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1.
PLoS One ; 16(10): e0258721, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34710119

RESUMO

In daylight, cone photoreceptors in the retina are responsible for the bulk of visual perception, yet compared to rods, far less is known quantitatively about their biochemistry. This is partly because it is hard to isolate and purify cone proteins. The issue is also complicated by the synergistic interaction of these parameters in producing systems biology outputs, such as photoresponse. Using a 3-D resolved, finite element model of cone outer segments, here we conducted a study of parameter significance using global sensitivity analysis, by Sobol indices, which was contextualized within the uncertainty surrounding these parameters in the available literature. The analysis showed that a subset of the parameters influencing the circulating dark current, such as the turnover rate of cGMP in the dark, may be most influential for variance with experimental flash response, while the shut-off rates of photoexcited rhodopsin and phosphodiesterase also exerted sizable effect. The activation rate of transducin by rhodopsin and the light-induced hydrolysis rate of cGMP exerted measurable effects as well but were estimated as relatively less significant. The results of this study depend on experimental ranges currently described in the literature and should be revised as these become better established. To that end, these findings may be used to prioritize parameters for measurement in future investigations.


Assuntos
GMP Cíclico/metabolismo , Transdução de Sinal Luminoso , Luz , Células Fotorreceptoras Retinianas Cones/fisiologia , Transducina/fisiologia , Visão Ocular/fisiologia , Animais , Camundongos , Camundongos Knockout
2.
Hum Mol Genet ; 29(6): 881-891, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31960909

RESUMO

Rhodopsin is the G protein-coupled receptor in rod photoreceptor cells that initiates vision upon photon capture. The light receptor is normally locked in an inactive state in the dark by the covalently bound inverse agonist 11-cis retinal. Mutations can render the receptor active even in the absence of light. This constitutive activity can desensitize rod photoreceptor cells and lead to night blindness. A G90D mutation in rhodopsin causes the receptor to be constitutively active and leads to congenital stationary night blindness, which is generally thought to be devoid of retinal degeneration. The constitutively active species responsible for the night blindness phenotype is unclear. Moreover, the classification as a stationary disease devoid of retinal degeneration is also misleading. A transgenic mouse model for congenital stationary night blindness that expresses the G90D rhodopsin mutant was examined to better understand the origin of constitutive activity and the potential for retinal degeneration. Heterozygous mice for the G90D mutation did not exhibit retinal degeneration whereas homozygous mice exhibited progressive retinal degeneration. Only a modest reversal of retinal degeneration was observed when transducin signaling was eliminated genetically, indicating that some of the retinal degeneration occurred in a transducin-independent manner. Biochemical studies on purified rhodopsin from mice indicated that multiple species can potentially contribute to the constitutive activity causing night blindness.


Assuntos
Mutação , Cegueira Noturna/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/fisiologia , Transducina/fisiologia , Animais , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Cegueira Noturna/etiologia , Degeneração Retiniana/etiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
3.
Hum Mol Genet ; 29(3): 394-406, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31925423

RESUMO

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αßγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.


Assuntos
Morte Celular , Luz , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/metabolismo , Transducina/fisiologia , cis-trans-Isomerases/fisiologia , Animais , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Retinose Pigmentar , Transdução de Sinais
4.
Exp Gerontol ; 113: 64-73, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30243894

RESUMO

To investigate aging-dependent changes in taste sensitivities, we performed behavioral tests regarding taste sensitivity among young and old SAMP1 mice. In this senescence-accelerated mice model, dramatic changes in taste sensitivities were observed at least 70 weeks old. As for in a brief access test, old mice showed significantly increased taste sensitivity to bitter, salty, sweet, and umami tastes. On the other hand, in a two-bottle test, avoidance of bitter and salty tastes increased, while preference for umami decreased with aging. To investigate the participation of peripheral taste detection systems in the observed changes, we analyzed both the expression of representative taste-related molecules and also turnover rates of taste bud cells. The mRNA expressions of the bitter taste receptor Tas2r105 and its coupled G protein gustducin were significantly decreased with aging. However, the majority of molecules tested did not show significant expression changes. In addition, no significant differences in the turnover rates of taste bud cells were observed between the two age groups. These results suggest that the changes in taste sensitivity of SAMP1 mice due to aging are caused by factors other than the deterioration of taste detection systems in the oral cavity.


Assuntos
Envelhecimento/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Papilas Gustativas/fisiologia , Paladar/fisiologia , Transducina/fisiologia , Animais , Preferências Alimentares , Masculino , Camundongos , Camundongos Mutantes , Receptores Acoplados a Proteínas G/genética , Papilas Gustativas/metabolismo , Percepção Gustatória/fisiologia , Transducina/deficiência
5.
Brain Behav Immun ; 71: 23-27, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29678794

RESUMO

Inflammatory bowel disease (IBD) is a debilitating immune-related condition that affects over 1.4 million Americans. Recent studies indicate that taste receptor signaling is involved in much more than sensing food flavor, and taste receptors have been localized in a variety of extra-oral tissues. One of the newly revealed functions of taste receptors and downstream signaling proteins is modulation of immune responses to microbes and parasites. We previously found that components of the taste receptor signaling pathway are expressed in subsets of the intestinal epithelial cells. α-Gustducin, a key G-protein α subunit involved in sweet, umami, and bitter taste receptor signaling, is expressed in the intestinal mucosa. In this study, we investigated the role of α-gustducin in regulation of gut mucosal immunity and inflammation using α-gustducin knockout mice in the dextran sulfate sodium (DSS)-induced IBD model. DSS is a chemical colitogen that can cause intestinal epithelial damage and inflammation. We analyzed DSS-induced colitis in α-gustducin knockout versus wild-type control mice after administration of DSS in drinking water. Our results show that the knockout mice had aggravated weight loss, diarrhea, intestinal bleeding, and inflammation over the experimental period compared to wild-type mice, concurrent with augmented immune cell infiltration and increased expression of TNF and IFN-γ but decreased expression of IL-13 and IL-5 in the colon. These results suggest that the taste receptor signaling pathway may play critical roles in regulating gut immune balance and inflammation.


Assuntos
Mucosa Intestinal/metabolismo , Transducina/metabolismo , Transducina/fisiologia , Animais , Colite/fisiopatologia , Colo/metabolismo , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/fisiopatologia , Interferon gama/metabolismo , Mucosa Intestinal/imunologia , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologia , Paladar/fisiologia , Papilas Gustativas/metabolismo , Transducina/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
J Proteome Res ; 16(12): 4391-4402, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28853286

RESUMO

Despite evidence for sex-specific cardiovascular physiology and pathophysiology, the biological basis for this dimorphism remains to be explored. Apart from hormonal factors, gender-related characteristics may reside in the function of sex chromosomes during cardiac development. In this study, we investigated the differential expression of the male-specific region of the Y chromosome (MSY) genes and their X counterparts during cardiac differentiation of human embryonic stem cells (hESC). We observed alterations in mRNA and protein levels of TBL1Y, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y1, DDX3Y, PRY, XKRY, BCORP1, RBMY, HSFY, and UTY, which accompanied changes in intracellular localization. Of them, the abundance of a Y chromosome missing protein, TBL1Y, showed a significant increase during differentiation while the expression level of its X counterpart decreased. Consistently, reducing TBL1Y cellular level using siRNA approach influenced cardiac differentiation by reducing its efficacy as well as increasing the probability of impaired contractions. TBL1Y knockdown may have negatively impacted cardiogenesis by CtBP stabilization. Furthermore, we presented compelling experimental evidence to distinguish TBL1Y from TBL1X, its highly similar X chromosome homologue, and proposed reclassification of TBL1Y as "found missing protein" (PE1). Our results demonstrated that MSY proteins may play an important role in cardiac development.


Assuntos
Cromossomos Humanos Y/genética , Miocárdio/citologia , Transducina/fisiologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Coração/crescimento & desenvolvimento , Humanos , Masculino , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo
7.
Dev Biol ; 431(1): 77-92, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28347645

RESUMO

We examine the molecular phylogeny of the proteins underlying the activation steps of vertebrate phototransduction, for both agnathan and jawed vertebrate taxa. We expand the number of taxa analysed and we update the alignment and tree building methodology from a previous analysis. For each of the four primary components (the G-protein transducin alpha subunit, GαT, the cyclic GMP phosphodiesterase, PDE6, and the alpha and beta subunits of the cGMP-gated ion channel, CNGC), the phylogenies appear consistent with expansion from an ancestral proto-vertebrate cascade during two rounds of whole-genome duplication followed by divergence of the agnathan and jawed vertebrate lineages. In each case, we consider possible scenarios for the underlying gene duplications and losses, and we apply relevant constraints to the tree construction. From tests of the topology of the resulting trees, we obtain a scenario for the expansion of each component during 2R that accurately fits the observations. Similar analysis of the visual opsins indicates that the only expansion to have occurred during 2R was the formation of Rh1 and Rh2. Finally, we propose a hypothetical scenario for the conversion of an ancestral chordate cascade into the proto-vertebrate phototransduction cascade, prior to whole-genome duplication. Together, our models provide a plausible account for the origin and expansion of the vertebrate phototransduction cascade.


Assuntos
Evolução Molecular , Visão Ocular/genética , Visão Ocular/fisiologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Duplicação Gênica , Humanos , Modelos Genéticos , Opsinas/genética , Opsinas/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Filogenia , Transducina/genética , Transducina/fisiologia , Vertebrados/genética , Vertebrados/crescimento & desenvolvimento , Vertebrados/fisiologia
8.
J Biol Chem ; 290(31): 19044-54, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26070566

RESUMO

G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína-Arginina N-Metiltransferases/fisiologia , Transducina/fisiologia , Transporte Ativo do Núcleo Celular , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Metilação , Sinais de Localização Nuclear , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteólise , Ubiquitinação
9.
Proc Natl Acad Sci U S A ; 110(30): 12468-73, 2013 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-23836670

RESUMO

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca(2+) current, suggesting interactions of transducin with the synaptic machinery.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transmissão Sináptica/fisiologia , Transducina/metabolismo , Animais , Escuridão , Luz , Camundongos , Camundongos Transgênicos , Transporte Proteico , Degeneração Retiniana/prevenção & controle , Células Fotorreceptoras Retinianas Bastonetes/citologia , Transducina/fisiologia
10.
J Neurosci ; 33(12): 5182-94, 2013 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-23516284

RESUMO

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gß3 and Gγt2 subunits. The function of Gßγ in this cascade has not been examined. Here, we investigate the role of Gß3 by assessing cone structure and function in Gß3-null mouse (Gnb3(-/-)). We found that Gß3 is required for the normal expression of its partners, because in the Gnb3(-/-) cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3(-/-) cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gß subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3(-/-) cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gß3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Transducina/genética , Visão Ocular/fisiologia , Animais , Cor , Feminino , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas de Fluorescência Verde/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Estimulação Luminosa , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Transducina/fisiologia , Raios Ultravioleta
11.
J Biol Chem ; 288(8): 5257-67, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23288843

RESUMO

The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gß(1)γ(1), 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/fisiologia , Animais , DNA Complementar/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Luz , Transdução de Sinal Luminoso , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Modelos Biológicos , Opsinas/metabolismo , Pigmentação , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Rodopsina/metabolismo , Transdução de Sinais , Transducina/genética
12.
Proc Natl Acad Sci U S A ; 108(5): 2094-9, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21245306

RESUMO

Ghrelin is a hunger hormone with gastroprokinetic properties but the factors controlling ghrelin secretion from the stomach are unknown. Bitter taste receptors (T2R) and the gustatory G proteins, α-gustducin (gust) and α-transducin, are expressed in the gut and are involved in the chemosensation of nutrients. This study aimed to investigate whether T2R-agonists affect (i) ghrelin release via α-gustducin and (ii) food intake and gastric emptying via the release of ghrelin. The mouse stomach contains two ghrelin cell populations: cells containing octanoyl and desoctanoyl ghrelin, which were colocalized with α-gustducin and α-transducin, and cells staining for desoctanoyl ghrelin. Gavage of T2R-agonists increased plasma octanoyl ghrelin levels in WT mice but the effect was partially blunted in gust(-/-) mice. Intragastric administration of T2R-agonists increased food intake during the first 30 min in WT but not in gust(-/-) and ghrelin receptor knockout mice. This increase was accompanied by an increase in the mRNA expression of agouti-related peptide in the hypothalamus of WT but not of gust(-/-) mice. The temporary increase in food intake was followed by a prolonged decrease (next 4 h), which correlated with an inhibition of gastric emptying. The delay in emptying, which was partially counteracted by ghrelin, was not mediated by cholecystokinin and GLP-1 but involved a direct inhibitory effect of T2R-agonists on gastric contractility. This study is unique in providing functional evidence that activation of bitter taste receptors stimulates ghrelin secretion. Modulation of endogenous ghrelin levels by tastants may provide novel therapeutic applications for the treatment of weight -and gastrointestinal motility disorders.


Assuntos
Comportamento Alimentar/fisiologia , Esvaziamento Gástrico/fisiologia , Grelina/metabolismo , Papilas Gustativas/fisiologia , Transducina/fisiologia , Animais , Sequência de Bases , Colecistocinina/fisiologia , Primers do DNA , Grelina/sangue , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Liso/fisiologia , Reação em Cadeia da Polimerase , Radioimunoensaio , Papilas Gustativas/efeitos dos fármacos
13.
PLoS One ; 5(11): e15009, 2010 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-21124784

RESUMO

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4(-/-)) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/-) Gnat1(-/-) Cnga3(-/-)). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.


Assuntos
Aprendizagem da Esquiva/efeitos da radiação , Luz , Atividade Motora/efeitos da radiação , Opsinas de Bastonetes/fisiologia , Animais , Atropina/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Channelrhodopsins , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Eletrorretinografia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Análise de Regressão , Retina/metabolismo , Opsinas de Bastonetes/genética , Transducina/genética , Transducina/fisiologia , Córtex Visual/metabolismo , Córtex Visual/efeitos da radiação
14.
J Physiol ; 588(Pt 17): 3231-41, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20603337

RESUMO

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.


Assuntos
Proteínas do Olho/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/genética , Tempo de Reação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/genética , Animais , Regulação para Baixo/genética , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Estimulação Luminosa/métodos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Tempo de Reação/genética , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sensibilidade e Especificidade , Transducina/biossíntese , Transducina/fisiologia , Regulação para Cima/genética
15.
J Neurophysiol ; 104(2): 896-901, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20519578

RESUMO

Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the alpha-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels.


Assuntos
Trifosfato de Adenosina/metabolismo , Papilas Gustativas/citologia , Papilas Gustativas/fisiologia , Paladar/fisiologia , Transducina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Benzamidinas/farmacologia , Carbenoxolona/farmacologia , Relação Dose-Resposta a Droga , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Quinina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Glutamato de Sódio/farmacologia , Edulcorantes/farmacologia , Paladar/efeitos dos fármacos , Papilas Gustativas/efeitos dos fármacos , Tetrodotoxina/farmacologia , Transducina/genética
16.
Cell Mol Life Sci ; 67(5): 817-28, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19946729

RESUMO

Malignant melanoma is one of the most aggressive human neoplasms which develop from the malignant transformation of normal epithelial melanocytes and share the lineage with retinal cells. cGMP-phosphodiesterase 6 (PDE6) is one of the cancer-retina antigens newly identified in melanoma cells. Normally, PDE6 hydrolyzes the photoreceptor second messenger cGMP allowing the visual signal transduction in photoreceptor cells. cGMP also play an important signaling role in stimulating melanogenesis in human melanocytes. Here, we present evidence that PDE6 is a key enzyme regulating the cGMP metabolism in melanoma cells. Decrease in intracellular cGMP leads to calcium accumulation in melanoma cells. In these cells, cGMP-phosphodiesterase 6 can be activated by another cancer-retina antigen, transducin, through Wnt5a-Frizzled-2 cascade, which leads to a lowering of cGMP and an increase in intracellular calcium mobilization. Thus, the aberrant expression of PDE6 may control cGMP metabolism and calcium homeostasis in melanoma cells.


Assuntos
Cálcio/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/fisiologia , Receptores Frizzled/fisiologia , Melanoma/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Neoplasias Cutâneas/metabolismo , Transducina/fisiologia , Proteínas Wnt/fisiologia , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Homeostase/efeitos dos fármacos , Homeostase/genética , Homeostase/fisiologia , Humanos , Melanoma/genética , Modelos Biológicos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/genética , Transducina/genética , Transducina/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a
17.
PLoS One ; 4(8): e6616, 2009 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-19672311

RESUMO

Pathogenesis in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65(-/-) mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65(-/-) mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65(-/-)/Gnat1(-/-) mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65(-/-)/Bax(-/-) mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65(-/-) mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65(-/-)/Bax(-/-) mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.


Assuntos
Apoptose/fisiologia , Amaurose Congênita de Leber/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Doenças Retinianas/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteína X Associada a bcl-2/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Amaurose Congênita de Leber/complicações , Camundongos , Camundongos Knockout , Transducina/genética , Transducina/fisiologia , Visão Ocular , Proteína X Associada a bcl-2/genética , cis-trans-Isomerases
18.
Ann N Y Acad Sci ; 1170: 91-4, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19686115

RESUMO

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that underlies the augmented insulin release from the pancreas in response to glucose in the gut lumen more than to intravenous injected glucose (the "incretin effect"). GLP-1, found in enteroendocrine L cells of the gut, regulates appetite and gut motility and is released from L cells in response to glucose. GLP-1-expressing duodenal L cells also express T1r taste receptors, alpha-gustducin, and many other taste transduction elements. Knockout mice lacking alpha-gustducin or T1r3 have deficiencies in secretion of GLP-1 and in the regulation of plasma levels of insulin and glucose. Gut-expressed taste-signaling elements underlie multiple chemosensory functions of the gut including the incretin effect. Modulating hormone secretion from gut "taste cells" may provide novel treatments for obesity, diabetes, and malabsorption.


Assuntos
Trato Gastrointestinal/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transducina/fisiologia , Animais , Trato Gastrointestinal/fisiologia , Camundongos , Camundongos Knockout , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/fisiologia
19.
Biochemistry ; 47(28): 7567-71, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18563917

RESUMO

Rhodopsin is the photosensitive pigment in the rod photoreceptor cell. Upon absorption of a photon, the covalently bound 11- cis-retinal isomerizes to the all- trans form, enabling rhodopsin to activate transducin, its G protein. All -trans-retinal is then released from the protein and reduced to all -trans-retinol. It is subsequently transported to the retinal pigment epithelium where it is converted to 11- cis-retinol and oxidized to 11- cis-retinal before it is transported back to the photoreceptor to regenerate rhodopsin and complete the visual cycle. In this study, we have measured the effects of all -trans- and 11- cis-retinals and -retinols on the opsin's ability to activate transducin to ascertain their potentials for activating the signaling cascade. Only 11- cis-retinal acts as an inverse agonist to the opsin. All -trans-retinal, all -trans-retinol, and 11- cis-retinol are all agonists with all -trans-retinal being the most potent agonist and all -trans-retinol being the least potent. Taken as a whole, our study is consistent with the hypothesis that the steps in the visual cycle are optimized such that the rod can serve as a highly sensitive dim light receptor. All -trans-retinal is immediately reduced in the photoreceptor to prevent back reactions and to weaken its effectiveness as an agonist before it is transported out of the cell; oxidation of 11- cis-retinol occurs in the retinal pigment epithelium and not the rod photoreceptor cell because 11- cis-retinol can act as an agonist and activate the signaling cascade if it were to bind an opsin, effectively adapting the cell to light.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Retinaldeído/farmacologia , Rodopsina/metabolismo , Transducina/fisiologia , Visão Ocular/fisiologia , Vitamina A/farmacologia , Animais , Células COS , Bovinos , Chlorocebus aethiops , Haplorrinos , Células Fotorreceptoras/fisiologia , Retinaldeído/química , Retinaldeído/metabolismo , Vitamina A/química
20.
Vision Res ; 48(20): 2052-61, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18456304

RESUMO

Signal transduction in outer segments of vertebrate photoreceptors is mediated by a series of reactions among multiple polypeptides that form protein-protein complexes within or on the surface of the disk and plasma membranes. The individual components in the activation reactions include the photon receptor rhodopsin and the products of its absorption of light, the three subunits of the G protein, transducin, the four subunits of the cGMP phosphodiesterase, PDE6 and the four subunits of the cGMP-gated cation channel. Recovery involves membrane complexes with additional polypeptides including the Na(+)/Ca(2+), K(+) exchanger, NCKX2, rhodopsin kinases RK1 and RK7, arrestin, guanylate cyclases, guanylate cyclase activating proteins, GCAP1 and GCAP2, and the GTPase accelerating complex of RGS9-1, G(beta5L), and membrane anchor R9AP. Modes of membrane binding by these polypeptides include transmembrane helices, fatty acyl or isoprenyl modifications, polar interactions with lipid head groups, non-polar interactions of hydrophobic side chains with lipid hydrocarbon phase, and both polar and non-polar protein-protein interactions. In the course of signal transduction, complexes among these polypeptides form and dissociate, and undergo structural rearrangements that are coupled to their interactions with and catalysis of reactions by small molecules and ions, including guanine nucleotides, ATP, Ca(2+), Mg(2+), and lipids. The substantial progress that has been made in understanding the composition and function of these complexes is reviewed, along with the more preliminary state of our understanding of the structures of these complexes and the challenges and opportunities that present themselves for deepening our understanding of these complexes, and how they work together to convert a light signal into an electrical signal.


Assuntos
Transdução de Sinal Luminoso/fisiologia , Proteínas de Membrana/metabolismo , Segmento Externo da Célula Bastonete/fisiologia , Membrana Celular/metabolismo , Humanos , Ligação Proteica/fisiologia , Rodopsina/metabolismo , Termodinâmica , Transducina/fisiologia
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