Richard N. Lolley

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.

Protein phosphorylation in rod outer segments from bovine retina: Cyclic nucleotide-activated protein kinase and its endogenous substrate

Abstract Protein kinase activity of isolated rod outer segments from bovine retinas is activated by cGMP when in a soluble form, and it is cyclic nucleotide independent when associated with the rod outer segment membranes. The soluble protein kinase phosphorylates in a cyclic nucleotide-dependent manner only a single endogenous protein with an apparent molecular weight of 30,000 daltons. The 30,000-dalton phosphoprotein is localized specifically in the visual cells of the retina. It is proposed that the light-induced changes in cGMP levels that occur in rod outer segments in vivo are linked by the cyclic nucleotide-dependent protein kinase to alterations in the content of the 30,000-dalton phosphoprotein.

Cyclic nucleotide phosphodiesterases in dystrophic rat retinas: Guanosine 3′,5′ cyclic monophosphate anomalies during photoreceptor cell degeneration

Abstract The kinetic characteristics and enzyme activities of cyclic nucleotide phosphodiesterase (PDE), using cyclic-AMP (cyclic-AMP-PDE) or cyclic-GMP (cyclic-GMP-PDE) as substrate, have been measured in developing retinas of control and Royal College of Surgeons (RCS) rats. The latter are afflicted with an inherited degeneration of the retinal photoreceptor cells. Two classes of cyclic-AMP-PDE are localized in the inner layers and one is in the photoreceptor layer of both control and RCS retinas. In RCS retina, the activity of the photoreceptor-specific cyclic-AMP-PDE surpasses that of the control retina during most of the period when rod outer segment debris accumulates, and falls below that of the control retina when the photoreceptor population is depleted. These data suggest that the accumulated debris has the capacity to hydrolyze cyclic-AMP. One class of cyclic-GMP-PDE is localized in the inner layers and one is in the photoreceptor layer of both control and RCS retinas. The kinetic characteristics of cyclic-GMP-PDE in the layers of the RCS retina are similar to those of control retina for the first 14 days of life but, during the period when debris accumulates, the cyclic-GMP-PDE of the inner layers is not demonstrable and that of the photoreceptor layers shows altered kinetics. Thereafter, the latter disappears due to the depletion of the photoreceptor population, and cyclic-GMP-PDE of the inner layers is again observable. The anomalous kinetics and activities of cyclic-GMP-PDE apparently result from the association of cyclic-GMP-PDE enzymes with a heat-denaturable, non-dialyzable material which is derived from the RCS photoreceptor cells and/or accumulated debris. The specificity of the material for cyclic-GMP-PDE suggests that cyclic-GMP metabolism may be altered, in situ, before the RCS photoreceptor cells degenerate.

Cyclic GMP: proposed role in visual cell function.

The cyclic nucleotides are known to act in a variety of tissues as the second messengers of neurotransmitter or hormone action. Our studies of the inherited retinal degenerative diseases of rd mice (Farber and Lolley, 1974, 1976) RCS rats (Lolley and Farber, 1975, 1976) and Irish Setter dogs (unpublished observations) demonstrate that an abnormality in cyclic GMP metabolism within visual cells is an early biochemical defect that precedes morphological pathology. Moreover, our recent investigations of isolated rod outer segments (ROS) from bovine retinas show that ROS possess the necessary biochemical components for translating light-induced changes in cyclic GMP concentration into a physiologically significant message, i.e. an ROS-specific phosphoprotein (Lolley, Brown and Farber, 1977). The work with the inherited disorders implies that cyclic GMP is serving a fundamentally important role in visual cell metabolism, and the biochemical studies of isolated ROS suggest that this role may be linked directly to the control of ion permeability in the ROS and visual cells. A model showing a relationship between rhodopsin, cyclic GMP modulation of enzyme activity, and ion permeability of the plasmalemma (Fig. 1) summarizes our current understanding of ROS biochemistry. If one disregards the effects of light, it shows that the level of cyclic GMP in darkness ,is high (Krishna er al., 1976). This results from low, basal activity of cyclic GMP-phosphodiesterase @‘DE)--the enzyme that hydrolyzes cyclic GMP to S’GMP (Bitensky, Miki, Keirns, Keirns, Baraban, Freeman, Wheeler, Lacy and Marcus, 1975). The high concentration of cyclic GMP activates and drives a soluble, cyclic GMPdependent protein kinase (PK,). This PK, has characteristics that are similar to those reported for cyclic nucleotidedependent protein kinases of other tissues, i.e. the holoenzyme is composed of regulatory and catalytic subunits (Krebs, 1972). For example, cyclic GMP binds to the regulatory component and releases an active catalytic subunit (PK: + PK:). The PK: catalyzes the phosphorylation of a single soluble protein of the ROS with an apparent molecular weight of 30,000 daltons (30 K) (Lolley et al., 1977). Biochemical studies of PK, and of a soluble phosphoprotein phosphatase (PPP) (unpublished observations) suggest that the 30,OOOdalton protein is maintained primarily in the phosphorylated form (30 K-P > 30 K) in the dark. Since all of the known actions of cyclic nucleotides are expressed through the activation of protein kinases which phosphorylate proteins selectively, we propose that the 30,OOOdalton phosphoprotein (30K-P) is responsible for the physiological action of cyclic GMP. We propose further that interactions of the 30K-P with the ROS plasmalemma and/or with a calcium-sequestering system will facilitate the passage of sodium ions across the membrane and provide the mechanism for sustained depolarization of the ROS in darkness. The proposed mechanism is similar to that suggested for the transient depolarization of neurons at certain postsynaptic junctions (Greengard, 1975), except that the condition in ROS is sustained in the dark state. When light impinges upon an ROS (see model, Fig. l), the bleaching of rhcdopsin initiates a decrease in the level of cyclic GMP which causes a reduction in the content of. the 30,000-dalton phosphoprotein (30 K-P). The resulting shift toward dephosphorylation of the 30 K-P, i.e. increase in 30 K/30 K-P ratio, facilitates the closure of Na+ channels in the ROS plasmalemma, i.e. hyperpolarization. In biochemical terms, the bleaching of rhodopsin activates cyclic GMP-phosphodiesterase (Bitensky et al., 1975) causing a rapid (detectable in milliseconds) fall in cyclic GMP levels (Woodruff, Bownds, Green, Morrisey and Shedlovsky, 1977). Several thousand cyclic GMP molecules may be hydrolyzed per rhodopsin bleached. The reduction in cyclic GMP concentration will reduce the activity of the soluble protein kinase (PK: + PK:) and slow the rate of phosphorylation of the 3O,OOOdalton protein (30 K). Consequently, the concentration of the soluble phosphoprotein (30K-P) will fall, as it is dephosphorylated by the phosphoprotein phosphatase (PPP), and its interaction with the ROS plasmalemma and/or calcium metabolism will be diminished. The net effect of light will be to reverse the dark state and, through closure of sodium ion channels, cause hyperpolarization of the ROS and visual cell. This model provides for amplification (lOOO-2OCNl molecules of cyclic GMP hydrolyzed/molecule of rhodopsin bleached) of the visual signal and for a link (protein kinase/3O,Oodalton phosphoprotein complex) between rhodopsin bleaching in the ROS disk membranes and ion channels in the ROS plasmalemma. It proposes simply that cyclic GMP is a second messenger of the visual signal.

A proposed link between debris accumulation, guanosine 3′,5′ cyclic monophosphate changes and photoreceptor cell degeneration in retina of RCS rats

Abstract The content of cyclic-AMP and cyclic-GMP, the activity of cyclic-GMP phosphodiesterase (PDE), and the influence of RCS debris upon the kinetics of cyclic-GMP-PDE were measured in developing retina of control and RCS rats. Additionally, the distribution of cyclic-GMP within the retina was determined in microdissected layers of control and RCS retina. The content of cyclic-GMP in RCS retina increases like that of the control for the first 10–15 days, and then it becomes stable at a level which is below that of the control. The activity of cyclic-GMP-PDE changes during development, following a pattern similar to that of the cyclic-GMP concent. Experiments in which control and RCS homogenates were mixed and assayed for PDE activity show that RCS debris is capable of modifying the kinetic characteristics of cyclic-GMP-PDE both from the control and the RCS retina. The temporal order in which abnormalities occur in the developing RCS retina suggest that a link exists between the accumulation of debris, the induced abnormality in cyclic-GMP metabolism, and the cause of photoreceptor degeneration in the RCS retina.

Calcium and magnesium content of rodent photoreceptor cells as inferred from studies of retinal degeneration

Abstract The content of calcium and magnesium was determined in control and degenerative retinas of mice and rats. The level of both cations was reduced in these disorders during the period of photoreceptor cell degeneration. The number of cells lost from these retina was estimated from DNA measurements, and the divalent cation content of an average photoreceptor cell was computed. From these data and the literature, it is suggested that an average photoreceptor cell of a mouse or rat retina contains 2–3 fmol of calcium, 3–4 fmol of magnesium and about 0·1 fmol of rhodopsin.

Metabolic and Anatomical Specialization Within the Retina

The retina comprises the photosensitive and neural integrative portion of the eye. It develops embryologically as an ectopic portion of the primitive fore-brain, and it never loses its similarity to the central nervous system. The inner layers of the mature retina show a lamanellar organization comparable to that of the gray matter in the cortex. The optic nerve shows the compartmentation by pia and the absence of sheaths of Schwann that characterize white matter in brain(1); thus, the optic nerve has the characteristics of a central nervous system fiber tract.

Cyclic GMP and visual cell degeneration in the inherited disorder of rd mice: a progress report

Abstract Cyclic GMP accumulates in visual cells of rd (retinal degeneration) mice before the onset of morphological pathology. Observations are presented which support the hypothesis that elevated levels of cyclic GMP initiate visual cell degeneration in some early-onset disorders causing blindness. The accumulation of cyclic GMP in rd visual cells results apparently from defective mechanisms that regulate cyclic GMP metabolism.

Influence of visual cell maturation or degeneration on cyclic AMP content of retinal neurons.

NEUROWTARGET cell interactions have been studied in a variety of tissues. In the peripheral nervous system. the devclopniciii :tnd maintenance of taste huds (G[’Ttl. 1974) serves as one exaiiiplc. . i i d 111 111s C’KS the use of chimeric animals has furthered our understanding of neuron neuron interactions. In addition to these and other (MULLEN, 1975) morphological works. BLACK & MYTILINEOU (1976) have provided solid biochemical evidence for reciprocal regulatory relationships between cholinergic and adrenergic neurons at the synapse during development. These observations have motivated us to consider whether adenosine 3’,5‘-monophosphate (cyclic AMP) metabolism is altered in the inner layers of the retina by the loss of photoreceptor cells. Using rd (retinal degeneration) mice and RCS (Royal College of Surgeons) rats as models of inherited retinal degenerative diseases, we were able to evaluate cyclic AMP metabolism in retinae where photoreceptor degeneration occurred during or after synapses were established in the inner synaptic layer of the retina. For example, visual cell degeneration occurs during the period of synapse formation in the rd retina (BLANKS et a/., 19744, and i t occurs after the maturation process is completed in the RCS retina (DOWLING & SIDMAN, 1962). In the retinae of C3H/HeJ mice. which possess the rd gene, photoreceptor degeneration begins at about 10 postnatal days, and i t is essentially complete by 20 days (NOELL, 1965). During this time period, the visual cells of the retina of normal mice form synapses with horizontal and bipolar cells (BLANKS er a/.. 1974a). and the retina begins to exhibit an ERG pattern with adult characteristics (NOELL, 1965). In C3H mice, a synapse between photoreceptor and bipolar cells does not develop fully (BLAWS et a/., 1974b) and the ERG is greatly attenuated (NOELL, 1965). In the inherited retinal disorder of RCS rats, the visual cells are relatively mature morphologically and physiologically (DOWLING & SIVMAK, 1962) when they begin to degenerate. Several lines of biochemical evidence now have demonstrated that the metabolism of cyclic A M P in the retina occurs primarily within its inner neural layers. The photoreceptor cells are particularly deficient in cyclic AMP

Changes in levels of ATPase activity in developing retinae of normal (DBA) and mutant (C3H) mice

Abstract The levels of activity of Mg 2+ -ATPase and Na + -K + -activated ATPase were determined in retinae of developing DBA/1J and C3H/HeJ mice. When levels of ATPase activity were expressed per unit of dry weight, no apparent difference between normal and mutant retinae was noted during postnatal development. When levels of ATPase activity were expressed per complete retina, a decrease in Mg 2+ - and Na + -K + -activated ATPase was observed in C3H retinae which correlated temporally with the development of cellular degeneration. It is concluded that the ATPase enzyme system is not involved in the etiology of the inherited disease.