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1.
DEN Open ; 3(1): e150, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35898842

RESUMO

Although there are many reports of hemostasis with covered self-expandable metal stent (CSEMS) for bleeding from the papilla of Vater and the intrapapillary and distal bile duct, there are rare reports of its use for hemostasis in the perihilar bile duct. We report the case of a patient undergoing supportive care for perihilar cholangiocarcinoma with acute cholecystitis after side-by-side placement of uncovered SEMS for perihilar bile duct obstruction. Percutaneous transhepatic gallbladder aspiration was performed upon admission, and hematemesis occurred the next day. Since computed tomography scanning showed a pseudoaneurysm in the right uncovered SEMS, hemostasis by interventional radiology (IVR) was performed thrice for massive bleeding; however, hemostasis could not be achieved. When endoscopic retrograde cholangiopancreatography was performed for scrutiny and treatment of melena and increased hepatobiliary enzyme, the endoscopic visual field could not be secured by bleeding, and changes in hemodynamics were observed; thus, IVR was required, but it was difficult to perform. Since bleeding from the right bile duct was expected, hemostasis was performed using CSEMS. This is the first report of hemostasis performed by placing a covered SEMS for bleeding from a pseudoaneurysm of the intrahepatic bile duct.

2.
Ther Adv Gastrointest Endosc ; 14: 26317745211044009, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34595474

RESUMO

INTRODUCTION: While the Tokyo Guidelines 2018 suggest primary stone removal for mild to moderate cholangitis, a guideline for severe acute cholangitis is not mentioned. We, therefore, investigated the clinical outcomes of patients with severe acute cholangitis to confirm the usefulness and safety of primary stone removal. METHOD: This study included 104 severe acute cholangitis patients without gallstone pancreatitis diagnosed at our institution between January 2014 and December 2020. Patients with percutaneous transhepatic biliary drainage as the primary drainage, bile duct stenosis, and endoscopically unidentified bile duct stones were excluded from this study. The clinical results of 14 patients with primary stone removal (primary group) and 23 patients with elective stone removal (elective group) were then retrospectively examined (excluding abnormal values due to underlying diseases). RESULTS: Upon comparing the patient characteristics between groups, the elective group had significantly higher cardiovascular dysfunction (57% vs 7%; p = 0.004), septic shock (39% vs 0%; p = 0.006), disseminated intravascular coagulation treatment (57% vs 14%; p = 0.016), and positive blood cultures (91% vs 43%; p = 0.006) than those in the primary group. Endoscopic sphincterotomy for naïve papilla (90% vs 21%; p = 0.01) and endoscopic nasobiliary drainage (50% vs 9%; p = 0.014) were higher in the primary group, while endoscopic biliary stenting (7% vs 87%; p < 0.001) was lower than that in the elective group. DISCUSSION: There were no significant differences in adverse events or complete stone removal rates between the two groups. In the primary group, the period from the first endoscopic retrograde cholangiopancreatography to stone removal (0 days vs 12 days; p < 0.001) and hospitalization period (12 days vs 26 days; p = 0.012) were significantly shorter and the hospitalization cost ($7731 vs $18758; p < 0.001) was significantly lower than those in the elective group. CONCLUSION: If patients are appropriately selected, bile duct stones may be safely removed for the treatment of severe acute cholangitis.

3.
Sci Rep ; 8(1): 7455, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748592

RESUMO

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.


Assuntos
Proteínas de Anfíbios/genética , Extremidades/fisiologia , Regeneração , Salamandridae/fisiologia , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Sequência de Bases , Agregação Eritrocítica , Eritrócitos/metabolismo , Feminino , Masculino , Salamandridae/sangue , Salamandridae/genética , Transcriptoma
4.
J Neurophysiol ; 114(5): 3002-13, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26378200

RESUMO

Neurons in the silkmoth antennal lobe (AL) are well characterized in terms of their morphology and odor-evoked firing activity. However, their intrinsic electrical properties including voltage-gated ionic currents and synaptic connectivity remain unclear. To address this, whole cell current- and voltage-clamp recordings were made from second-order projection neurons (PNs) and two morphological types of local interneurons (LNs) in the silkmoth AL. The two morphological types of LNs exhibited distinct physiological properties. One morphological type of LN showed a spiking response with a voltage-gated sodium channel gene expression, whereas the other type of LN was nonspiking without a voltage-gated sodium channel gene expression. Voltage-clamp experiments also revealed that both of two types of LNs as well as PNs possessed two types of voltage-gated potassium channels and calcium channels. In dual whole cell recordings of spiking LNs and PNs, activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. However, simultaneous recording of a nonspiking LN and a PN showed that activation of the nonspiking LN induced hyperpolarization responses in the PN. We also observed bidirectional synaptic transmission via both chemical and electrical coupling in the pairs of spiking LNs. Thus our results indicate that there were two distinct types of LNs in the silkmoth AL, and their functional connectivity to PNs was substantially different. We propose distinct functional roles for these two different types of LNs in shaping odor-evoked firing activity in PNs.


Assuntos
Bombyx/fisiologia , Interneurônios/fisiologia , Potenciais da Membrana , Neurônios/fisiologia , Condutos Olfatórios/fisiologia , Animais , Bombyx/citologia , Bombyx/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Vias Neurais/citologia , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/fisiologia
5.
Chem Commun (Camb) ; 50(22): 2958-61, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24509495

RESUMO

To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone.


Assuntos
Proteínas de Insetos/metabolismo , Receptores Odorantes/metabolismo , Receptores de Feromônios/metabolismo , Animais , Bombyx , Álcoois Graxos/metabolismo , Proteínas de Fluorescência Verde , Proteínas de Insetos/genética , Receptores Odorantes/genética , Receptores de Feromônios/genética , Atrativos Sexuais/metabolismo
6.
Proc Natl Acad Sci U S A ; 110(38): 15455-60, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24006366

RESUMO

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.


Assuntos
Bombyx/fisiologia , Interneurônios/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Atrativos Sexuais/metabolismo , Olfato/fisiologia , Animais , Animais Geneticamente Modificados , Primers do DNA/genética , Vetores Genéticos , Luz , Masculino , Neurônios Receptores Olfatórios/metabolismo , Optogenética , Rodopsina/genética , Rodopsina/fisiologia , Limiar Sensorial/fisiologia , Fatores de Tempo
7.
J Neurosci ; 33(3): 987-1001, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23325237

RESUMO

Correlated spiking activity prevails in immature cortical networks and is believed to contribute to neuronal circuit maturation; however, its spatiotemporal organization is not fully understood. Using wide-field calcium imaging from acute whole-brain slices of rat pups on postnatal days 1-6, we found that correlated spikes were initiated in the anterior part of the lateral entorhinal cortex and propagated anteriorly to the frontal cortex and posteriorly to the medial entorhinal cortex, forming traveling waves that engaged almost the entire cortex. The waves were blocked by ionotropic glutamatergic receptor antagonists but not by GABAergic receptor antagonists. During wave events, glutamatergic and GABAergic synaptic inputs were balanced and induced UP state-like depolarization. Magnified monitoring with cellular resolution revealed that the layer III neurons were first activated when the waves were initiated. Consistent with this finding, layer III contained a larger number of neurons that were autonomously active, even under a blockade of synaptic transmission. During wave propagation, the layer III neurons constituted a leading front of the wave. The waves did not enter the parasubiculum; however, in some cases, they were reflected at the parasubicular border and propagated back in the opposite direction. During this reflection process, the layer III neurons in the medial entorhinal cortex maintained persistent activity. Thus, our data emphasize the role of layer III in early network behaviors and provide insight into the circuit mechanisms through which cerebral cortical networks maturate.


Assuntos
Potenciais de Ação/fisiologia , Ondas Encefálicas/fisiologia , Córtex Cerebral/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Ondas Encefálicas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Carbenoxolona/farmacologia , Córtex Cerebral/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Ratos , Transmissão Sináptica/efeitos dos fármacos
8.
Neurosci Lett ; 528(1): 61-6, 2012 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22981347

RESUMO

Kenyon cells (KCs), which are present in the mushroom bodies (MBs) of the insect brain, play an important role in olfactory information processing and associative learning. However, the intrinsic electrophysiological properties of KCs in silkmoth (Bombyx mori) MBs remain unknown. Here, we use whole-cell patch-clamp recordings to elucidate the functional parameters of membrane voltage and voltage-activated ionic currents of KCs in silkmoth MBs. KCs generated action potentials in response to stepping pulses of depolarizing current, and application of GABA-receptor blocker abolished inhibitory synaptic inputs and depolarized resting membrane potential. Pharmacological isolation of KC voltage-gated ionic currents revealed that KCs express a range of voltage-activated channels, including transient and non-inactivating potassium, sodium, and calcium channels. Our results provide the first electrophysiological characterization of KCs in silkmoth MBs and represent an important step toward understanding neuronal computation that underlies olfactory information processing in silkmoths.


Assuntos
Bombyx/fisiologia , Corpos Pedunculados/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Potenciais de Ação/fisiologia , Animais
9.
Zoolog Sci ; 29(8): 477-80, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22873803

RESUMO

Ciliated and microvillar olfactory receptor cells have been reported in many fish species, including teleosts and elasmobranchs. Morphological studies have suggested that microvillar cells are the only olfactory receptor cells in the elasmobranchs; however, there is no direct evidence for this hypothesis. Here we used a cat shark (Scyliorhinus torazame) to determine the cell type of the olfactory receptor cells in elasmobranchs. Retrograde labeling with a fluorescent dye, Dil, labeled only cells in the second layer from the surface of the olfactory epithelium, suggesting that ciliated cells located in the surface layer are not olfactory receptor cells. In addition, electron microscopic observation revealed that the labeled cells in the second layer have a thin dendritic knob extending from the cell body to the free surface of the epithelium. A part of the dendritic knob facing the mucous layer did not have ciliary structures. These results provide evidence that the aciliate cells are the only olfactory receptor cells in the cat shark olfactory organ.


Assuntos
Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Tubarões/anatomia & histologia , Tubarões/fisiologia , Animais , Corantes Fluorescentes , Coloração e Rotulagem
10.
PLoS One ; 7(4): e35611, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558175

RESUMO

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Iris/metabolismo , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Iris/citologia , Transdução de Sinal Luminoso , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Rodopsina/genética , Rodopsina/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
11.
Photochem Photobiol ; 88(6): 1356-61, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22417174

RESUMO

All-trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all-trans retinal is reduced within the outer segment to all-trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all-trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light-induced oxidation was measured with fluorescence imaging using an oxidation-sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light-induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all-trans retinal. Furthermore, light-induced oxidation increased with the concentration of exogenously added all-trans retinal. The results show that the all-trans retinal generated during the detection of light can mediate light-induced oxidation. Its removal through reduction to all-trans retinol protects photoreceptor outer segments against light-induced oxidative damage.


Assuntos
Luz , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Retinaldeído/metabolismo , Animais , Estrutura Molecular , Oxirredução/efeitos da radiação , Rana pipiens , Retinaldeído/química , Rodopsina/metabolismo
12.
Zoolog Sci ; 27(4): 369-73, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20377357

RESUMO

Amphibians are capable of smelling both volatile and water-soluble (e.g., amino acids) odorants. Adult Japanese newts, Cynops pyrrhogaster, live mostly in water, except during hibernation, but sometimes on land. To examine olfactory responses of the newts to adaptation to a short-term stay on land (land adaptation), we measured the magnitude of the olfactory response at five different time points (land adaptation time: 0, 30, 54, 90, and 114 h after transfer from an aquatic to a terrestrial habitat by using electro-olfactogram (EOG) recordings. Statistical analysis by the weighted linear model (P < 0.05) indicated that the time to land adaptation had a significant effect on the magnitude of the EOG induced by 1 microM and 10 microM amino acid mixtures. Further, the slope estimates of the weighted linear model were significantly positive (P < 0.05). These results indicate that the magnitude of the EOG response to amino acid mixtures (arginine, alanine, proline, and glutamic acid) significantly increases with land adaptation time. On the other hand, we observed no significant relationship between the magnitude of the EOG response induced by an 0.05% volatile odorant mixture (isoamyl acetate, n-amyl acetate, cineole, and limonene) and land adaptation time. Our results indicate that olfactory sensitivity to amino acids significantly increases with land adaptation time in adult Japanese newts.


Assuntos
Adaptação Fisiológica/fisiologia , Aminoácidos/farmacologia , Comportamento Animal/fisiologia , Ecossistema , Odorantes , Salamandridae/fisiologia , Potenciais de Ação , Animais , Masculino , Nervo Olfatório/efeitos dos fármacos
13.
Neurosci Res ; 66(1): 99-105, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19808063

RESUMO

TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.


Assuntos
Canais de Cálcio/metabolismo , Dissulfetos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Biofísica , Canais de Cálcio/química , Canais de Cálcio/genética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Sequestradores de Radicais Livres/farmacologia , Isotiocianatos/farmacologia , Microinjeções , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Oocistos , Técnicas de Patch-Clamp/métodos , Ácidos Sulfínicos/farmacologia , Sulfóxidos , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Xenopus
14.
J Gen Physiol ; 130(1): 21-40, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17591985

RESUMO

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.


Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Pigmentos da Retina/genética , Pigmentos da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Eletrofisiologia , Duplicação Gênica , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Fatores de Tempo , Visão Ocular/fisiologia
15.
J Biol Chem ; 282(9): 6677-84, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17194706

RESUMO

Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Visão Ocular , Animais , Arrestina , Meia-Vida , Cinética , Camundongos , Mutação de Sentido Incorreto , Fótons , Rodopsina/genética
16.
J Physiol ; 553(Pt 1): 125-35, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14500766

RESUMO

Magnesium ions (Mg2+) play an important role in biochemical functions. In vertebrate photoreceptor outer segments, numerous reactions utilize MgGTP and MgATP, and Mg2+ also regulates several of the phototransduction enzymes. Although Mg2+ can pass through light-sensitive channels under certain conditions, no clear extrusion mechanism has been identified and removing extracellular Mg2+ has no significant effect on the light sensitivity or the kinetics of the photoresponse. We have used the fluorescent Mg2+ dye Furaptra to directly measure and monitor the free Mg2+ concentration in photoreceptor outer segments and examine whether the free Mg2+ concentration changes under physiological conditions. Resting free Mg2+ concentrations in bleached salamander rod and cone photoreceptor cell outer segments were 0.86 +/- 0.06 and 0.81 +/- 0.09 mM, respectively. The outer segment free Mg2+ concentration was not significantly affected by changes in extracellular pH, Ca2+ and Na+, excluding a significant role for the respective exchangers in the regulation of Mg2+ homeostasis. The resting free Mg2+ concentration was also not significantly affected by exposure to 0 Mg2+, suggesting the lack of significant basal Mg2+ flux. Opening the cGMP-gated channels led to a significant increase in the Mg2+ concentration in the absence of Na+ and Ca2+, but not in their presence, indicating that depolarization can cause a significant Mg2+ influx only in the absence of other permeant ions, but not under physiological conditions. Finally, light stimulation did not change the Mg2+ concentration in the outer segments of dark-adapted photoreceptors. The results suggest that there are no influx and efflux pathways that can significantly affect the Mg2+ concentration in the outer segment under physiological conditions. Therefore, it is unlikely that Mg2+ plays a significant role in the dynamic modulation of phototransduction.


Assuntos
Ambystoma/fisiologia , Fura-2/análogos & derivados , Magnésio/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Cálcio/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , GMP Cíclico/fisiologia , Corantes Fluorescentes , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Larva , Estimulação Luminosa , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Sódio/fisiologia
17.
Biophys J ; 82(2): 728-39, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11806915

RESUMO

Calcium (Ca(2+)) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca(2+) enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na(+)/Ca(2+),K(+) exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca(2+). This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca(2+) concentration propagates through the rod outer segment depends on the Ca(2+) diffusion coefficient. We have used the fluorescent Ca(2+) indicator fluo-3 and confocal microscopy to measure the profile of the Ca(2+) concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 +/- 1 microm(2)s(-1) for the radial Ca(2+) diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L.Lagnado, L. Cervetto, and P.A. McNaughton, 1992, J. Physiol. 455:111-142) and with a buffering capacity of approximately 20 for rods in darkness(S. Nikonov, N. Engheta, and E.N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7-37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca(2+). Also, because of baffling by the disks, the longitudinal Ca(2+) diffusion coefficient will be in the order of 2 microm(2)s(-1), which is much smaller than the longitudinal cGMP diffusion coefficient (30-60 microm(2)s(-1); ). Therefore, the longitudinal decline of Ca(2+) lags behind the longitudinal spread of excitation by cGMP.


Assuntos
Cálcio/metabolismo , Difusão , Células Fotorreceptoras Retinianas Bastonetes/química , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Anilina/farmacologia , Animais , Corantes Fluorescentes/farmacologia , Cinética , Microscopia Confocal , Modelos Biológicos , Modelos Teóricos , Potássio/metabolismo , Sódio/metabolismo , Fatores de Tempo , Urodelos , Xantenos/farmacologia
18.
Adv Exp Med Biol ; 514: 1-20, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12596912

RESUMO

Visual phototransduction, the conversion of incoming light to an electrical signal, takes place in the outer segments of the rod and cone photoreceptor cells. Light reduces the concentration of cGMP, which, in darkness, keeps open cationic channels present in the plasma membrane of the outer segment. Ca2+ plays an important role in phototransduction by modulating the cGMP-gated channels as well as cGMP synthesis and breakdown. Ca2+ is involved in a negative feedback that is essential for photoreceptor adaptation to background illumination. The effects of Ca2+ on the different components of rod phototransduction have been characterized and can quantitatively account for the steady state responses of the rod cell to background illumination. The propagation of the Ca2+ feedback signal from the periphery toward the center of the outer segment depends on the Ca2+ diffusion coefficient, which has a value of 15 +/- 1 microm2 s(-1). This value shows that diffusion of Ca2+ in the radial direction is quite slow providing a significant barrier in the propagation of the feedback signal. Also, because the diffusion coefficient of Ca2+ is much smaller than that of cGMP, the decline of Ca2+ in the longitudinal direction lags behind the propagation of excitation by the decline of cGMP.


Assuntos
Cálcio/metabolismo , Luz , Animais , GMP Cíclico/metabolismo , Eletrofisiologia , Transdução de Sinais , Fatores de Tempo
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