The renewal of protein in retinal rods and cones

The renewal of protein in retinal rods and cones has been analyzed by quantitative electron microscope radioautography in adult frogs injected with a mixture of radioactive amino acids. Protein synthesis occurs predominantly in the ergastoplasm, localized in the myoid region of the photoreceptor cells. Much of the newly formed protein next flows through the Golgi complex. In rods, a large proportion of the protein then moves past the mitochondria of the ellipsoid segment, passes through the connecting cilium into the outer segment, and is there assembled into membranous discs at the base of that structure. Discs are formed at the rate of 36 per day in red rods and 25 per day in green rods at 22.5° C ambient temperature. In cones, a small proportion of the protein is similarly displaced to the outer segment. However, no new discs are formed. Instead, the protein becomes diffusely distributed throughout the cone outer segment. Low levels of radioactivity have been detected, shortly after injection, in the mitochondria, nucleus, and synaptic bodies of rods and cones. Nevertheless, in these organelles, the renewal process also appears to involve the utilization of protein formed in the ergastoplasm of the myoid.     

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods

The Ca2+ dependence of the kinetics and light sensitivity of light-activated phosphodiesterase was studied with a pH assay in toad and bovine rod disk membranes (RDM), and in a reconstituted system containing GTP-binding protein, phosphodiesterase and rhodopsin kinase. Three statistics, peak hydrolytic velocity, turnoff time, and time to peak velocity, were measured. ATP decreased phosphodiesterase light sensitivity nearly 10-fold and accelerated the dim-flash kinetics of cGMP hydrolysis when compared to those with GTP alone. CA2+ reversed all of the effects of ATP, Ca2+ increased peak velocity, turnoff time, and time to peak velocity, to the values obtained with GTP alone. The Ca2+ dependence of peak velocity and turnoff time can be characterized as hyperbolic saturation functions with a K0.5 for Ca2+ of 1.0-1.5 mM in toad RDM. In bovine RDM the Ca2+ dependence of peak velocity and turnoff time has a K0.5 of 0.1 mM Ca2+. The Ca2+ dependence in the reconstituted system is similar to that in bovine RDM for peak velocity (K0.5 = 0.1 mM Ca2+) but differs for turnoff time (K0.5 = 2.5 mM Ca2+). We tested the hypothesis that a soluble modulator, normally required to confer submicromolar Ca2+ sensitivity, was too dilute in our assay by comparing data obtained at one RDM concentration with those obtained at 10-fold higher RDM, and therefore a constituent protein, concentration. We observe no difference and present a formal analysis of these data that excludes the hypothesis that the soluble modulator binds its target protein with Kd less than 5 microM. The lack of submicromolar Ca2+ dependence of any of the steps in the cGMP cascade that underlie cGMP phosphodiesterase activation and inactivation in vitro argues against Ca2+ regulation of these steps having a significant role in the light adaptation of the intact rod.     

Light and dark active phosphodiesterase regulation in salamander rods

We studied the activation of 3',5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) by using a cell-permeant enzyme inhibitor. Rods of Ambystoma tigrinum held in a suction electrode were jumped into a stream of 3-isobutyl-1-methylxanthine (IBMX), 0.01-1 mM. Initial transient light-sensitive currents fit the notion that dark and light-activated forms of PDE contributed independently to metabolic activity and were equivalently inhibited by IBMX (apparent Ki 30 microns). Inhibition developed within 50 ms, producing a step decrease of enzyme velocity, which could be offset by activation with flashes or steps of light. The dark PDE activity was equivalent to light activation of enzyme by 1,000 isomerization rod-1s-1, sufficient to hydrolyze the free cGMP pool (1/e) in 0.6 s. Steady light activated PDE in linear proportion to isomerization rate, the range from darkness to current saturation amounting to a 10-fold increase. The conditions for simultaneous onset of inhibitor and illumination to produce no net change of membrane current defined the apparent lifetime of light-activated PDE, TPDE* = 0.9 s, which was independent of both background illumination and current over the range 0-3 x 10(5) isomerization s-1, from 50 to 0 pA. Adaptation was a function of current rather than isomerization: jumps with different proportions of IBMX concentration to steady light intensity produced equal currents, and followed the same course of adaptation in maintained light, despite a 10-fold difference of illumination. Judged from the delay between IBMX- and light-induced currents, the dominant feedback regulatory site comes after PDE on the signal path. The dark active PDE affects the hydrolytic flux and cytoplasmic diffusion of cGMP, as well as the proportional range of the cGMP activity signal in response to light.     

Recoverin regulates light-dependent phosphodiesterase activity in retinal rods

The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin.     

Rods are rods and cones cones, and (never) the twain shall meet.

More than 100 photopigment G protein-coupled receptors (opsins) have been sequenced and organized into six classes. Rod photoreceptors in various species have been found to express an opsin from one of the two rhodopsin classes, while cones express an opsin from one of the four remaining classes. It has now been discovered that salamander short-wavelength sensitive cones and green rods express the same opsin, while manifesting other features that classically distinguish rods from cones.     

Differential sensitivity of rods and cones in Xenopus retina to hemicholinium-3

Summary Hemicholinium-3 (HC-3), a drug which prevents synthesis of acetylcholine in neurons, when injected intraperitoneally in doses as low as 2×5 mg/kg produces marked ultrastructural changes and damage in rod but not in cone photoreceptors. In rods it causes a reduction in cytoplasmic back-ground density, swelling of the endoplasmic reticulum, ballooning of the outer membrane of the nucleus, leaching of the nucleoplasm and clumping of the nuclear chromatin. In dark-adapted rods HC-3 produces some loss of cytoplasmic synaptic vesicles but no reduction in numbers of those vesicles which lie adjacent to the synaptic ribbons. In light-adapted rods the drug does not cause such an apparent reduction of synaptic vesicles but does induce a considerable reduction in numbers of vesicles associated with the ribbons. These structural changes are discussed in the light of what is known about the pharmacology of HC-3 and neurotransmitter release from vertebrate photoreceptors.The authors wish to thank SRC for a grant in support of this work     

Energy-dependent activation and magnesium-dependent in activation of hepatocyte hormone-sensitive phosphodiesterase

Incubation of solubilized hormone-activated phosphodiesterase from isolated hepatocytes, under conditions likely to favour a dephosphorylation reaction, did not cause a loss of the hormone activation. If, however, the enzyme was incubated with Mg²⁺ (10 mM) while still associated with its membrane, and subsequently solubilized, the activity of the hormone-stimulated enzyme declined to the level seen in control cells.Diminution of hepatocyte ATP levels to about 20% of contol values, by incubation with fructose, blunted the effect of glucagon and abolished the effect of insulin on phosphodiesterase. More severe ATP depletion caused by dinitrophenol abolished the stimulation of the enzyme by both hormones. These effects were not considered likely to be due to altered hormone-binding and are consistent with the involvement of an energy-dependent step in the hormonal activation of phosphodiesterase.     

Electron microscope observations on synaptic vesicles in synapses of the retinal rods and cones

The submicroscopic organization of the rod and cone synapses of the albino rabbit has been investigated with the use of the electron microscope. The most common rod synapse consists of an enlarged expansion of the rod fiber (the so called spherule) into which the dendritic postsynaptic fiber of the bipolar cell penetrates and digitates. The membrane surrounding the terminal consists of a double layer, the external of which is interpreted as belonging to the intervening glial cells. The synaptic membrane has a pre- and a postsynaptic layer with a total thickness of 180 to 300 A. The presynaptic layer is frequently denser and is intimately associated with the adjacent synaptic vesicles. The synaptic membrane shows processes constituted by foldings of the presynaptic layer. The entire spherule is filled with synaptic vesicles varying in diameter between 200 and 650 A with a mean of 386 A. In addition, the spherule contains a few large vacuoles near the rod fiber, interpreted as endoplasmic reticulum, and a matrix in which with high resolution a fine filamentous material can be observed. The postsynaptic fiber is homogeneous and usually does not show synaptic vesicles. In animals maintained in complete darkness for 24 hours vesicles appear to accumulate near the synaptic membrane and its processes. After 9 days there is a sharp decrease in size of the synaptic vesicles. A special rod synapse in which the dendritic postsynaptic expansion penetrates directly into the rod cell body has been identified. In line with Cajal's classification this type of synapse could be considered as a somatodendritic one. The cone synapse has a much larger terminal with a more complex relationship with the postsynaptic fiber. However, the same components recognized in the rod synapse can be observed. In animals maintained for 9 days in complete darkness there is also a considerable diminution in size of the synaptic vesicles.     

Like night and day: rods and cones have different pigment regeneration pathways

Synapse adhesion molecules play a key role in specifying and facilitating the recognition of axodendritic contacts. New studies by and reported in this issue of Neuron reveal multiple functions for the cadherin-catenin complex. This adhesion complex regulates synaptogenesis and coordinates synaptic strength with presynaptic and postsynaptic organization, including the shape of dendritic spines.     

Spatiotemporal cGMP dynamics in living mouse rods

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ~2 μm.     

Imaging adhesion and signaling dynamics in Xenopus laevis growth cones

Xenopus laevis provides a robust model system to study cellular signaling and downstream processes during development both in vitro and in vivo. Intracellular signals must function within highly restricted spatial and temporal domains to activate specific downstream targets and cellular processes. Combining the versatility of developing Xenopus neurons with advances in fluorescent protein biosensors and imaging technologies has allowed many dynamic cellular processes to be visualized. This review will focus on the techniques we use to visualize and measure cell signaling, motility and adhesion by quantitative fluorescence microscopy in vitro and in vivo.     

Molecular mechanism of spontaneous pigment activation in retinal cones

Spontaneous current and voltage fluctuations (dark noise) in the photoreceptor cells of the retina limit the ability of the visual system to detect dim light. We recorded the dark current noise of individual salamander L cones. Previous work showed that the dark noise in these cells arises from thermal activation of the visual pigment. From the temperature dependence of the rate of occurrence of elementary noise events, we found an Arrhenius activation energy E(a) of 25 +/- 7 kcal/mol (mean +/- SD). This E(a) is similar to that reported for the thermal isomerization of 11-cis retinal in solution, suggesting that the cone pigment noise results from isomerization of the retinal chromophore. E(a) for the cone noise is similar to that previously reported for the "photon-like" noise of rods, but the preexponential factor is five orders of magnitude higher. To test the hypothesis that thermal isomerization can only occur in molecules whose Schiff base linkage is unprotonated, we changed the pH of the solution bathing the cone outer segment. This had little effect on the rate of occurrence of elementary noise events. The rate was also unchanged when the cone was exposed to Ringer solution made up from heavy water, whose solvent isotope effect should reduce the probability, that the Schiff base nitrogen is naked.