of rhodopsin from the bleached state is also studied but cannot be used to discriminate old from new rhodopsin because the point of entry of regeneration retinoids and/or their concentrations cannot be controlled. However, a new insight into pigment regeneration in the living toad eye is gained: regeneration is faster in the basal disks than in the distal.     

Rhodopsin and Porphyropsin Fields In the Adult Bullfrog Retina

Though it had been supposed earlier that the bullfrog undergoes a virtually complete metamorphosis of visual systems from vitamin A₂ and porphyropsin in the tadpole to vitamin A₁ and rhodopsin in the adult, the present observations show that the retina of the adult frog may contain as much as 30–40% porphyropsin, all of it segregated in the dorsal zone. The most dorsal quarter of the adult retina may contain 81–89% porphyropsin mixed with a minor amount of rhodopsin; the ventral half contains only rhodopsin. Further, the dorsal zone contains a two to three times higher concentration of visual pigments than the ventral retina. The pigment epithelium underlying the retina contains a corresponding distribution of vitamins A₁ and A₂, predominantly vitamin A₂ in the dorsal pigment epithelium, exclusively vitamin A₁ in the ventral zone. The retina accepts whatever vitamin A the pigment epithelium provides it with, and turns it into the corresponding visual pigment. Thus, a piece of light-adapted dorsal retina laid back on ventral pigment epithelium regenerates rhodopsin, whereas a piece of light-adapted ventral retina laid back on dorsal pigment epithelium regenerates predominantly porphyropsin. Vitamin A₂ must be made from vitamin A₁, by dehydrogenation at the 3,4-bond in the ring. This conversion must occur in the pigment epithelium, presumably through the action of a vitamin A-3,4-dehydrogenase. The essential change at metamorphosis is to make much less of this dehydrogenase, and to sequester it in the dorsal pigment epithelium. Some adult bullfrogs, perhaps characteristically taken in the summer, contain very little porphyropsin—only perhaps 5%—still sequestered in the dorsal retina. The gradient of light over the retinal surface has little if any effect on this distribution. The greater density of visual pigments in the dorsal retina, and perhaps also—although this is less clear—the presence of porphyropsin in this zone, has some ecological importance in increasing the retinal sensitivity to the dimmer and, on occasion, redder light received from below.     

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning³¹P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.     

Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors [published erratum appears in J Gen Physiol 1987 May;89(5):following 837]

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.     

Rhodopsin lateral diffusion as a function of rod outer segment disk membrane axial position

Rhodopsin lateral diffusion was measured at three points along the axis of frog outer segments using the method of absorbance recovery after photobleaching. Mean recovery times were slightly longer in distal disk membranes than in proximal disks. A small reduction of pigment mobility with disk age may reflect subtle changes in membrane composition.     

THE VISUAL CELLS AND VISUAL PIGMENT OF THE MUDPUPPY, NECTURUS

Electron microscopy of the visual cells of the mudpuppy Necturus have revealed several new or hitherto neglected features of organization: (a) A system of deeply staining micelles in virtually crystalline array, probably located in the lamellae of the rod outer segments. These particles may contain the visual pigment, porphyropsin. Counts of the micelles, and microspectrophotometric measurements of porphyropsin in the retina and single rods yield the estimate that each lamellar micelle may contain about 50 molecules of porphyropsin. (b) Systems of about 30 cytoplasmic filaments (here called dendrites), continuous with the cytoplasm of the inner segment, and standing like a palisade about the outer segments of the rods and cones. In the rods, one such filament stands in the mouth of each of the approximately 30 deep fissures that carve the outer segment into a radial array of lobules. (c) A system of deeply staining particles in the membranes of the dendrites, and another in the membranes of the pigment epithelial processes. It is suggested that these may have a part in interchanges of material with the outer segments. The ciliary process is found to penetrate more deeply than is commonly supposed into the outer segments of the rods and cones. The edge of each double-membrane disc in rods forms a differentiated rim structure, both around the disc circumference and bordering the fissures. These anatomical arrangements are summarized in Figs. 13 and 14, and the relevant measurements in Table I. The dilution of visual pigment in Necturus rods and cones and a general consideration of their microstructures make it seem unlikely that such typically solid state processes as exciton migration or photoconduction can transport the effects of light far from the site of absorption. Excitation must, therefore, be conveyed to the receptor as a whole by some axial structure. Among axial structures, the plasma membrane is most likely to be the site of nervous excitation. The ciliary process probably plays its main role in the embryogenesis and regeneration of outer segments; and the dendrites and pigment epithelial processes in exchanges of material with the outer segments and perhaps with one another.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

ATP-dependent calcium uptake activity associated with a disk membrane fraction isolated from bovine retinal rod outer segments

Ca2+ sequestration and release from disks of rod outer segments may play a critical role in visual transduction. An ATP-dependent Ca2+ uptake activity has been identified in association with purified disks of bovine rod outer segments. A crude preparation of osmotically active disks was obtained from rod outer segments by hypoosmotic shock and subsequent flotation on a 5% Ficoll 400 solution. These "crude" disks were further purified by separation into two distinct components by centrifugation in a linear Ficoll gradient. Disks comprised the major component; at least 60% of the protein was rhodopsin. This fraction also contained a Ca2+ uptake activity stimulated approximately 4-fold by ATP. The initial rate was approximately 0.21 nmol of Ca2+ (mg of protein)-1 min-1. Most of the ATP-dependent accumulation of 45Ca2+ was released by the calcium ionophore A23187. The uptake activity was sensitive to vanadate (Ki approximately 20 microM) and insensitive to the mitochondrial Ca2+ uptake inhibitor ruthenium red (10 microM). The ATP-dependent Ca2+ uptake exhibited Michaelis-Menten activation kinetics with respect to [Ca2+] (Km approximately 6 microM). The osmotic properties of the sealed disk membranes were exploited to determine whether the association of Ca2+ transport activity with the disks was merely coincidental. The sedimentation properties of these disks, upon centrifugation on a second Ficoll linear density gradient, varied with the osmolarity of the gradient solution. In several separate gradient solutions of differing osmotic and ionic strengths, the Ca2+ uptake activity always comigrated with the disks. These results indicate that the ATP-dependent Ca2+ uptake activity was physically associated with sealed native disk membranes. The characteristics of the Ca2+ uptake activity suggest that it may play a major role in the regulation of cytosolic Ca2+ levels in rod cells in vivo.     

A Simplified Mass-Transfer Model for Visual Pigments in Amphibian Retinal-Cone Outer Segments

When radiolabeled precursors and autoradiography are used to investigate turnover of protein components in photoreceptive cone outer segments (COSs), the labeled components—primarily visual pigment molecules (opsins)—are diffusely distributed along the COS. To further assess this COS labeling pattern, we derive a simplified mass-transfer model for quantifying the contributions of advective and diffusive mechanisms to the distribution of opsins within COSs of the frog retina. Two opsin-containing regions of the COS are evaluated: the core axial array of disks and the plasmalemma. Numerical solutions of the mass-transfer model indicate three distinct stages of system evolution. In the first stage, plasmalemma diffusion is dominant. In the second stage, the plasmalemma density reaches a metastable state and transfer between the plasmalemma and disk region occurs, which is followed by an increase in density that is qualitatively similar for both regions. The final stage consists of both regions slowly evolving to the steady-state solution. Our results indicate that autoradiographic and cognate approaches for tracking labeled opsins in the COS cannot be effective methodologies for assessing new disk formation at the base of the COS.     

On the visual pigments of deep-sea fish

The retinal visual pigments of 52 species of deep-sea fish were measured by partial bleaching of detergent extracts. The retinae of 45 species contained only a single rhodopsin with maximum absorbance (λ_max) at a wavelength between 474 and 490 nm, matching both the region of highest intensity downwelling sunlight and the maximum emission of most deep-sea bioluminescence. Seven species were shown to have more than one visual pigment within their retinae and these had λ_max values that generally fell outside the usual range. One of these, Bonapartia pedaliota , was particularly interesting as, unlike most such multipigment species, it had one rhodopsin and one porphyropsin pigment, apparently based on different opsins. The relative proportions of the visual pigments in the seven multipigment species are presented.     

The 3, 4-didehydroretinal chromophore of goldfish porphyropsin

The isomeric configuration of the 3,4-didehydroretinal chromophore of goldfish porphyropsin was determined by high performance liquid chromatography (HPLC) and by the regeneration of this visual pigment with authentic isomers of 3,4-didehydroretinal. A nonisomerizing, quantitative method using hydroxylamine and methylene chloride was employed to extract the 3,4-didehyroretinal chromophore from the rod outer segment membrane (containing the porphyropsin). When this extracted chromophore was injected into the HPLC, only a single major peak was observed and this peak coeluted with the authentic 11-cis 3,4-didehydroretinyl oxime. This suggests that the chromophore of goldfish porphyropsin is 11-cis 3,4-didehydroretinal. When the bleached rod outer segments (containing the opsin) were incubated with different 3,4-didehydroretinal isomers (13-cis, 11-cis, 9-cis, and all-trans), only the 11-cis isomer resulted in the degeneration of porphyropsin. This also suggests that the porphyropsin chromophore exists in the 11-cis configuration.     

Modeling the role of incisures in vertebrate phototransduction

Phototransduction is mediated by a G-protein-coupled receptor-mediated cascade, activated by light and localized to rod outer segment (ROS) disk membranes, which, in turn, drives a diffusion process of the second messengers cGMP and Ca2+ in the ROS cytosol. This process is hindered by disks-which, however, bear physical cracks, known as incisures, believed to favor the longitudinal diffusion of cGMP and Ca2+. This article is aimed at highlighting the biophysical functional role and significance of incisures, and their effect on the local and global response of the photocurrent. Previous work on this topic regarded the ROS as well stirred in the radial variables, lumped the diffusion mechanism on the longitudinal axis of the ROS, and replaced the cytosolic diffusion coefficients by effective ones, accounting for incisures through their total patent area only. The fully spatially resolved model recently published by our group is a natural tool to take into account other significant details of incisures, including their geometry and distribution. Using mathematical theories of homogenization and concentrated capacity, it is shown here that the complex diffusion process undergone by the second messengers cGMP and Ca2+ in the ROS bearing incisures can be modeled by a family of two-dimensional diffusion processes on the ROS cross sections, glued together by other two-dimensional diffusion processes, accounting for diffusion in the ROS outer shell and in the bladelike regions comprised by the stack of incisures. Based on this mathematical model, a code has been written, capable of incorporating an arbitrary number of incisures and activation sites, with any given arbitrary distribution within the ROS. The code is aimed at being an operational tool to perform numerical experiments of phototransduction, in rods with incisures of different geometry and structure, under a wide spectrum of operating conditions. The simulation results show that incisures have a dual biophysical function. On the one hand, since incisures line up from disk to disk, they create vertical cytoplasmic channels crossing the disks, thus facilitating diffusion of second messengers; on the other hand, at least in those species bearing multiple incisures, they divide the disks into lobes like the petals of a flower, thus confining the diffusion of activated phosphodiesterase and localizing the photon response. Accordingly, not only the total area of incisures, but their geometrical shape and distribution as well, significantly influence the global photoresponse.     

Degradation of rod outer segment proteins by cathepsin D.

The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3.4.23.5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the porteine of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.     

Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light

The rate of disk addition to rod outer segments (ROS) varies widely in Xenopus laevis tadpoles kept in cyclic light (12L:12D). When measured as radioactive band (3H-band) displacement during the 2nd day after injection of [3H]leucine, 75% of the daily increment of displacement occurred during the first 8 h of light. During the same interval, the number of open disks at the ROS base increased more than threefold. During the last 8 h of darkness, 3H-band displacement was undetectable and the number of open disks was reduced. These observations suggest the possibility that disk addition may occur discontinuously. During the 3rd and 4th days after injection of [3H]leucine, maximal displacement of the 3H-band occurred later in the day than on the 2nd day, its movement no longer corresponding to the increase in open disks. This delay in 3H-band displacement may reflect a time delay as a result of propagation of compressive stress in an elastic ROS system. Maximal disk loss from ROS as reflected in counts of phagosomes in the pigment epithelium occurred within 1 h of light exposure, and phagosome counts remained high for 4 h before declining to a low level in darkness. Modified lighting regimes affected the daily rhythms of shedding and disk addition differently, suggesting that control mechanisms for the two processes are not directly coupled. During 3 days in darkness, disk addition was reduced 50% compared to controls (12L:12D), whereas shedding was reduced by about 40%. Although reduced in level, shedding occurred as a free-running circadian rhythm. There was no evidence of rhythmicity of disk addition in darkness. In constant light, the rate of disk addition was not different from controls, but shedding was reduced by about 80% after the 1st day. This resulted in a 21% increase in ROS length. Among animals kept on a 2.5L:21.5D cycle, the rate of disk addition was reduced by 40% while shedding was maintained near control levels, resulting in a slight decrease in ROS length. These observations indicate that normal shedding requires alternating light and darkness, and that the daily rhythm of disk addition is due primarily to daily stimulation by light.     

The metamorphosis of visual systems in the sea lamprey

The life cycle of the sea lamprey, Petromyzon marinus, includes two metamorphoses. At the end of a period spent as a blind larva, buried in the mud of streams, a first metamorphosis prepares it to migrate downstream to the sea or a lake for its growth phase. Then, following a second metamorphosis, it migrates upstream as a sexually mature adult to spawn and die. The downstream migrants have a visual system based upon rhodopsin and vitamin A₁, whereas that of the upstream migrants is based upon porphyropsin and vitamin A₂. The livers contain vitamin A₁ at all stages. The sea lamprey therefore exhibits a metamorphosis of visual systems, like those observed earlier among amphibia. The presence of porphyropsin in this member of the most primitive living group of vertebrates, as in fishes and amphibia, supports the notion that porphyropsin may have been the primitive vertebrate visual pigment. Its association with fresh water existence throughout this range of organisms also is consistent with the view that the vertebrate stock originated in fresh water. The observation that in the life cycle of the lamprey rhodopsin precedes porphyropsin is not at variance with the idea that porphyropsin is the more primitive pigment, since this change is part of the second metamorphosis, marking the return to the original environment. The observation that in lampreys, fishes, and amphibia, porphyropsin maintains the same general association with fresh water, and rhodopsin with marine and terrestrial habit, suggests that a single genetic mechanism may govern this association throughout this wide span of organisms.     

Spectral sensitivity of the weakly discharging electric fish Gnathonemus petersi using its electric organ discharges as the response measure

The spectral sensitivity of the weakly electric mormyrid fish Gnathonemus petersi was investigated under dark- and light-adapted conditions using a transient change (startle) in its electric organ discharge (EOD) rate as response measure. The startle was resistant to habituation and graded with light intensity. Under both lighting conditions, the fish responded optimally to a monochromatic light of 525 nm. A porphyropsin pigment (520–540₂) appears to mediate spectral sensitivity over most of the visible spectrum. However, G. petersi responded more strongly to 625- and 675-nm lights (dark- and light-adapted fish) and a 725-nm light (light-adapted fish only) than predicted by the presence of a single rod pigment. These data suggest that at least one additional visual pigment (most likely of cone cells) maximally absorbing long wavelength light (600 nm or longer) is present. The spectral sensitivity data are consistent with the sensitivity hypothesis in that heightened sensitivity to long wavelength light is predicted for fish living in blackwater habitats which are characterized typically by low light levels and transmission of predominantly long wavelengths. Histology of the retina showed photoreceptors grouped into bundles and ensheathed by pigment epithelial cells. Our results demonstrated a functional visual sense in a species of fish much better known and studied for its electrosensory and electromotor abilities.     

Linkage of Retinal to Opsin

RHODOPSIN, the visual pigment of vertebrate rods, has been shown to consist of a chromophore (11-cis retinal) bound to a protein (opsin)^1–2. It has been proposed that the linkage is a Schiff base between phosphatidyl ethanolaniine (PE) and retinal and that when exposed to light, the retinal migrates from PE to the ε-amino-group of a lysine residue in opsin^3–7. Most of the support for this theory comes from the observation that N-retinylidenephosphatidylethanolamine (N-RPE) can be extracted in the dark from rod outer segments (ROS)^3,4. Furthermore, N-retinylphosphatidylethanolamine (N-RH₂PE) has been extracted from ROS preparations after treating the visual pigment with acid and NaBH₄—conditions which are assumed fix retinal to its “native” binding site through a secondary amine linkage⁷. All these studies, however, were carried out on crude extracts of ROS in various detergents. These crude extracts contain large amounts of phospholipid and retinal which is not bound to opsin. Thus, the isolation of N-RPE from crude ROS extracts does not necessarily point to its involvement in the binding of retinal to opsin. In contradiction to these reports are findings that purified visual pigment contains no phospholipid^9,10 and that the molar concentration of N-RPE in bovine ROS is less than that of rhodopsin¹¹. We have taken advantage of the observation that visual pigment in the outer segment disks is continually being renewed¹² to label the rhodopsin with ³H-retinal and to show in yet another way that N-RPE does not exist in purified visual pigment.     

The cone photoreceptors and visual pigments of chameleons

Visual pigments, oil droplets and photoreceptor types in the retinas of four species of true chameleons have been examined by microspectrophotometry. The species occupy different photic environments: two species of Chamaeleo are from Madagascar and two species of Furcifer are from Africa and the Arabian Peninsula. In addition to double cones, four spectrally distinct classes of single cone were identified. No rod photoreceptors were observed. The visual pigments appear to be mixtures of rhodopsins and porphyropsins. Double cones contained a pale oil droplet in the principle member and both outer segments contained a long-wave-sensitive visual pigment with a spectral maximum between about 555 nm and 610 nm, depending on the rhodopsin/porphyropsin mixture. Long-wave-sensitive single cones contained a visual pigment spectrally identical to the double cones, but combined with a yellow oil droplet. The other three classes of single cone contained visual pigments with maxima at about 480–505, 440–450 and 375–385 nm, combined with yellow, clear and transparent oil droplets respectively. The latter two classes were sparsely distributed. The transmission of the lens and cornea of C. dilepis was measured and found to be transparent throughout the visible and near ultraviolet, with a cut off at about 350 nm.