RESUMEN
Anatomical, neurological and behavioural research has suggested differences between the brains of right- and non-right-handed individuals, including differences in brain structure, electroencephalogram patterns, explicit memory and sleep architecture. Some studies have also found decreased longevity in left-handed individuals. We therefore aimed to determine whether handedness independently affects the relationship between volatile anaesthetic concentration and the bispectral index, the incidence of definite or possible intra-operative awareness with explicit recall, or postoperative mortality. We studied 5585 patients in this secondary analysis of data collected in a multicentre clinical trial. There were 4992 (89.4%) right-handed and 593 (10.6%) non-right-handed patients. Handedness was not associated with (a) an alteration in anaesthetic sensitivity in terms of the relationship between the bispectral index and volatile anaesthetic concentration (estimated effect on the regression relationship -0.52 parallel shift; 95% CI -1.27 to 0.23, p = 0.17); (b) the incidence of intra-operative awareness with 26/4992 (0.52%) right-handed vs 1/593 (0.17%) non-right-handed (difference = 0.35%; 95% CI -0.45 to 0.63%; p = 0.35); or (c) postoperative mortality rates (90-day relative risk for non-right-handedness 1.19, 95% CI 0.76-1.86; p = 0.45). Thus, no change in anaesthetic management is indicated for non-right-handed patients.
Asunto(s)
Anestésicos/farmacología , Lateralidad Funcional , Despertar Intraoperatorio , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Electroencefalografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mortalidad , Periodo PosoperatorioRESUMEN
We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.
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Miosina Tipo I/clasificación , Terminología como Asunto , Animales , Humanos , Miosina Tipo I/genéticaAsunto(s)
Ritmo Circadiano/efectos de la radiación , Luz , Retina/fisiología , Retina/efectos de la radiación , Adenosina/fisiología , Animales , AMP Cíclico/fisiología , Dopamina/fisiología , Espacio Extracelular/fisiología , Membranas Intracelulares/fisiología , Células Fotorreceptoras/fisiología , Epitelio Pigmentado Ocular/fisiologíaRESUMEN
Two class III myosins have been identified to date: NINAC from Drosophila melanogaster and MyoIII(Lim) from Limulus polyphemus. Both have N-terminal kinase domains and are expressed exclusively in photoreceptors. Mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival. We report the cloning and chromosomal localization of a human class III myosin, MYO3A, from retina and a retinal pigment epithelial cell line. Human MYO3A (which we will refer to simply as MYO3A) possesses an N-terminal kinase domain and three consensus calmodulin-binding (IQ) motifs, two in the neck and one in the tail domain. We detected two MYO3A splice variants differing by 52 amino acids near the kinase/myosin junction. On Northern blots, MYO3A probes detected a 6. 5-kb transcript in human and monkey retina, in a cultured human RPE cell line (RPE-19), and at much lower levels in human pancreas. A somatic hybrid panel PCR screen localized MYO3A to human chromosome 10, and a radiation hybrid screen further localized it proximal to marker D10S197, which is located at 10p11.1 on the human cytogenetic map. Since mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival, the human homologue MYO3A may also play a role in photoreceptor function and/or maintenance.
Asunto(s)
Mapeo Cromosómico , Clonación Molecular , Proteínas de Drosophila , Cadenas Pesadas de Miosina/genética , Anciano , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Células Cultivadas/metabolismo , Cartilla de ADN/química , ADN Complementario/análisis , Proteínas del Ojo/genética , Femenino , Humanos , Macaca mulatta , Persona de Mediana Edad , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/biosíntesis , Miosina Tipo III , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Retina/citología , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Several forms of sensory deficit have been associated with unconventional myosin defects in humans and other animals. Normal hearing in mammals has been shown to require functional myosin VI (Avraham et al., 1995) and myosin VIIA (Gibson et al., 1995; Liu et al., 1997), and the combined blindness and deafness of Usher syndrome type IB has been shown to be produced by specific defects in myosin VIIA (Weil et al., 1997). Here we report the cloning and characterization of two distinct myosin VI isoforms (FMVIA and FMVIB) initially identified in a degenerate PCR screen of retinal cDNA from the striped bass, Morone saxatilis. Open reading frames for FMVIA and FMVIB encode predicted proteins of 1304 and 1270 amino acids respectively, which are 83% identical at the amino acid level. Both fish isoforms are likewise approximately 83-86% identical to mammalian class VI myosins (Hasson and Mooseker, 1994). Northern blot analysis revealed that FMVIA mRNA is broadly expressed and most abundant in kidney, a pattern similar to that previously reported for mammalian myosin VI. FMVIB expression is dramatically more abundant in retina than in any other tissue examined. Antibodies directed against pig myosin VI (Hasson and Mooseker, 1994) detect a doublet at approximately 150 kDa in bass retina and RPE. Since both fish VIA and VIB isoforms share high sequence identity with pig myosin VI within the domain used for antibody production, it seems likely that this antibody crossreacts with both FMVIA and FMVIB. Immunocytochemistry with this same affinity-purified rabbit anti-myosin VI antibody shows that myosin VI isoforms are primarily localized in photoreceptors, horizontal cells and Müller cells in both fish and primate retinas. This report is the first demonstration that two myosin VI genes are expressed in the same organism and the same cell type (RPE). The relatively high abundance of FMVIB expression in retina suggests that it may play an important role in retinal motility events.
Asunto(s)
Lubina/metabolismo , Miosinas/análisis , Retina/química , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Inmunohistoquímica , Microscopía Confocal , Datos de Secuencia Molecular , Miosinas/genética , Epitelio Pigmentado Ocular/química , Isoformas de Proteínas/análisis , ARN Mensajero/análisis , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
The heterotrimeric microtubule motor kinesin II has been shown to be required for morphogenesis and maintenance of both motile flagella and immotile sensory cilia. Recently, we showed that the KIF3A subunit of kinesin II is concentrated in the inner segment and connecting cilium of fish photoreceptors. Here we report the gene structure of human KIF3A (HsKIF3A) and describe its localization in human and monkey retina. We also describe the localization of both KIF3A and KIF3B kinesin II subunits in Xenopus retina. Using a portion of HsKIF3A we had amplified from adult human retinal cDNA, we found by a GenBank database search that an identical sequence had already been obtained by the Human Genome Center at Lawrence Berkeley National Laboratories in a direct sequencing analysis of 680 kb of human chromosome 5q31. By comparing the genomic sequence of HsKIF3A to the open reading frame (ORF) of the highly homologous mouse Kif3A, we determined that the HsKIF3A gene has 17 exons and an ORF of approximately 2.1 kb, predicting a protein of 80.3 kDa. Antibodies against sea urchin KRP85, a KIF3A homologue, bound to a single band of approximately 85 kDa in immunoblots of total retina protein from human, monkey and Xenopus. In these same samples, a single band of approximately 95 kDa is recognized by antibodies against Xklp3, a Xenopus KIF3B homologue. In sections of Xenopus retina, both antibodies strongly labelled photoreceptor inner segments and the outer limiting membrane. Both antibodies also labelled photoreceptor axonemes. The axonemal localization of kinesin II subunits suggests that kinesin II may play a role in transport of materials from the photoreceptor cell body to the outer segment.
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Cinesinas/análisis , Macaca mulatta/metabolismo , Células Fotorreceptoras de Vertebrados/química , Xenopus laevis/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Exones , Humanos , Técnicas para Inmunoenzimas , Intrones , Cinesinas/genética , Microscopía Confocal , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Many forms of intracellular transport are mediated by microtubule-dependent motors of the kinesin superfamily (KIFs). To identify kinesins expressed in human retina and RPE, we used degenerate primer RT-PCR to amplify a approximately 440 bp kinesin motor domain fragment from human retinal and RPE messenger RNAs. Four distinct kinesins were detected: one C-kinesin (HsKIFC3); one kinesin from the unc104/KIF1 family [HsKIF1A]; and the ubiquitous and neuronal forms of conventional kinesin heavy chain [HsuKHC and HsnKHC]. The C-kinesin HsKIFC3 comprised 33.3% of the retinal clones and was 60% identical to FKIF2, the most abundant kinesin detected in a previous screen of fish retina and 95% identical to a fragment of MmKifC3 recently amplified from mouse brain. Elsewhere we have reported the sequence of HsKIFC3 and shown that it maps to the same locus on chromosome 16q13-q21 as Bardet-Biedl syndrome Type II, a hereditary retinal degeneration. We describe here the kinesin PCR screen of human retina and RPE and examine the tissue and subcellular distribution of KIFC3 in both fish and human retina using an antibody raised against a peptide conserved between FKIF2 and HsKIFC3. This peptide antibody identified a single approximately 80 kDa band in Western blots of fish and human retina and RPE. In both fish and human retina this antibody strongly labeled photoreceptor terminals in the outer plexiform layer, suggesting that FKIF2/KIFC3 may play some role in the photoreceptor synapse.
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Lubina/metabolismo , Cinesinas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Retina/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Southern Blotting , Western Blotting , Citoesqueleto/metabolismo , Humanos , Técnicas para Inmunoenzimas , Cinesinas/química , Cinesinas/inmunología , Datos de Secuencia Molecular , Células Fotorreceptoras de Vertebrados/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment-cone outer segment preparations (CIS-COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2-like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS-COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory "dark signal" influencing photoreceptor metabolism and function in the fish retina.
Asunto(s)
Adenosina/farmacología , Receptores Purinérgicos P1/fisiología , Células Fotorreceptoras Retinianas Conos/citología , Adenosina/análogos & derivados , Adenosina Trifosfato/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Oscuridad , Luz , Fármacos Neuroprotectores/farmacología , Perciformes , Fenetilaminas/farmacología , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/fisiología , Teobromina/análogos & derivados , Teobromina/farmacologíaRESUMEN
Kinesins are a large superfamily of microtubule motors that mediate specific motile processes. In a previous study, we identified 11 kinesin family members in the retina and retinal pigment epithelium (RPE) of the striped bass, Morone saxatilus. We have now identified, cloned, and sequenced the human homologue (KIFC3) of the most abundantly expressed retinal kinesin from that study, the C-terminal kinesin FKIF2. An antibody raised against an FKIF2 peptide cross-reacted with an approximately 80-kDa protein in human retina, RPE, kidney, and lung. Since microtubule-dependent processes are critical to the function and morphogenesis of the photoreceptors and RPE, the abundantly expressed KIFC3 was considered to be a potential candidate gene for causing human retinal degeneration. Chromosomal localization of the KIFC3 gene revealed that it maps to chromosome 16q13-q21, within the critical region for a Bardet-Biedl syndrome locus (BBS2). Bardet-Biedl syndrome is a genetically heterogeneous, autosomal recessive disorder characterized by retinal dystrophy, polydactyly, obesity, hypogonadism, renal abnormalities, and mental retardation. The chromosomal localization and expression pattern of KIFC3 suggest that it may be an excellent candidate for families linked to BBS2.
Asunto(s)
Cromosomas Humanos Par 16/genética , Genes/genética , Cinesinas/genética , Síndrome de Laurence-Moon/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Humanos , Células Híbridas , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Kinesins comprise a large superfamily of microtubule-based motor proteins, individual members of which mediate specific types of motile processes. To identify kinesin family members (KIFs) that are critical to retinal function and thus to vision, a reverse transcriptase polymerase chain reaction (RT-PCR) cloning strategy was used to isolate putative KIFs expressed in the neural retina and retinal pigmented epithelium (RPE) of the striped bass, Morone saxatilus. Eleven fish KIFs (FKIFs) were isolated from neural retina and six of the same FKIFs were also isolated from RPE. One of the KIFs identified in this screen, FKIF2, was the most prevalent clone detected both in the retina (41% of clones) and RPE (72% of clones). Based on predicted amino acid sequence homology within the motor domain, seven of the FKIFs have been tentatively assigned to known kinesin families: the kinesin heavy chain family (FKIF1, 5 and 9), the unc104/KIF1 family (FKIF3 and 8), the KIF2 family (FKIF4), and the cKIF family (FKIF2). Northern blot analysis revealed that each detectable FKIF exhibited a unique tissue-specific expression pattern. Since FKIF2 was more highly expressed in retina than in any other tissue tested, including brain, and was the most abundant KIF message expressed in both retina and RPE, it was examined in more detail and the complete approximately 2.3 kb open reading frame for FKIF2 was cloned and sequenced. The predicted amino acid sequence indicates that FKIF2 has a C-terminal motor domain, and thus is a member of the cKIF family. FKIF2 is only 36.5% identical at the amino acid level to the most closely related cKIF in the database, suggesting that FKIF2 may be a novel member of this family. Antibodies raised against a unique peptide specific to FKIF2 recognize an approximately 80 kd protein in homogenates of retina, RPE, brain and kidney. The pronounced expression of FKIF2 in retina and RPE suggests that FKIF2 may play an important role in microtubule-dependent motile events in these two tissues.
Asunto(s)
Lubina/metabolismo , Cinesinas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Lubina/genética , Northern Blotting , Western Blotting , Pollos , ADN , Humanos , Cinesinas/genética , Datos de Secuencia Molecular , Perciformes , Reacción en Cadena de la Polimerasa , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
In the teleost retinal pigment epithelium (RPE), melanin pigment granules disperse into long apical projections in the light and reaggregate into the cell body in the dark. To investigate the cytoskeletal mechanisms responsible for these movements, we have examined the effects of cytoskeletal inhibitors on pigment granule transport in cultured, dissociated RPE cells using time-lapse video microscopy. The kinetics of pigment granule transport during normal aggregation and dispersion are quite distinct: during aggregation, all pigment granules undergo simultaneous, nonsaltatory centripetal movement (mean velocity 3.6 microm/min); during dispersion, individual granules undergo independent, bidirectional saltatations (mean velocities 3.7 microm/min centrifugal; 1.1 microm/min centripetal). Nocodazole disruption of microtubules within the RPE apical projections had little effect on the kinetics of pigment granule movement, and essentially no effect on extent of pigment granule aggregation or dispersion, or on maintenance of the fully aggregated or fully dispersed states. In contrast, cytochalasin D (CD) treatment blocked net aggregation and dispersion of pigment granules, and compromised maintenance of the fully aggregated and dispersed states. These observations suggest that the actin cytoskeleton plays an important role in both centripetal and centrifugal transport of pigment granules in teleost RPE cells.
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Actinas/fisiología , Gránulos Citoplasmáticos/efectos de los fármacos , Peces/metabolismo , Microtúbulos/fisiología , Epitelio Pigmentado Ocular/citología , Pigmentos Retinianos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Citocalasina D/farmacología , Microscopía por Video , Nocodazol/farmacología , Epitelio Pigmentado Ocular/efectos de los fármacosRESUMEN
The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Perciformes/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Calmodulina/antagonistas & inhibidores , División Celular/fisiología , Tamaño de la Célula/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Células Fotorreceptoras Retinianas Bastones/enzimologíaRESUMEN
Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.
Asunto(s)
Cilios/química , Cinesinas/análisis , Segmento Externo de la Célula en Bastón/química , Animales , Anticuerpos Monoclonales , Microscopía Fluorescente , Microscopía Inmunoelectrónica , PerciformesRESUMEN
In the eyes of teleosts and amphibians, melanin pigment granules of the retinal pigment epithelium (RPE) migrate in response to changes in light conditions. In the light, pigment granules disperse into the cells' long apical projections, thereby shielding the rod photoreceptor outer segments and reducing their extent of bleach. In darkness, pigment granules aggregate towards the base of the RPE cells. In vitro, RPE pigment granule aggregation can be induced by application of nonderivatized cAMP, and pigment granule dispersion can be induced by cAMP washout. In previous studies based on RPE-retina co-cultures, extracellular calcium was found to influence pigment granule migration. To examine the role of calcium in regulation of RPE pigment granule migration in the absence of retinal influences, we have used isolated RPE sheets and dissociated, cultured RPE cells. Under these conditions depletion of extracellular or intracellular calcium ([Ca2+]o, [Ca2+]i) had no effect on RPE pigment granule aggregation or dispersion. Using the intracellular calcium dye fura-2 and a new dye, fura-pe3, to monitor calcium dynamics in isolated RPE cells, we found that [Ca2+]i did not change from basal levels when pigment granule aggregation was triggered by cAMP, or dispersion was triggered by cAMP washout. Also, no change in [Ca2+]i was detected when dispersion was triggered by cAMP washout in the presence of 10 microM dopamine, a treatment previously shown to enhance dispersion. In addition, elevation of [Ca2+]i by addition of ionomycin neither triggered pigment movements, nor interfered with pigment granule motility elicited by cAMP addition or washout. Since other studies have indicated that actin plays a role in both pigment granule dispersion and aggregation in RPE, our findings suggest that RPE pigment granule migration depends on an actin-based motility system that is not directly regulated by calcium.
Asunto(s)
Calcio/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Pigmentos Biológicos/metabolismo , Animales , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Fura-2/análogos & derivados , Fura-2/metabolismo , Perciformes , Epitelio Pigmentado Ocular/citologíaRESUMEN
Teleost rod photoreceptors elongate in the light and shorten in darkness. We are investigating the role of cAMP-dependent protein kinase (PKA), phosphatases and target phosphoproteins in the regulation of photoreceptor cell shape. Preparations of rod fragments, consisting of the motile inner segment with attached photosensory outer segment (RIS-ROS), undergo light-stimulated elongation in culture. The PKA-selective inhibitor, H89, enhanced RIS-ROS elongation in both light and darkness, suggesting that elongation is associated with dephosphorylation of PKA substrates. Okadaic acid and calyculin A, inhibitors of type 1 and 2A phosphatases, blocked light-dependent and light-independent elongation with relative potencies suggesting that elongation requires dephosphorylation by type 1 phosphatase in light and type 2A phosphatase in darkness. To identify targets of PKA and phosphatases, RIS-ROS were isolated from retinas prelabeled with 32P-orthophosphate, and then incubated in the presence of kinase inhibitors or phosphatase inhibitors. Two phosphoproteins, PP33 and PP35, were phosphorylated by PKA and dephosphorylated by type 1 or 2A phosphatases in light- and dark-cultured RIS-ROS. PP35 (but not PP33) was immunoprecipitated by an antibody to phosducin, a PKA-regulated modulator of phototransduction (Lee et al., 1992); PP35 was also phosphorylated in vitro by a Ca2+ calmodulin-activated kinase. PP33 further differed from PP35 in its phosphopeptide maps and phosphorylation by PKC. We conclude that RIS-ROS elongation is correlated with the dephosphorylation of PKA substrates by type 1 or 2A phosphatases. Candidate mediator proteins include PP35, a fish phosducin homolog, and PP33, a newly described photoreceptor phosphoprotein.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas del Ojo/fisiología , Perciformes/fisiología , Fosfoproteínas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Segmento Externo de la Célula en Bastón/citología , Segmento Externo de la Célula en Bastón/fisiología , Adaptación Ocular , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fenómenos Fisiológicos Celulares , Adaptación a la Oscuridad , Reguladores de Proteínas de Unión al GTP , Luz , Movimiento , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Fosforilación , Pruebas de Precipitina , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Segmento Externo de la Célula en Bastón/efectos de la radiaciónRESUMEN
In the retinas of teleost fish, cone photoreceptors change shape in response to light and circadian signals. They elongate in the dark, contract in the light, and under conditions of constant darkness undergo appropriate movements at expected dusk and dawn. Dopamine induces cones to contract, thus mimicking the effect of light or expected dawn. To identify the receptor subtype responsible for mediating dopamine regulation of cone retinomotor movements, we have carried out pharmacological studies using isolated fragments of teleost cones consisting of cone inner segments-cone outer segments (CIS-COS). Isolated CIS-COS retain the ability to elongate in dark culture and contract when subsequently exposed to light or dopamine. We report that dark-induced elongation of CIS-COS was inhibited by dopamine and its agonists with an effectiveness ranking of dopamine = quinpirole > bromocriptine >>> SKF-38393. After 60 min of elongation in dark culture, CIS-COS myoids contracted when subsequently cultured in the dark with dopamine or quinpirole. Quinpirole-induced inhibition of elongation and quinpirole-induced contraction were completely blocked by clozapine at 1 microM or by sulpiride at 100 microM. These effectiveness profiles for dopamine agonists and antagonists suggest that dopamine regulation of cone retinomotor movement is mediated by a D4-like receptor.
Asunto(s)
Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Dopamina/farmacología , Receptores Dopaminérgicos/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Colforsina/farmacología , Oscuridad , Ergolinas/farmacología , Técnicas In Vitro , Luz , Perciformes , Quinpirol , Receptores Dopaminérgicos/clasificaciónRESUMEN
To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.
Asunto(s)
Expresión Génica , Cinesinas/fisiología , Perciformes , Epitelio Pigmentado Ocular/química , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Lubina/fisiología , Clonación Molecular , Gránulos Citoplasmáticos/química , Immunoblotting , Inmunohistoquímica , Cinesinas/análisis , Microinyecciones , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Myosin-I is a nonfilamentous motor protein associated with the actin cytoskeleton and cellular membranes in several cell types. The occurrence and subcellular distribution of myosin-I in mammalian and fish RPE were investigated to examine the possible role of myosin-I in retinal pigment epithelium (RPE) motility processes. METHODS: Antibodies directed against myosin-I proteins from bovine adrenal medulla or chicken intestinal brush border were used to examine cultured fetal human RPE cells and freshly isolated bovine or green sunfish RPE by Western immunoblots and immunocytochemistry, using both conventional and confocal fluorescence microscopy. RESULTS: The monoclonal antibody directed against bovine adrenal myosin-I identified a single strong immunoreactive band at 116 kD in Western blots of homogenates of cultured human RPE cells and 114 kD in bovine RPE sheets. An immunoreactive band of similar molecular weight was also observed in bovine and rabbit retina. Cell fractionation studies of bovine RPE cells revealed that myosin-I was present in all fractions that included cell membranes. The polyclonal antibody directed against chicken brush border myosin-I identified doublet immunoreactive bands at 115/110 kD in Western blots of homogenates of fish retina but identified a strong predominant immunoreactive band at 140 kD in fish RPE and brain homogenates; minor bands at 115/110 kD were identified in fish RPE homogenates. Immunocytochemistry of cultured human RPE cells using the bovine adrenal myosin-I antibody revealed a broad distribution of myosin-I that appeared to be most concentrated along the length of the lateral membranes; no colocalization was seen with actin-rich stress fibers. CONCLUSIONS: Proteins immunoreactive with myosin-I antibodies are present in both RPE and retina of mammals and green sunfish. In confluent cultures of human RPE cells, myosin-I is concentrated along the lateral cell membranes of the cuboidal cells.