Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature.

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.

The ciliary base is marked by a transition zone in which Y-shaped cross-linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y-shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA-binding component seen in cytochemical experiments.

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.

Methoxyindoles and photoreceptor metabolism: activation of rod shedding.

Using an in vitro eye-cup preparation, we have evaluated a potential relationship between methoxyindole metabolism and photoreceptor disk shedding. Melatonin, 6-chloromelatonin, and 5-methoxytryptophol all activate rod disk shedding in culture. The effect is compound specific since serotonin and N-acetylserotonin are without effect, and it is similar to shedding in vivo because it is evoked by light and is quantitatively comparable to a normal intact animal response. The results suggest the involvement of 5-methoxyindoles in the control of rhythmic photoreceptor metabolism.

Photoreceptor outer segments: accelerated membrane renewal in rods after exposure to light.

The rate of rod outer segment renewal in Rana pipiens tadpoles under constant light and under diurnal conditions of 12 or 2 hours light per day is significantly increased compared to that in animals in darkness. Furthermore, during 24 hours in light after 6 days in darkness the rate of renewal is three to four times that in darkness. In Xenopus laevis tadpoles the rate of renewal is more than five times greater during the first 8 hours of a normal diurnal cycle than during the following 16 hours. These observations demonstrate that bursts of renewal activity occur as a response to light, and suggest that a normal pattern of light alternating with darness plays a fundamental role in the regulation of rod outer segment turnover.

Circadian clock functions localized in xenopus retinal photoreceptors.

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.

Phase shifting the retinal circadian clock: xPer2 mRNA induction by light and dopamine.

A circadian clock is located in the retinal photoreceptors of the African clawed frog Xenopus laevis. These photoreceptor clocks are thought to govern a wide variety of output rhythms, including melatonin release and gene expression. Both light and dopamine phase shift the retinal clock in a phase-dependent manner. Two homologs of the Drosophila period gene have been cloned in Xenopus, and one of these (xPer2) is acutely regulated by light. Light and dopamine induce xPer2 mRNA in a similar manner. In addition, the increase of xPer2 mRNA in response to light and dopamine is the same at all times of day tested. In contrast, xPer1 mRNA exhibits circadian oscillations but is relatively insensitive to phase-shifting treatments of light or dopamine. Our data suggest that xPer2 functions as the molecular link between the light/dark cycle and the circadian clock.

Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length.

In lower vertebrates, cone retinomotor movements occur in response to changes in lighting conditions and to an endogenous circadian clock. In the light, cone myoids contract, while in the dark, they elongate. In order to test the hypothesis that melatonin and dopamine may be involved in the regulation of cone movement, we have used an in vitro eyecup preparation from Xenopus laevis that sustains light- and dark-adaptive cone retinomotor movement. Melatonin mimics darkness by causing cone elongation. Dark- and melatonin-induced cone elongation are blocked by dopamine. Dopamine also stimulates cone contraction in dark-adapted eyecups. The effect of dopamine appears to be mediated specifically by a dopamine receptor, possibly of the D2 type. The dopamine agonist apomorphine and the putative D2 agonist LY171555 induced cone contraction. In contrast, the putative D1 agonist SKF38393-A and specific alpha 1-, alpha 2-, and beta-adrenergic receptor agonists were without effect. Furthermore, the dopamine antagonist spiroperidol not only blocked light-induced cone contraction, but also stimulated cone elongation in the light. These results suggest that dopamine is part of the light signal for cone contraction, and that its suppression is part of the dark signal for cone elongation. Melatonin may affect cone movement indirectly through its influence on the dopaminergic system.

Effects of cyclic adenosine 3',5'-monophosphate on photoreceptor disc shedding and retinomotor movement. Inhibition of rod shedding and stimulation of cone elongation.

As a test of the hypothesis that cyclic nucleotides play a role in the regulation of retinomotor movements and disc shedding in the photoreceptor-pigment epithelial complex, we have used an in vitro eyecup preparation that sustains both disc shedding and cone retinomotor movements, Eyecups were prepared in white light from animals in which both shedding and cone movement had been blocked by 4 d of constant-light treatment. In eyecups incubated for 3 h in light, disc shedding was negligible and cones remained in the light-adapted (contracted) position. In eyecups incubated in darkness, however, a massive shedding response (dominated by rod photoreceptors) was induced, and at the same time cone photoreceptors elongated to their dark-adapted position. In eyecups incubated in light dbcAMP promoted cone elongation and thus mimicked darkness; the dbcAMP effect was potentiated by the phosphodiesterase inhibitors papaverine and 3-isobutylmethylxanthine. In eyecups incubated in darkness, on the other hand, both phosphodiesterase inhibitors and dbcAMP reduced the phagosome content of the pigment epithelium. The effects of dbcAMP on the cone elongation and rod shedding appear to be specific in that dbcGMP, adenosine, and adenosine 5'-monophosphate had no significant effect. Our results suggest that cAMP plays a role in the regulation of both retinomotor movements and disc shedding.

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse.

BACKGROUND: Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. RESULTS: cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. CONCLUSION: The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

Transgene expression in Xenopus rods.

The photoreceptors of the vertebrate retina express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod- and cone-specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219-226). The mechanisms that regulate gene expression in a rod/cone-specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99-126, Wiley-Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173-3183) can be used to study cell-specific expression in the retina. We have used a 5.5 kb 5' upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179-3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina and pineal, which is apparent by 4 days post-nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.

D2-like dopamine receptors in amphibian retina: localization with fluorescent ligands.

Dopamine induces several light adaptive changes in amphibian retina via receptors with D2-like pharmacology, but the identity of the primary target cells has not been determined. Using a fluorescent probe consisting of a selective D2 antagonist, N-(p-aminophenethyl)-spiperone (NAPS), derivatized with the fluorophore Bodipy (NAPS-Bodipy), we identified the distribution of dopamine binding sites in the retina of two amphibians, post-metamorphic Xenopus laevis and larval Ambystoma tigrinum. Specific labeling was defined as staining that was displaced by D2 selective ligands (eticlopride or sulpiride), but insensitive to D1 selective drugs (SCH 23390), adrenergic catecholamines (epinephrine or norepinephrine), or serotoninergic analogues (ketanserin). Both rod and cone cells showed specific dopamine D2-like binding sites arranged in clustered arrays on discrete membrane domains of the inner segment. Labeling of photoreceptor outer segments was continuous and was not displaced by competition with D2 selective ligands; this labeling was considered nonspecific. In addition, in both species, clustered binding of the D2-probe was found on Müller cells and on a subset of inner retinal cells with the morphology of amacrine/interplexiform cells. Our data provide direct evidence for D2 receptors on both rods and cones, and suggest that the receptors may be clustered into patches within a discrete cellular domain, the inner segment.

Stimulation of photoreceptor disc shedding and pigment epithelial phagocytosis by glutamate, aspartate, and other amino acids.

It has been reported that aspartate and glutamate selectively impair the structure (Olney, '82) and function (e.g., Furakawa and Hanawa, '55) of second- and third-order retinal neurons while leaving the photoreceptor unaffected. Either amino acid may mimic the endogenous photoreceptor neurotransmitter (Ehinger, '82). We report here that excitatory amino acids also induce massive rod photoreceptor disc shedding in eyecups of Xenopus laevis maintained in vitro. Disc shedding is the process whereby photoreceptors eliminate effete discs. It involves interaction between the distal outer segment and pigment epithelium. Millimolar L-aspartate and L-glutamate, as well as micromolar kainic acid, a glutamate analog, stimulate disc shedding three- to fivefold higher than normal light-evoked shedding levels and result in extensive inner retinal damage. Fifty-millimolar KCl, 1.0 microM ouabain, and replacement of sodium with choline also stimulate disc shedding and alter retinal structure. Extensive neurotoxicity appears unrelated to disc shedding since other amino acids having no significant or marginal effects on retinal structure also stimulate shedding. While the site and mechanism of action of these effectors, and in particular the excitatory amino acids, is now undefined, the data show that amino acids thought to act directly and specifically on inner retinal neurons can also markedly alter photoreceptor and pigment epithelial metabolism.

Circadian regulation of retinomotor movements: II. The role of GABA in the regulation of cone position.

Cone photoreceptor movements in lower vertebrates are regulated by the interaction of the light-dark cycle and an endogenous circadian clock. We have suggested that melatonin and dopamine interact to regulate dark- and light-adaptive movements, respectively, and that melatonin affects cones indirectly by inhibiting dopamine release. In fact, any factor modulating dopaminergic neurons in the retina may have effects on either cone elongation or contraction. We have utilized an in vitro eyecup preparation from the African clawed frog, Xenopus laevis, to evaluate a possible role of the neurotransmitter GABA, which is thought to tonically suppress dopamine release. GABA agonists mimic the effects of darkness and induce cone elongation; the effects of GABA agonists are blocked by dopamine. Muscimol-induced cone elongation occurs at low light intensity but is inhibited by bright light in eyecups prepared from cyclic-light-maintained animals. Although neither melatonin nor muscimol stimulates cone elongation in bright light, simultaneous application of both drugs induces elongation. The GABA antagonist picrotoxin induces cone contraction which is blocked by the dopamine receptor antagonist spiroperidol, which suggests that GABA may affect cone movement in Xenopus by regulating dopamine neurons. Consistent with this, picrotoxin-induced cone contraction is Ca+2 dependent and is blocked by high Mg+2 or the Ca+2 antagonist nifedipine. Pharmacological analysis suggests that the effects of GABA may result from its action at more than one receptor subtype. Our results support the hypothesis that dopamine is part of the light signal for cone contraction and that its suppression is part of the signal for cone elongation.

Light-evoked and kainic-acid-induced disc shedding by rod photoreceptors: differential sensitivity to extracellular calcium.

In order to study the light and Ca2+ dependence of disc shedding by rod photoreceptors, we have used eyecups prepared from adult Rana pipiens frogs that had been kept in constant light for 4 days. Disc shedding was initiated by a treatment involving 1 hour of darkness followed by exposure to light or by treatment with kainic acid. Maximal L-evoked disc shedding occurred quickly (within 30-60 minutes) after light onset and could be triggered by brief (15 minutes) exposure to light. L-evoked disc shedding was completely blocked by omission of Ca2+ from culture medium or by treatment with 3mM Co2+ or 12 mM Mg2+ in the presence of Ca2+ (2 mM). The response was also blocked by the organic Ca2+ antagonist nifedipine. Experiments designed to distinguish between Ca2+ dependence of the dark- or light-dependent processes necessary for shedding suggest that voltage-sensitive channels mediate a Ca2+-dependent process involved in light-triggering. Kainic acid caused a dose-dependent stimulation of disc shedding under lighting conditions (continuous culture in light or darkness) that did not normally result in a significant response in the absence of the drug. Disc shedding induced by kainic acid was similar in time course and magnitude to that induced by light. However, kainic-acid-induced disc shedding was not inhibited by medium Ca2+ reduction or by the presence of Co2+. The latter observation suggests that kainic acid activates disc shedding directly, by-passing the Ca2+-dependent process involved in the L-evoked response. The Ca2+-dependent process may involve release of an effector of disc shedding that is mimicked by kainic acid.

Effects of kynurenate and other excitatory amino acid antagonists as blockers of light- and kainate-induced retinal rod photoreceptor disc shedding.

Photoreceptor disc shedding in the retina involves detachment of discs from distal outer segments and phagocytosis of those discs by adjacent pigment epithelial cells. The disc-shedding process balances the continuous renewal of photosensitive membrane. In amphibians, rod disc shedding normally is light-stimulated. However, excitatory amino acids such as kainate stimulate disc shedding independent of a dark-light transition. Excitatory amino acid-induced disc shedding is accompanied by toxic changes within the retina. To evaluate the possible role of an endogenous excitatory amino acid in the regulation of light-evoked disc shedding, we examined the effects of excitatory amino acid antagonists on kainate-induced and light-evoked disc shedding and on retinal toxicity. Using eyecups from Rana pipiens, we found that kynurenate, D-O-phosphoserine, and cis-2,3-piperidine dicarboxylic acid (cis-PDA) all block both the neurotoxic and disc-shedding effects of kainate. Kynurenate and D-O-phosphoserine, but not cis-PDA, also block light-evoked disc shedding. Our analysis suggests that kynurenate blocks the mechanism by which light "triggers" disc shedding rather than directly inhibiting disc detachment and phagocytosis. The observation that cis-PDA antagonizes the effects of kainate, but not light, suggests that the receptor mediating the kainate effect on disc shedding may not be involved in the normal initiation of the response by light. In contrast, our data on kynurenate suggest that it antagonizes an endogenous mechanism involved in the normal control of disc shedding. Thus, analysis of the differences between cis-PDA and kynurenate as antagonists in the retina may yield important insight into the mechanism by which light initiates disc shedding.

Partial characterization of lectin binding sites of retinal photoreceptor outer segments and interphotoreceptor matrix.

We have used cytochemistry together with exoglycosidase digestion and polyacrylamide gel electrophoresis to partially characterize lectin binding sites of the interphotoreceptor matrix and of photoreceptor outer segments. In order to obtain uniform access of reagents to all regions of the preparation, we have used a procedure in which plastic sections are etched with sodium ethoxide prior to cytochemical analysis. Neuraminidase pretreatment of plastic-embedded sections of Xenopus laevis eyecups leads to a loss of wheat germ agglutinin binding and a concomitant appearance of Ricinus communis agglutinin binding to the interphotoreceptor matrix. In contrast, wheat germ agglutinin binding to the outer segments is not altered by the neuraminidase pretreatment. These results suggest that wheat germ agglutinin binding sites of interphotoreceptor matrix are sialoglyconjugates and that outer segment binding sites are not sialoglycoconjugates. Enzyme digestions followed by lectin cytochemistry of matrix polypeptides separated by polyacrylamide gel electrophoresis do not identify a likely candidate to give rise to the cytochemical staining patterns. Lectin cytochemistry of retinas from which the interphotoreceptor matrix has been extracted do not show a loss of wheat germ agglutinin binding sites to the matrix. These results suggest that the major wheat germ agglutinin binding sites in the interphotoreceptor matrix are to as yet unidentified sialoglycoconjugates.

Melatonin deacetylase activity in the pineal gland and brain of the lizards Anolis carolinensis and Sceloporus jarrovi.

Melatonin modulates a variety of rhythmic processes in vertebrates, and is synthesized in both the retina and pineal gland. We have shown previously that retinal melatonin is deacetylated generating 5-methoxytryptamine, which is then deaminated by monoamine oxidase, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. This process occurs within the eyes of a variety of vertebrates including the iguanid lizard Anolis carolinensis. To determine whether melatonin deacetylase activity also occurs in the pineal organ or in other parts of the lizard brain, pineals and brains of Anolis carolinensis and Sceloporus jarrovi were cultured in the presence of [3H-methoxy]-melatonin. High-performance liquid chromatography of the resulting culture media and tissues revealed the generation of radiolabeled 5-methoxytryptamine and 5-methoxyindoleacetic acid. These two methoxyindoles were the only radiolabeled metabolites detectable, and together accounted for all melatonin lost. Both the loss of melatonin and the production of melatonin metabolites were inhibited by inclusion of 100 microM eserine, an inhibitor of the melatonin deacetylase. Pargyline, a monoamine oxidase inhibitor, reduced the production of 5-methoxyindoleacetic acid and increased the production of 5-methoxytryptamine relative to control incubations. Similar effects of eserine and pargyline were seen in eyecup, brain and pineal gland, but the specific activity of melatonin deacetylation in cultured pineal glands was much greater than in either brains or eyecups. These results indicate that pineal glands of both Anolis carolinensis and Sceloporus jarrovi can rapidly catabolize melatonin by a mechanism very similar to that in the eye, that the melatonin deacetylation pathway exists elsewhere in the iguanid brain, and also extend our previous observations of ocular melatonin deacetylation to an additional species.

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors.

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.