Biomedical subjects

J C Besharse

Publications and source records attributed to J C Besharse.

At least 55 records · Page 3Linked to original sources

PubMed1989

Cytochalasin D inhibits L-glutamate-induced disc shedding without altering L-glutamate-induced increase in adhesiveness.

Excitatory amino acid-stimulated disc shedding is correlated with the appearance of microfilament-rich ensheathing processes of the retinal pigment epithelium (RPE) and increased apparent adhesiveness between photoreceptors and RPE in explanted eyecups of Xenopus laevis. We have compared the time course of disc shedding and increased retinal adherence during L-glutamate treatment. Increased adherence was measured on the basis of the tendency of the apical RPE domains to partition with isolated neural retinas. In medium supplemented with L-glutamate (12 mM) or kainate (100 microM), a glutamate analog, the time course of increased partitioning of melanin pigment-rich cell fragments which contain ensheathing processes differs, even though the kinetics of induced disc shedding is the same in either case. Co-treatment with cytochalasin D (5 microM) completely blocks L-glutamate-induced disc shedding, as well as formation of microfilament-rich ensheathing processes, even though it has little effect upon apparent adhesiveness. The virtually complete dissociation of the effects of L-glutamate on disc shedding from that on increased adhesiveness of photoreceptors to RPE suggests that increased retinal adherence and pseudopod formation may be unrelated causally.

Adhesiveness↗

PubMed1989

Retinal melatonin is metabolized within the eye of xenopus laevis.

Retinal synthesis of melatonin, a potent modulator of rhythmic retinal processes, is elevated at night as a result of regulation by a circadian clock. Despite high nocturnal synthetic capacity, both melatonin content and release are low in the retina of the frog Xenopus laevis. We report here that cultured eyecups from Xenopus have the capacity for rapid metabolic breakdown of melatonin. Pharmacological analysis indicates that the initial step in this degradation pathway is deacetylation of melatonin by the enzyme aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13). This produces 5-methoxytryptamine, which is then deaminated by monoamine oxidase [amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4], producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Inhibition of aryl acylamidase with eserine dramatically increases the release of endogenous melatonin by eyecups cultured at night, indicating that this pathway is the normal fate of retinal melatonin. Metabolism within the eye suggests a local neuromodulatory role for retinal melatonin, in contrast to the hormonal role of pineal melatonin.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

Dopamine modifies the balance of rod and cone inputs to horizontal cells of the Xenopus retina.

Dopamine (greater than or equal to 2 microM) increased the cone input and suppressed the rod input to axon-bearing horizontal cells of the Xenopus retina. Dopamine (10 microM) also depolarized the horizontal cell by about 9 mV. The D2-dopamine antagonists spiperone and metoclopramide had the opposite action to dopamine, whereas the D1-dopamine antagonist SCH 23390 was without effect. None of the agents tested modified the light-evoked responses of rods.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

Distribution of acetylated alpha-tubulin in retina and in vitro-assembled microtubules.

We have used the mouse monoclonal antibody 6-11 B-1, specific for acetylated alpha-tubulin, to determine the distribution of acetylated alpha-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated alpha-tubulin. Immunofluorescence, using 6-11 B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for alpha-tubulin, demonstrated the colocalization of both alpha-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated alpha-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.

Acetylation↗

PubMed1988

Excitatory amino acids and rod photoreceptor disc shedding: analysis using specific agonists.

L-Glutamate and L-aspartate stimulate photoreceptor disc shedding. In order to evaluate the possible involvement of a receptor, we examined the effects of specific excitatory amino acid agonists on rod photoreceptor disc shedding and neural retinal toxicity. Using eyecups from both Xenopus laevis and Rana pipiens, we found that kainate, quisqualate, and N-methyl-D-aspartate (NMDA) were all neurotoxic, but that kainate caused a more extensive inner retinal lesion. Kainate also caused disc shedding at concentrations as low as 10 microM; dihydrokainate, a structural analogue, was at least 100-fold less potent. In contrast, quisqualate induced disc shedding only at concentrations above 5.0 mM, and NMDA had no effect on disc shedding at any concentration examined. Our results suggest that excitatory amino acids act via a receptor of the kainate type to effect disc shedding. The mechanism in the retina or photoreceptor-pigment epithelial complex by which an excitatory amino acid receptor system influences disc shedding remains to be identified.

Amino Acids↗

PubMed1987

Membrane turnover in rod photoreceptors: ensheathment and phagocytosis of outer segment distal tips by pseudopodia of the retinal pigment epithelium.

We have documented the ultrastructural changes that occur within the photoreceptor outer segment and the retinal pigment epithelium (RPE) during photosensitive membrane turnover. We employed an in vitro eyecup preparation from Xenopus laevis in which a large shedding event was induced by adding the excitatory amino acid L-aspartate (Greenberger & Besharse 1985; J. comp. Neurol. 239, 361-372). We found that during L-aspartate-induced shedding the RPE cells formed, on their apical domains, previously undescribed processes that were directly involved in disc phagocytosis. These processes are structurally similar to processes formed by macrophages during phagocytosis and are accordingly referred to as pseudopodia. Pseudopodia were distinguishable from the apical villous process normally extended from the RPE in that they were closely applied to the surface of the outer segment, had a cytoplasmic matrix of low electron density that was devoid of most cellular organelles and were enriched in thin (7 nm diameter) filaments. Filament size, specific pseudopodial staining with the actin-specific probe rhodamine phalloidin and inhibition of pseudopod formation by cytochalasin D suggested that the thin filaments were composed of actin. Pseudopodial formation also occurs during a normal light-initiated shedding event. However, the low frequency of shedding, the asynchrony of the individual shedding events and the transient appearance of the pseudopodia prevented a full appreciation of their role during normal disc shedding. Associated with massive shedding and pseudopodial formation, there was an increased adherence between retina and RPE. During L-aspartate treatment, the apical portions of the RPE cells partitioned with the distal outer segment during retinal isolation. This effect was directly related to the development of pseudopodia and may reflect alteration of surface features of the rod outer segment (ROS)-RPE interface related to phagocytosis. Our observations show that transiently forming pseudopodia are the organelles of phagocytosis and that they may play a role in disc detachment as well.

Animals↗

PubMed1987

Melatonin and rhythmic photoreceptor metabolism: melatonin-induced cone elongation is blocked at high light intensity.

We have proposed a model for circadian regulation of cone position in Xenopus laevis that involves interaction of melatonin and dopamine as signals for darkness and light respectively. One problem, however, is that the effects of melatonin have not been detected in eye cups prepared from animals maintained on a cyclic light schedule. Since melatonin's effect would be expected to occur in low light intensity at night, we have investigated the relationship among melatonin, light intensity, and cone length. We report that melatonin mimics the effects of darkness and stimulates cone elongation in eye cups from cyclic light animals incubated at low but not at high light intensities.

Animals↗

PubMed1987

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.

Animals↗

PubMed1986

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.

Animals↗

PubMed1986

Dopamine receptor-mediated inhibition of serotonin N-acetyltransferase activity in retina.

The possible involvement of catecholamines in the regulation of serotonin N-acetyltransferase (NAT) activity in retina of the African clawed frog was investigated using an in vitro eye cup preparation. Dopamine (10 microM) and norepinephrine (50 microM) had no significant effect on NAT activity of eye cups incubated in the light. However, dopamine inhibited the increase of retinal NAT activity that occurs in eye cups incubated in darkness; the ED50 for dopamine was 0.3 microM. The effect of dopamine on NAT activity was mimicked by the dopamine receptor agonists apomorphine and bromocriptine, but not by agonists of alpha 1-, alpha 2- or beta-adrenergic receptors. Dopamine-mediated inhibition of NAT activity was antagonized by spiroperidol and by alpha-flupenthixol, but not by beta-flupenthixol, phentolamine or timolol. Benztropine, an inhibitor of dopamine reuptake, also decreased NAT activity in eye cups incubated in the dark. The inhibitory effect of benztropine was antagonized by spiroperidol, suggesting that it was mediated by an increase in the extracellular concentration of endogenous dopamine. These studies indicate that the regulation of NAT activity in the retina is subject to modulation by a dopamine receptor-mediated mechanism and suggest that dopamine may play a role in the inhibition of NAT activity by light.

Acetyltransferases↗

PubMed1986

Cyclic AMP stimulates serotonin N-acetyltransferase activity in Xenopus retina in vitro.

The possible involvement of cyclic AMP in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in a defined medium. Addition of dibutyrylcyclic AMP (dbcAMP) increased retinal NAT activity in eye cups cultured in light. Addition of adenosine or 5'-AMP under identical conditions was without effect. 3-Isobutylmethylxanthine (IBMX) increased both retinal cyclic AMP levels and NAT activity in light-exposed eye cups. Forskolin also increased the concentration of cyclic AMP and the activity of NAT, and the effect of forskolin on both of these parameters was synergistically enhanced by IBMX. The effects of forskolin and of dbcAMP did not require the addition of calcium to the medium; thus, Ca2+ -dependent synaptic transmission does not appear to be required for the response to these drugs. Incubation conditions that activate cyclic AMP-dependent protein kinase in retinal homogenates had no effect on NAT activity, suggesting that direct phosphorylation of NAT was probably not involved in the response to elevating cyclic AMP in situ. The effect of dbcAMP was blocked by protein synthesis inhibitors. These results suggest that cyclic AMP increases retinal NAT activity by a mechanism that involves protein synthesis, and support a role for cyclic AMP in the nocturnal increase of NAT activity in darkness.

1-Methyl-3-isobutylxanthine↗

PubMed1986

Involvement of calcium in the regulation of serotonin N-acetyltransferase in retina.

The possible involvement of calcium in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in defined medium. Omitting CaCl2 from the culture medium completely inhibited the dark-dependent increase of NAT activity at night. Approximately 10(-4)-10(-3) M free Ca2+ was found to be required for the maximal increase of NAT activity in the dark. Other divalent cations--Ba2+, Sr2+, and Mn2+--did not substitute for Ca2+. Antagonists of voltage-sensitive calcium channels, including nifedipine, methoxyverapamil (D600), Co2+, and Mg2+, were found to be effective inhibitors of the dark-dependent increase of retinal NAT activity. Trifluoperazine also decreased retinal NAT activity. These studies indicate that the increase of retinal NAT activity in the dark is mediated by a specific Ca2+-dependent process and that Ca2+ influx through voltage-sensitive calcium channels is involved.

Acetyltransferases↗

PubMed1986

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.

Animals↗

PubMed1986

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. II. Quantitative freeze-fracture analysis.

Tunicamycin (TM), a selective inhibitor of dolichylphosphate-dependent oligosaccharide biosynthesis, effectively blocks glycosylation, but not synthesis, of opsin, the rod visual pigment apoglycoprotein. In parallel with this inhibition, vesicular membrane material accumulates in the compartment between rod inner and outer segments (the intersegmental space) in TM-treated retinas (Fliesler et al, J Cell Biol 100:574-587, 1985). Freeze-fracture analysis was applied to isolated Xenopus laevis retinas which were incubated in the presence or absence of TM in order to clarify the relationship of those intersegmental membranous structures to others in the rod outer segment (ROS) assembly pathway. The membranes in the intersegmental space display characteristics similar to those of the ROS, but distinct from those of the inner segment. They exhibit densely particulate convex protoplasmic face (PF)-leaflets and relatively particle-free concave exoplasmic face (EF)-leaflets, similar to comparable leaflets of the ROS plasmalemma and nascent (immature) disc membranes. Quantitative analysis further demonstrates that the size distribution and densities of intramembrane particles (IMPs) in PF-leaflets of the intersegmental membranes are indistinguishable from those of ROS membranes, suggesting that both membrane systems contain similar integral proteins (i.e., opsin). Finally, the topology of the intersegmental membranes is most closely related to that of the ROS plasmalemma or nascent disc membranes, suggesting that they arise as a result of aberrant disc morphogenesis, rather than by breakdown of mature discs. Overall, the data support the conclusion that the tubulo-vesicular membranes in the intersegmental space represent newly assembled, opsin-containing material which has been efficiently compartmentalized in preparation for disc morphogenesis, but is incapable of forming normal, topologically closed discs.

Animals↗

PubMed1985

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.

Amino Acids↗

PubMed1985

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.

Animals↗