Measurement of fast light-induced disc shrinkage within bovine rod outer segments by means of a light-scattering transient

A fast light-induced light-scattering transient, previously found in rod outer segment suspension, the so-called P-signal (Hofmann, K.P., Uhl, R., Hoffmann, W. and Kreutz, W. (1976) Biophys. Struct. Mechanism 2, 61–77), is described in more detail.The effect has the same action spectrum as rhodopsin bleaching. It is not regenerated with 11-cis retinal.The response is not linear with light-intensity for flashes which bleach more than 2.0% of rhodopsin; it saturates at an intensity corresponding to 15% rhodopsin bleaching.The wavelength- and scattering angle dependence lead to the conclusion that the change in light-scattering reflects a shrinkage of an osmotic compartment of the rod outer segment.The only compartment which we found to be intact in our rod outer segment preparations was the disc or rod sac; therefore, the effect must be attributed to a light-induced shrinkage of the rhodopsin-containing disc organelles.The overall effect (15% of rhodopsin is bleached) is in the range of 0.5–1.5% of the original volume.A light-induced passive cation-efflux from the disc, e.g. of Ca²⁺, can be ruled out as a possible molecular origin of the disc-shrinkage in our preparations.     

Calcium-hydrogen exchange in isolated bovine rod outer segments

We have measured Ca-H exchange in rod photoreceptors with different preparations of rod outer segments isolated from bovine retinas (ROS). One preparation contained ROS with an intact plasma membrane (intact ROS), and in the other preparation, the plasma membrane was leaky to small solutes (leaky ROS) and the cytoplasmic space was freely accessible to externally applied solutes. Addition of Ca2+ to Ca2+-depleted ROS (both intact and leaky) resulted in uptake of Ca2+ that was accompanied by the release of protons when catalytic amounts of the ionophore A23187 were present. This ionophore mediates Ca-H exchange transport across ROS membranes and serves to gain access to the intracellular compartment where Ca-H exchange appears to take place. Two protons were ejected for each calcium ion taken up. Conversely, when protons were added to Ca2+-enriched ROS, Ca2+ was released in the presence of A23187. The majority of this Ca-H exchange was observed only when A23187 was present in both intact and leaky ROS. We conclude that Ca-H exchange occurs predominantly in the intradiskal space and at the surface of the disk membrane rather than across the disk membrane. These exchange binding sites can accommodate 10 mol of Ca2+/mol of rhodopsin at physiological pH. We were unable to detect any Ca2+ release when a proton gradient was rapidly established across the disk membrane in the absence of A23187. These results are discussed in relation to the hypothesis that protons produced by the light-induced hydrolysis of cGMP cause the release of Ca2+ into the cytoplasm of rod photoreceptor cells.     

Measurements of fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells

Flash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles. We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin. The following results are discussed:

1. The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin).
2. At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course:

N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane. Ni: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect. P: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. “P” may reflect an influence of the light-induced transmitter release on disc shape and/or mass.     

Light-activated calcium release from sonicated bovine retinal rod outer segment disks.

Calcium trapped within sonicated and resealed bovine rod outer segment disks is released upon light exposure with a stoichiometry of 0.75 +/- 0.05 calcium for each rhodopsin bleached. The amount of calcium liberated is proportional to the amount of bleaching in the range of 20 to 100% bleaching and is relatively insensitive to the internal trapped calcium concentration. The results are obtained using a flow system in which the disk membrane vesicles are adsorbed on glass particle supported by a filter. The external calcium is washed away and subsequent calcium release is monitored by collecting fractions of the effluent before, during, and after light exposure. Disks that are sonicated and allowed to reseal prior to incubation with 45Ca show no change in calcium efflux upon bleaching. The light-activated calcium release is also eliminated if disks sonicated in the presence of 45Ca are treated with a calcium ionophore prior to bleaching. The results demonstrate that the light-released calcium comes from the disks and not from the external disk surface. Lowering temperature to 3--4 degrees C surpresses the light-stimulated release, implicating a transition after the formation of metarhodopsin I in the transport process. The resluts suggest a model for the disk in which each bleached rhodopsin functions as a "one-shot carrier" to transport a single calcium ion across the membrane.     

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.     

Interaction of bovine rhodopsin with calcium ions. II: calcium release in bovine rod outer segments upon bleaching.

The calcium content of bovine rod outer segment (ROS) suspensions was determined by flame spectrophotometry to be about 0.2 Ca2+ per molecule rhodopsin. After bleaching of rhodopsin, a release of 0.01--0.1 Ca2+ per molecule rhodopsin from ROS into the solution was observed. These figures agree with some data in the literature (Appendix). A measured absorption increase of the Ca2+-indicator phthalein purple (10 degrees C, 562 nm, pH 9.3) occurs apparently simultaneously with the formation of metarhodopsin ii in ROS. This indicates that a light induced Ca2+-release of 12 calcium ions per photoactivated rhodopsin is coupled in time with the formation of metarhodopsin II.     

Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.     

Superoxide dismutase catalyzes activation of synaptosomal soluble guanylate cyclase from rat brain

The hydrogen ion changes resulting from the photolysis of the rod visual pigment, rhodopsin, were investigated at acidic pH (5.2–6.5). After light-induced proton uptake, slow proton release occurred both in the dark and in the light. It was found that the amount of proton release in the dark was not equal to that in the light; about 0.9 proton remained bound to rhodopsin bleached in the dark, while all the bound protons were released in the light. Furthermore, the time course of proton release in the dark is not related to the decay of metarhodopsin II₃₈₀, but is closely related to the formation of metarhodopsin III₄₆₅.     

The correlation of the receptor potential with the light induced transient increase in intracellular calcium-concentration measured by absorption change of arsenazo III injected into Limulus ventral nerve photoreceptor cell

The light-induced membrane voltage response (receptor potential, ReP) and the absorption change of the intracellularly injected calcium indicator arsenazo III (arsenazo response) were recorded simultaneously in Limulus ventral nerve photoreceptor cells. A double pulse technique was applied for stimulation. After pressure injection of the indicator into the cell absorption changes were measured at 646 nm to obtain a measure of the changes of the intracellular calcium ion concentration.

1. The size of the arsenazo response increases with increasing intensity of the light stimulus. The intensity dependence of the size of the arsenazo response δA_max shows almost no correlate with the peak amplitude of the ReP, but correlates rather well with the time integral of the ReP.
2. Decreasing light adaptation (caused by prolongation of the repetition time of the pulse pairs) leads to an increase in size of the arsenazo response. Also here δA_max correlates better with the time integral of the ReP than with its peak amplitude.
3. Lowering the calcium concentration in the superfusate (from 10 mmol/l to ca. 40 Μmol/l) causes after ca. 10 min a drastical diminution of the arsenazo response to values below the noise level, and a less marked reduction in size of the ReP. The falling phase of the ReP is slower. After return to normal calcium concentration the arsenazo response recovers partly in ca. 50 min, while the ReP recovers faster.

The results show two opposite correlations between ReP and arsenazo response: Increase in size and duration of the ReP causes a greater transient increase of the intracellular calcium ion concentration. This in turn tends to reduce and shorten the ReP. Which effect dominates obviously depends on the conditions of the experiment: when the calcium concentration in the superfusate is reduced to ca. 40 Μmol/l a slow decrease of the ReP is accompanied by a small increase of the intracellular calcium ion concentration.     

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.     

On the relation between rapid light-induced Ca2+ release and proton uptake in rod outer segment disk membranes

In this paper we review our experiments on the light-induced Ca2+ release and proton uptake at the rod outer segment (ROS) disk membrane using flash-spectrophotometry and the indicating dyes arsenazo III and bromcresol purple. We used three different ROS preparations in order to locate the intracellular site of Ca2+ release. The ionophore A23187 was required to communicate the Ca2+ release to the indicator located in the external medium in both ROS with an intact and with a leaky plasma membrane. A23187 was also required to observe the Ca2+ released in the interior of vesicles prepared by sonication of ROS. From this we conclude that the site of Ca2+ release is located at the luminal side of the disk membrane, whereas this Ca2+ was not transported across the disk membrane under our experimental conditions and on the time scale of our experiments (20 s). Light-induced Ca2+ release was inhibited by electrolysis in the suspension medium provided that the electrolytes gained access to the compartment where Ca2+ was released. The effectivity to inhibit Ca2+ release markedly increased from monovalent to divalent to trivalent cations. The results strongly suggest that electrolytes (cations) act by screening the electrostatic potential at the disk membrane surface due to the presence of a net fixed negative surface charge. The surface potential controls the free Ca2+ concentration at the membrane surface and, therefore, controls the amount of Ca2+ bound to the disk membrane. The kinetics of light-induced Ca2+ release and proton uptake showed a similar dependence on the structural status of the ROS. In sonicated ROS almost linear Arrhenius plots were observed for metarhodopsin II formation, Ca2+ release and proton uptake (energy of activation 150 kJ/mol). In intact ROS both Ca2+ release and proton uptake showed a nonlinear Arrhenius plot with rate constants up to 30-fold slower than metarhodopsin II formation. At temperatures above 10 degrees C a process other than metarhodopsin II formation rate limited both ligh-induced proton uptake and Ca2+ release (energy of activation 42 kJ/mol). A model is discussed in which metarhodopsin II formation triggers the uptake of proton(s) into the disk membrane lowering the surface potential. A reduction potential of the surface in turn decreases the free Ca2+ concentration at the surface thereby causing the release of part of the bound Ca2+.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Cyclic GMP and photoreceptor function.

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Mathematical and computational modelling of spatio-temporal signalling in rod phototransduction

Rod photoreceptors are activated by light through activation of a cascade that includes the G protein-coupled receptor rhodopsin, the G protein transducin, its effector cyclic guanosine monophosphate (cGMP) phosphodiesterase and the second messengers cGMP and Ca2+. Signalling is localised to the particular rod outer segment disc, which is activated by absorption of a single photon. Modelling of this cascade has previously been performed mostly by assumption of a well-stirred cytoplasm. We recently published the first fully spatially resolved model that captures the local nature of light activation. The model reduces the complex geometry of the cell to a simpler one using the mathematical theories of homogenisation and concentrated capacity. The model shows that, upon activation of a single rhodopsin, changes of the second messengers cGMP and Ca2+ are local about the particular activated disc. In the current work, the homogenised model is computationally compared with the full, non-homogenised one, set in the original geometry of the rod outer segment. It is found to have an accuracy of 0.03% compared with the full model in computing the integral response and a 5200-fold reduction in computation time. The model can reconstruct the radial time-profiles of cGMP and Ca2+ in the interdiscal spaces adjacent to the activated discs. Cellular electrical responses are localised near the activation sites, and multiple photons sufficiently far apart produce essentially independent responses. This leads to a computational analysis of the notion and estimate of 'spread' and the optimum distribution of activated sites that maximises the response. Biological insights arising from the spatio-temporal model include a quantification of how variability in the response to dim light is affected by the distance between the outer segment discs capturing photons. The model is thus a simulation tool for biologists to predict the effect of various factors influencing the timing, spread and control mechanisms of this G protein-coupled, receptor-mediated cascade. It permits ease of simulation experiments across a range of conditions, for example, clamping the concentration of calcium, with results matching analogous experimental results. In addition, the model accommodates differing geometries of rod outer segments from different vertebrate species. Thus it represents a building block towards a predictive model of visual transduction.     

Incorporation of glucosamine into rhodopsin in isolated bovine retina

Radioactive glucosamine is incorporated into the outer segments of the rod cells of bovine retinas incubated in vitro. One component of the outer segment labeled in this process is rhodopsin which can be extracted with detergent, purified by sequential chromatography on calcium phosphate-Celite and agarose, and shown to be light sensitive by its altered chromatographic mobility. The radioactive component can be released from rhodopsin by acid hydrolysis and shown to migrate with glucosamine on paper chromatography. In double label experiments both glucosamine and leucine are incorporated into rhodopsin. The time course of glucosamine incorporation is similar to that of leucine. The system supports prolonged synthesis of both the polypeptide and oligosaccharide portions of the rhodopsin molecule in vitro.     

Influence of calcium on guanosine 3'',5''-cyclic monophosphate levels in frog rod outer segments

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.     

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.