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J C Besharse

Publications and source records attributed to J C Besharse.

At least 37 records · Page 2Linked to original sources
PubMed1994

Transport of phosphatidylcholine to Xenopus photoreceptor rod outer segments in the presence of tunicamycin.

Study of the dynamics of membrane protein and phospholipid transport from the inner to the outer segment of vertebrate photoreceptors has shown an interesting dissociation of the two components under a number of experimental treatments which inhibit protein synthesis or transport. Under conditions which block the addition of opsin to outer segments, various lipids continue to be synthesized and transported to the outer segment in the presence of monensin, puromycin, brefeldin A, tunicamycin and several general metabolic inhibitors. In the current study, isolated retinas from adult Xenopus laevis were incubated with or without 20 micrograms mg-1 of tunicamycin in total darkness or light for 2-12 h in the presence of [3H]choline to study the dependence of phosphatidylcholine synthesis and transport on protein transport to the outer segment. Phosphatidylcholine is a major bulk lipid of outer segments, comprising close to one half of the phospholipid of outer segment phospholipids, and blocking choline uptake in retinas is known to cause photoreceptor degeneration. Biochemical analysis demonstrates that tunicamycin does not block the synthesis of phosphatidylcholine in photoreceptor inner segments or transport of radiolabelled phosphatidylcholine to outer segments during 6 h incubations with [3H]choline in light or total darkness. Light and electron microscopic autoradiography and morphometric analysis show that [3H]choline radiolabelled phospholipid does not accumulate in a band of newly formed basal discs in the outer segment or in the tubulo-vesicular structures which accumulate in the intersegmental space of tunicamycin-treated retinas. We conclude that transport of phosphatidylcholine can occur independently of opsin transport to the outer segment but whether this represents two separable components of a single pathway or involves two distinct routes of transport to the outer segment is still unresolved.

Animals↗
PubMed1994

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.

5-Methoxytryptamine↗
PubMed1994

Tryptophan hydroxylase expression is regulated by a circadian clock in Xenopus laevis retina.

A circadian clock has been localized to the photoreceptor layer in the Xenopus laevis retina. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin synthesis in Xenopus laevis retina is tryptophan hydroxylase. A cDNA clone coding for Xenopus tryptophan hydroxylase was isolated, characterized, and used as a probe for analysis of tryptophan hydroxylase mRNA expression. Northern blot analyses of total retinal RNA show that the tryptophan hydroxylase message levels are low in the day and higher at night. The expression of tryptophan hydroxylase mRNA is under circadian control because rhythmic changes are also seen in constant darkness, with elevated levels during the subjective night. Nuclear run-on analysis during the first subjective day in constant darkness revealed that transcription initiation is low early in the day and increases throughout the day. Our observations suggest that the circadian clock modulates tryptophan hydroxylase gene expression. An understanding of how the circadian clock controls tryptophan hydroxylase expression may lead to a clearer understanding of the melatonin biosynthetic pathway, and possibly the clock itself.

Amino Acid Sequence↗
PubMed1993

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.

Ambystoma↗
PubMed1993

Tunicamycin does not inhibit transport of phosphatidylinositol to Xenopus rod outer segments.

Tunicamycin inhibits the dolichol pathway for N-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. Adult Xenopus retinas were preincubated in dim red light with 20 micrograms ml-1 of tunicamycin for one hour followed by incubation in the light for 2-6 h with tunicamycin plus either [3H]mannose, [3H]leucine, [2-(3)H]glycerol or [3H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [3H]leucine and [3H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [3H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [3H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [3H]glycerol and [3H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Solubilization and biochemical characterization of the melatonin deacetylase from Xenopus laevis retina.

Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N-acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.

Amidohydrolases↗
PubMed1993

Gamma-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia.

gamma-Tubulin, a newly discovered member of the tubulin superfamily required for microtubule nucleation, is associated with the centrosome(s) throughout the vertebrate cell cycle. We have used a polyclonal antibody, generated against a highly conserved segment of gamma-tubulin, to localize this protein in postmitotic, ciliated cells, in which the major microtubule organizing centers are the basal bodies. Single-cilium photoreceptor cells from bovine retina contained a strongly immunoreactive species, with molecular characteristics of gamma-tubulin, in association with a detergent-resistant, cytoskeletal fraction devoid of cytoplasmic microtubules. gamma-Tubulin was discretely localized throughout the basal body region, extending opposite to the axonemal shaft, in mechanically detached rod outer segments and whole-mounted, connecting cilium-derived axonemes. In multiciliated epithelia from bovine trachea and oviduct, gamma-tubulin immunoreactivity was detected at the base of the cilia, where basal bodies are located. These results suggest that this key centrosomal protein of mitotically active cells is also an integral component of microtubule organizing centers, required for the generation of the microtubule network in terminally differentiated cells.

Animals↗
PubMed1992

The "ON"-bipolar agonist, L-2-amino-4-phosphonobutyrate, blocks light-evoked cone contraction in xenopus eye cups.

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought to be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing "ON"-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups from Xenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.

Aminobutyrates↗
PubMed1992

Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups.

In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Light-evoked contraction of red absorbing cones in the Xenopus retina is maximally sensitive to green light.

To test the hypothesis that light-evoked cone contraction in eye cups from Xenopus laevis is controlled through a direct mechanism initiated by the cone's own photopigment, we conducted spectral-sensitivity experiments. We estimate that initiation of contraction of red absorbing cones (611 nm) is 1.5 log units more sensitive to green (533 nm) than red (650 nm) light stimuli. The difference is comparable to that predicted from the spectral-sensitivity function of the green absorbing, principal rod (523 nm). Furthermore, 480-nm and 580-nm stimuli which are absorbed nearly equally by the principal rod have indistinguishable effects on cone contraction. We also found that light blockade of nighttime cone elongation is much more sensitive to green than to red light stimuli. Our observations are inconsistent with the hypothesis tested, and suggest that light-regulated cone motility is controlled through an indirect mechanism initiated primarily by the green absorbing, principal rod.

Animals↗
PubMed1992

Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina.

Several lines of evidence indicate that retinal photoreceptors produce melatonin. However, there are other potential melatonin sources in the retina, and melatonin synthesis can be regulated by feedback from the inner retina. To analyze cellular mechanisms of melatonin regulation in retinal photoreceptors, we have developed an in vitro method for destruction of the inner retina that preserves functional photoreceptors in contact with the pigment epithelium. Eyecups, which include the neural retina, retinal pigment epithelium, choriod, and sclera were prepared. The vitreal surface of the retina in each eyecup was washed sequentially with 1% Triton X-100, water, and culture medium. This lysed the ganglion cells and neurons and glia of the inner nuclear layer, causing the retina to split apart within the inner nuclear layer. The damaged inner retina was peeled away, leaving photoreceptors attached to the pigment epithelium. The cell density of the inner nuclear layer was reduced 94% by this method, but there was little apparent damage to the photoreceptors. Lesioned eyecups produced normal melatonin levels in darkness at night, and melatonin production was inhibited by light. These results indicate that the inner retina is not necessary for melatonin production nor for regulation of photoreceptor melatonin synthesis by light. The lesion method used in this study may be useful for other physiological and biochemical studies of photoreceptors.

Animals↗
PubMed1991

Melatonin deacetylation: retinal vertebrate class distribution and Xenopus laevis tissue distribution.

Deacetylation is a rapid clearance mechanism for ocular melatonin. We have studied the distribution of retinal melatonin deacetylase activity among vertebrate classes. Exogenous radiolabeled melatonin is metabolized by ocular tissue prepared from the amphibian Xenopus laevis, the reptile Anolis carolinensis, the teleost fish Carassius auratus, and the bird Gallus domesticus. In contrast, we were unable to detect ocular melatonin breakdown in rat or pig. In each species exhibiting ocular melatonin breakdown, melatonin is first deacetylated to 5-methoxytryptamine, which is deaminated, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Deacetylation of melatonin is inhibited by eserine (physostigmine), causing a reduction in the levels of all 3 metabolites. Deamination of 5-methoxytryptamine is inhibited by the monoamine oxidase inhibitor pargyline, such that 5-methoxyindoleacetic acid and 5-methoxytryptophol levels are decreased while levels of 5-methoxytryptamine are increased. Incubation with the deacetylase inhibitor eserine increases endogenous melatonin levels in Xenopus and Carassius eyecups, indicating that endogenous melatonin is metabolized via the deacetylase. We also studied the tissue distribution of the deacetylase in Xenopus laevis. Melatonin deacetylation occurs in retina, retinal pigment epithelium, and skin, all of which are sites of melatonin action. These results indicate that among non-mammalian vertebrates, deacetylation is a common clearance mechanism for ocular melatonin, and may degrade melatonin at other sites of action as well. Melatonin deacetylation may help regulate local melatonin concentration, and generates other biologically active methoxyindoles.

Amidohydrolases↗
PubMed1991

Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock.

1. Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions. 2. Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species. 3. The development of suitable in vitro retinal preparations, particularly the eyecup from the African clawed frog, Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation. 4. Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotonin N-acetyltransferase, and a novel pathway for degradation of melatonin within the retina. 5. Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator. 6. A recently developed in vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms. 7. A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.

Animals↗
PubMed1991

Resetting the circadian clock in cultured Xenopus eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine receptors.

A circadian oscillator is located within the eye of Xenopus laevis. This oscillator regulates retinal melatonin synthesis, stimulating it at night. The primary goal of the studies reported here was to define input pathways to this circadian oscillator as a step toward identification of circadian clock mechanisms. A flow-through superfusion culture system was developed to monitor circadian rhythms of melatonin release from individual eyecups. This system was used to determine the effects of light and dopaminergic agents on melatonin production and on the phase of the circadian oscillator. Six hour light pulses suppressed melatonin production and reset the phase of the free-running melatonin rhythm. Light pulses caused phase delays when applied during the early subjective night, phase advances when applied during the late subjective night, and no phase shift when applied during the subjective day. Dopamine receptor agonists mimicked light in suppressing melatonin release and resetting the phase of the circadian rhythm. The phase-response relationship for phase shifts induced by quinpirole, a D2 dopamine receptor agonist, was similar to that for phase shifts induced by light. Pharmacological analysis with selective catecholamine receptor agonists and antagonists indicated that there are pathways to the melatonin-generating system and the circadian oscillator that include D2 dopamine receptors. A D2 receptor antagonist, eticlopride, completely blocked the effects of dopamine on melatonin release and on circadian phase. However, eticlopride did not alter similar effects induced by light, indicating that dopamine-independent pathways exist for light input to these systems. The effects of light and quinpirole on melatonin release and circadian phase were not additive, indicating that the pathways converge. These pathways to the circadian oscillator in the retina present new avenues for pursuit of cellular circadian clock mechanisms.

Animals↗
PubMed1990

Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.

Animals↗
PubMed1990

Circadian regulation of melatonin in the retina of Xenopus laevis: limitation by serotonin availability.

Treatments expected to increase retinal serotonin levels were found to stimulate melatonin production by cultured eyecups from Xenopus laevis. The monoamine oxidase inhibitor pargyline (100 microM) caused a sixfold increase in melatonin release, and the serotonin precursor 5-hydroxy-L-tryptophan (100 microM) caused a 70-fold increase. Both acted synergistically with eserine, an inhibitor of melatonin deacetylation in the retina. The effect of 5-hydroxytryptophan was dose dependent, with effects increasing from 1 to 100 microM. Increasing the tryptophan level in the culture medium had no effect on melatonin release. These results indicate that the rate-limiting step in retinal melatonin synthesis is 5-hydroxylation of tryptophan. Melatonin released from individual eyecups in superfusion culture in constant darkness with and without added 5-hydroxy-L-tryptophan was monitored over a 5-day period. Control eyecups released low levels of melatonin, with circadian rhythmicity persisting for 1-3 days. With 5-hydroxy-L-tryptophan added, melatonin levels were elevated 10-20-fold at all times, and rhythmicity was apparent for as long as five cycles. This provides a model system for studies of the circadian clock in the eye.

5-Hydroxytryptophan↗