Cyclic-nucleotide phosphodiesterase: an early defect in inherited retinal degeneration of C3H mice.

Cyclic nucleotides have been implicated in the differentiation and function of the vertebrate retina. In the normal retina of DBA mice, the specific activity of cyclic-nucleotide phosphodiesterase (PDE), with cyclic-AMP as the substrate (cAMP-PDE), increases eightfold between the 6th and 20th postnatal day. Kinetic analysis of retinae from newborn mice reveals a PDE with a single Michaelis constant (K(m)) value for cyclic-AMP (low K(m)-PDE). After the 6th postnatal day, a second PDE with a high K(m) for cyclic-AMP (high K(m)-PDE) can be demonstrated. The appearance and increasing activity of the high K(m)-PDE coincides with the differentiation and growth of photoreceptor outer segments. Additionally, the high K(m)-PDE is shown by microchemical techniques to be concentrated in the photoreceptor cell layer and the low K(m)-PDE within the inner layers of the normal retina. In C3H mice afflicted with an inherited degeneration of the photoreceptor layer, the postnatal increase in the specific activity of cAMP-PDE is substantially lower than in the normal retina. The postnatal increase in the specific activity of cAMP-PDE in two regions of the brain of C3H mice is the same as in the normal strain. A deficiency in high K(m)-PDE activity in the C3H retina is evident on the 7th postnatal day, when the activity of low K(m)-PDE, photoreceptor morphology, and rhodopsin content of these retina are essentially normal. In the adult C3H retina, the PDE activity with cyclic-GMP and cyclic-UMP as substrates is significantly below that of the normal retina. These data indicate that an alteration in cyclic-AMP metabolism occurs before photoreceptor cell degeneration in the retinae of C3H mice.

Cyclic guanosine monophosphate: elevation in degenerating photoreceptor cells of the C3H mouse retina.

As a result of an early deficiency in cyclic nucleotide phosphodiesterase activity, guanosine 3',5'-monophosphate accumulates in retinal photoreceptor cells before they begin to degenerate. It is suggested that degeneration of the photoreceptor cells is related to an imbalance in their metabolism or function which is caused by the elevated levels of cyclic guanosine monophosphate.

Cyclic GMP accumulation causes degeneration of photoreceptor cells: simulation of an inherited disease.

Guanosine 3',5'-monophosphate (cyclic GMP) metabolism in developing eye rudiments of Xenopus laevis embryos in culture is disrupted by the phosphodiesterase inhibitor isobutylmethylxanthine. At low concentrations of inhibitor the rudiments develop normally, but at higher concentrations of the inhibitor, cyclic GMP accumulates in the rudiments and the retinal photoreceptor cells degenerate selectively. The isobutylmethylxanthine-induced photoreceptor degeneration is associated with an accumulation of cyclic GMP and, in this respect, it stimulates an early biochemical defect in the inherited degenerative disease of rd mice.

Linkage of photoreceptor degeneration by apoptosis with inherited defect in phototransduction.

PURPOSE: To investigate the developing retina of normal and rd/rd mice to establish if the inherited defect in the retinal degeneration (rd) gene, encoding the beta subunit of the cascade phosphodiesterase, is associated with rd photoreceptor degeneration by apoptosis. METHODS: DNA content of developing normal and rd/rd retinas was measured spectrophotometrically and analyzed for differential loss during the course of photoreceptor degeneration. Degenerating rd photoreceptors were evaluated by electron microscopy for cytoplasmic features and chromosomal condensation. DNA fragmentation was analyzed by agarose gel electrophoresis at daily intervals during the developmental period in which rd/rd cell death occurs. RESULTS: DNA loss from developing rd/rd retinas is maximal between 10 and 15 postnatal days. Photoreceptor cells die individually throughout the postnatal period of degeneration, with pycnotic nuclei dispersed among morphologically normal rd photoreceptors. DNA fragmentation into 200 base pair multiples occurs maximally in rd/rd retinas between 10 and 15 postnatal days. CONCLUSION: Photoreceptor cell death in developing rd/rd retinas occurs by a mechanism that links a defect in the phototransduction cascade with a program for cell death, called apoptosis.

Rod photoreceptor cells dissociated from mature mice retinas.

Intact rod photoreceptors were dissociated from pronase-treated whole retinas of adult mice by repeated passage through a plastic pipette tip. Hemocytometer counts of the cell suspensions indicate that, during a series of ten dissociation steps, a total of about 1-2 million intact photoreceptor cells are dissociated from one adult mouse retina, with less than 5% contamination from Müller cells and neurons of the inner retina. Visual cells with rod outer segments (ROS) and synaptic terminals are released in each step, but they occur in the greatest number during the sixth to ninth steps; detached ROS are released most frequently in the early steps, and neurons of the inner retinal layers appear in the later steps of dissociation. Nuclei are found in each step. Cell intactness was estimated by Trypan blue and Erythrosin B exclusion and by microscopic analysis using differential interference optics or scanning electron microscopy. The cells bind lectins (concanavalin A, Ricinis communis, and wheat germ agglutinin but not peanut agglutinin), displaying surface topography like that observed in situ. The metabolic capacity of dissociated cells was assessed by measuring the utilization of 32P inorganic phosphate for the synthesis of phospholipids and for the light-dependent phosphorylation of rhodopsin. Mature photoreceptor cells were estimated to contain, on average, 6.4 X 10(-12) g DNA, 2.3 X 10(-12) g RNA and 42-64 X 10(-12) g protein. The dissociation procedure provides a population of photoreceptor cells that appears suitable for microscopic, electrophysiological, and biochemical analysis.

Distribution patterns of photoreceptors, protein, and cyclic nucleotides in the human retina.

The concentration of cGMP, cAMP, protein and the number of cone and rod photoreceptors have been measured in parallel arrays of punches, 3 mm in diameter, taken from each quadrant of normal human retinas. A separate punch containing the fovea and parafoveal region was also analyzed. Eyes were obtained from four male donors ranging in age from 35 to 67 yr. The retina thins considerably from the center to the periphery, and consequently the protein content forms a gradient in the same direction. Similar gradients were observed for cAMP and cGMP concentrations. In all eyes studied, the foveal-parafoveal region had higher levels of cAMP than cGMP. The data was analyzed with the aid of a computer in order to obtain three-dimensional maps of the patterns of distribution of the different parameters. A strong correlation between the areas of higher cone density, non-photoreceptor neurons, and cAMP, and an equally strong correlation between rod distribution and that of cGMP was observed. These maps will serve as baseline data in studies of pathological conditions such as retinitis pigmentosa.

Characterization of a phosphodiesterase-immunoreactive polypeptide from rod photoreceptors of developing rd mouse retinas.

In the inherited retinal degeneration of rd mice, cyclic GMP accumulates in affected rod photoreceptors prior to their degeneration. A deficiency in the activity of the visual cell phosphodiesterase apparently results in the accumulation of cyclic GMP. The cyclic GMP phosphodiesterase (PDE) of normal mouse photoreceptors is a heteromeric protein complex of about 170 kDa, consisting of the alpha beta catalytic unit and the gamma inhibitory unit. The isolated complex has low enzyme activity but it can be activated by incubation with histone. Affinity-purified polyclonal antibodies against the PDE complex of bovine rod outer segments were prepared and used to identify in retinas of both normal and rd mice PDE-immunoreactive polypeptides which comigrated on SDS-polyacrylamide gels with the large subunits (88 kDa) of the normal PDE complex. During development of normal retinas, the 88 kDa immunoreactive component of the PDE complex were detected by day 7, with immunoreactivity increasing throughout the second postnatal week. In rd retinas, the 88 kDa immunoreactivity increased after 9 postnatal days, decreased during rod photoreceptor degeneration, and was undetectable in mature rd retinas. Under nondenaturing conditions, the PDE-immunoreactive polypeptide of rd retinas sedimented on sucrose gradients with a sedimentation coefficient of 5.6S and an apparent molecular mass of about 105 kDa; no associated histone-activated PDE activity was detected. These findings show that PDE-immunoreactive polypeptides are synthesized in immature rd photoreceptors and that the PDE-immunoreactive polypeptides fail to form a PDE complex which is comparable to that of normal photoreceptors.

Phosphodiesterase-probes show distinct defects in rd mice and Irish setter dog disorders.

The phosphodiesterase from the visual cells of rd mice and affected Irish setter dogs has been analyzed, using biochemical, biophysical, and immunological techniques. The authors' findings demonstrate that the mechanisms that cause a deficiency in phosphodiesterase activity in rd mice and Irish setter dogs are distinctly different. Apparently, the phosphodiesterase complex is normal in affected Irish setter dogs but is abnormal in rd mice. The criteria used for determining the normalcy of the phosphodiesterase complex were sedimentation characteristics, immuno-cross-reactivity, and histone-activation, which is shown to be a unique characteristic of the visual cell enzyme. According to these criteria, the phosphodiesterase complex in the visual cells of rd mice is either absent or abnormal from the onset of visual cell differentiation until degeneration, because it exhibits no cross-reactivity with antibody to phosphodiesterase; it is not activated by histone; and if present, it exhibits abnormal sedimentation characteristics and perhaps subunit structure. On the other hand, phosphodiesterase from the visual cells of affected Irish setter dogs is normal by the same criteria, because it cross-reacts with antibody against phosphodiesterase; it is activated by histone; and it exhibits normal sedimentation and electrophoretic patterns. It is proposed that depressed levels of phosphodiesterase activity in affected setter photoreceptors are due, perhaps, to a defect in the light-initiated cascade which activates the enzyme normally, in situ.

Cyclic nucleotides of cone-dominant retinas. Reduction of cyclic AMP levels by light and by cone degeneration.

Dark-adapted retinas or whole eyes of 13-line ground squirrels (Citellus tridecemlineatus) and western fence lizards (Sceloporus occidentalis) contain higher levels of cyclic AMP than of cyclic GMP. In these cone-dominant retinas, light reduces cyclic AMP content selectively. Freezing of dark- or light-adapted retinas or eyes also reduces cyclic AMP content, with only minimal changes in cyclic GMP levels. In addition, exposure of frozen retinas of dark-adapted ground squirrel to light results in a significant decrease in cyclic AMP content. The destruction of cone visual cells of ground squirrel retina by iodoacetic acid injection decreases the cyclic nucleotide content of the dark-adapted retina. Considering the relative loss of cyclic nucleotides from cone degeneration, we estimate that the content of cyclic AMP in visual cells of ground squirrel retina is about four times greater than that of cyclic GMP.

Cyclic GMP metabolic defects in inherited disorders of rd mice and RCS rats.

The inherited diseases of rd mice and RCS rats are compared. The rd disorder is characterized by the failure of rod visual cells to differentiate fully, by the accumulation of cyclic GMP, which results from a reduced level of phosphodiesterase activity, and by the rapid degeneration of visual cells, which is unaffected by light. The RCS rat disorder is characterized by the failure of pigment epithelium cells to phagocytize shed membranes of rod outer segments, which accumulate as debris, by a debris-associated reduction in cyclic GMP content, and by the slow degeneration of visual cells, which is accelerated by light. Both disorders result in blindness and both display abnormalities in cyclic GMP metabolism which occur before visual cells degenerate. Identification of a role for cyclic GMP in visual cell metabolism or function may suggest how either elevated levels of cyclic GMP in rd retinas or reduced levels of cyclic GMP in RCS retinas fit into the pattern of visual cell degeneration.

Cyclic nucleotides in rod- and cone-dominant retinas.

Rod-and cone-dominant retinas differ in their relative content of cyclic AMP and cyclic GMP. Cyclic GMP is concentrated in retinas dominated by rods and is responsive to light; cyclic AMP is enriched in those having a majority of cones. Light reduces by about 50% the content of cyclic AMP in cone-dominant retinas, but the levels of cyclic GMP are affected only minimally. Microdissection of rod-dominant retinas shows that most of the cyclic GMP is localized in photoreceptor cells whereas cyclic AMP is evenly distributed throughout the retina. In contrast, our studies of cyclic nucleotides and the morphological changes that occur in cone visual cells of the ground squirrel retina, during hibernation and iodoacetate-induced cone degeneration, suggest that cyclic AMP is localized in cone visual cells. By analogy with the rod system, cyclic AMP may modulate the intracellular metabolism of cones.

Calcium modulation of cyclic GMP synthesis in rat visual cells.

The synthesis of cyclic GMP in dark-adapted rat retinas, retinal homogenates or isolated ROS is stimulated during incubation with medium containing low levels of Ca2+. The guanylate cyclase that is stimulated by low [Ca2+] is localized exclusively in visual cells of the retina because the stimulatory effect of low [Ca2+] is observed in developing retinas only after visual cells begin to differentiate, and it is lost in diseased retinas when the photoreceptor cells degenerate. The accumulation of cyclic GMP during incubation with low [Ca/+] is prevented by illumination; the effect of light stems apparently from the light-enhanced hydrolysis of cyclic GMP. Following light adaptation and transfer of the animals to darkness, retinas become progressively more responsive to low [Ca2+], and a maximal response is restored after about 30 min of dark adaptation in vivo. Incubated retinas accumulate cyclic GMP when exposed to media containing less than about 5 x 10(-9) M [Ca2+], whereas the synthesis of cyclic GMP in retinal homogenates or lysed ROS is stimulated at concentrations of less than 10(-6) M-Ca2+. These findings indicate that calcium acts as an inhibitory effector in the regulation of guanylate cyclase in rod photoreceptor cells, and they suggest that changes in intracellular [Ca2+] may regulate the synthesis of cyclic GMP in dark-adapted visual cells in situ.

Induction of experimental autoimmune uveitis by the retinal photoreceptor cell protein, phosducin.

Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell mediated inflammatory diseases of the retina and uveal tract of the eye and the pineal gland respectively. They can be induced in experimental animals by immunization with several well characterized retinal autoantigens. We induced a mild to moderate EAU and EAP in Lewis rats by immunization with phosducin, a 33K retinal phosphoprotein which is involved in the phototransduction of vision. In contrast to the severe EAU induced by other retinal antigens like S-antigen (SAg) or interstitial retinoid binding protein (IRBP), the clinical disease was late in onset, low grade in severity and predominantly affected the posterior segment of the eye. Our study demonstrates that another photoreceptor cell protein, phosducin, is capable of eliciting EAU and EAP.