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1.
J Photochem Photobiol B ; 167: 256-263, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28088107

RESUMO

BACKGROUND: Local anesthetics are used to relieve pre- and postoperative pain, acting on both sodium channels and nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction (NMJ). Bupivacaine acts as a non-competitive antagonist and has limitations, such as myotoxicity, neurotoxicity, and inflammation. Low-level laser therapy (LLLT) has anti-inflammatory, regenerative, and analgesic effects. The aim of the present study was to evaluate the effects of a gallium arsenide laser (GaAs) on the morphology of the NMJ and nAChRs after application of bupivacaine in the sternomastoid muscle. METHODS: Thirty-two adult male Wistar rats received injections of bupivacaine 0.5% (Bupi: right antimere) and 0.9% sodium chloride (Cl: left antimere). Next, the animals were divided into a Control group (C) and a Laser group (LLLT). The laser group received LLLT (GaAs 904nm, 50mW, 4,8J) in both antimeres for five consecutive days. After seven days, the animals were euthanized and the surface portion of the sternomastoid muscle was removed, frozen, and subjected to morphological and morphometric analyses of the NMJs (nonspecific esterase reaction), confocal laser scanning, and an ultrastructural analysis. The nAChRs were quantified by Western blotting. RESULTS: In the chloride group, the morphology and morphometry of the NMJs remained stable. The maximum diameters of the NMJs were lower in the Bupi (15.048±1.985) and LLLT/Bupi subgroups (15.456±1.983) compared to the Cl (18.502±2.058) and LLLT/Cl subgroups (19.356±2.522) (p<0.05). Ultrastructurally, LLLT reduced myonecrosis observed after application of bupivacaine, with recovery in the junctional folds and active zone. There was an increase in the perimeter of the LLLT/Bupi subgroup (150.33) compared to the Bupi subgroup (74.69) (p<0.01) observed by confocal microscopy. There was also an increase in the relative planar area of the NMJ after LBI (8.75) compared to CBupi (4.80) (p<0.01). An analysis of the protein expression of nAChRα1 showed no major differences in the groups studied. There was an increase in protein expression of the ε subunit after application of LLLT (13.055) compared to Bupi (0.251) (p<0.01). Taken together, the present experiments indicate that there was a positive association of the α and γ subunits (p<0.05). CONCLUSIONS: These results demonstrate that LLLT at the dose used in this study reduced structural alterations in the NMJ and molecular changes in nAChRs triggered by bupivacaine, providing important data supporting the use of LLLT in therapeutic protocols for injuries triggered by local anesthetics.


Assuntos
Anestésicos Locais/efeitos adversos , Bupivacaína/efeitos adversos , Lasers Semicondutores , Terapia com Luz de Baixa Intensidade , Junção Neuromuscular/efeitos da radiação , Receptores Nicotínicos/efeitos da radiação , Animais , Western Blotting , Masculino , Microscopia Confocal , Ratos , Ratos Wistar
2.
Chembiochem ; 17(14): 1323-7, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27123791

RESUMO

We describe the reversible photoactivation of the acid sensitive ligand-gated ion channel ASIC2a, a mammalian channel found throughout the central and peripheral nervous systems that is associated with vision and pain. We also show the activation of GLIC, an acid-sensitive prokaryotic homologue of the nicotinic acetylcholine receptor. Photoactivation was achieved by using visible light irradiation of a newly synthesized water-soluble merocyanine photoacid, 1, which was designed to remove adverse channel blocking effects of a related system. Activation of ASIC2a and GLIC occurs reversibly, in a benign manner, and only upon irradiation. Further studies using transient absorption spectroscopy showed that protonation of a colorimetric base occurred rapidly (ca. 100 µs) after excitation of 1. These results demonstrate that irradiation of 1 can induce rapid, local pH changes that can be used to investigate both biological and chemical proton transfer reactions.


Assuntos
Canais Iônicos Sensíveis a Ácido/efeitos da radiação , Luz , Canais Iônicos Sensíveis a Ácido/fisiologia , Animais , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Ligantes , Dor/fisiopatologia , Receptores Nicotínicos/efeitos da radiação , Visão Ocular/fisiologia
3.
Biochemistry ; 46(42): 11911-8, 2007 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-17910479

RESUMO

3-Azioctanol is a photoactivatable analogue of octanol that noncompetitively inhibits nicotinic acetylcholine receptors (nAChRs). Photolabeling studies using [3H]-3-azioctanol in Torpedo nAChR identified alphaE262 as a site of desensitization-dependent incorporation. However, it is unknown whether photolabeling of alphaE262 causes functional effects in nAChRs and what other roles this residue plays in gating, desensitization, and channel block. We used ultrafast patch-perfusion electrophysiology and ultraviolet (UV) irradiation to investigate the state-dependence of both reversible nAChR inhibition by 3-azioctanol and the irreversible effects of photoactivated 3-azioctanol. Channels with mutations at alphaE262 were studied to determine ACh EC50s, desensitization rates, and sensitivities to reversible and photoirreversible 3-azioctanol inhibition. Exposure to 3-azioctanol in the presence of 365 nm UV light produced irreversible inhibition of wild-type nAChRs. Desensitization with ACh dramatically increased the degree of irreversible inhibition by photoactivated 3-azioctanol. Mutations at alphaE262 that reduce diazirine photomodification decreased the irreversible inhibition induced by photoactivated 3-azioctanol. Hydrophobic mutations at alphaE262 significantly slowed rapid ACh-induced desensitization and dramatically slowed fast resensitization. In contrast, alphaE262 mutations minimally affected 3-azioctanol channel block, and a half blocking concentration of 3-azioctanol did not alter the rate of ACh-induced fast desensitization. Our results indicate that position alphaE262 on muscle nAChRs contributes to an allosteric modulator site that is strongly coupled to desensitization. Occupation of this pocket by hydrophobic molecules stabilizes a desensitized state by slowing resensitization.


Assuntos
Músculos/metabolismo , Antagonistas Nicotínicos/química , Octanóis/química , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efeitos da radiação , Substituição de Aminoácidos , Animais , Eletrofisiologia , Feminino , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Ativação do Canal Iônico/genética , Camundongos , Microinjeções , Músculos/química , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacologia , Octanóis/metabolismo , Octanóis/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Raios Ultravioleta , Xenopus laevis
4.
J Mol Neurosci ; 30(1-2): 3-4, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17192602

RESUMO

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.


Assuntos
Acetilcolina/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efeitos da radiação , Acetilcolina/química , Regulação Alostérica , Animais , Sítios de Ligação , Receptores Nicotínicos/química , Tetracaína/metabolismo , Raios Ultravioleta
5.
Neurosci Lett ; 336(2): 81-4, 2003 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-12499045

RESUMO

Light-exposure of the chick embryo induces development of asymmetry in the thalamofugal visual projections to the Wulst regions of the forebrain since the embryo is turned so that it occludes its left and not its right eye. This asymmetry can be reversed by occluding the embryo's right eye and exposing its left eye to light. Here we show that three sub-regions of the thalamus (two in the dorsolateral anterior thalami (DLA) and one more caudal) have differing asymmetries of contralateral and/or ipsilateral projections. Hence the effect of asymmetrical light stimulation is regionally specific within the thalamus. Lateralised light stimulation appears to promote the development of ipsilateral projections from DLA pars dorsolateralis pars anterioris and contralateral projections from the caudal regions (the nucleus superficialis parvocellularis especially) but it may suppress the development of contralateral projections from the nucleus dorsolateralis anterior thalami pars lateralis rostralis. We also show that the light stimulation causes lateralised expression of c-fos and receptors for neurotransmitters.


Assuntos
Lateralidade Funcional/efeitos da radiação , Luz , Telencéfalo/crescimento & desenvolvimento , Telencéfalo/efeitos da radiação , Tálamo/crescimento & desenvolvimento , Tálamo/efeitos da radiação , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/efeitos da radiação , Animais , Embrião de Galinha , Galinhas , Lateralidade Funcional/fisiologia , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos da radiação , Estimulação Luminosa , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/efeitos da radiação , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efeitos da radiação , Privação Sensorial/fisiologia , Telencéfalo/citologia , Telencéfalo/embriologia , Tálamo/citologia , Tálamo/embriologia , Vias Visuais/embriologia
6.
Chembiochem ; 3(7): 652-8, 2002 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-12324999

RESUMO

We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclohexadienoylpropyl)trimethylammonium ([(3)H]DCTA). We quantified the incorporation of radioactivity into the receptor subunits as a function of the mixing time of [(3)H]DCTA with the receptor by using a rapid-mixing device adapted with a photochemical quenching system. A saturable increase of the specific photolabeling on the alpha and gamma subunits was observed with a half-time of about 2 minutes. We further analyzed this photoincorporation either after rapid mixing for 500 ms or after equilibration for 50 minutes. Under these conditions, [(3)H]DCTA explored transient state(s) and the stable desensitized state, respectively. Comparative analyses showed that at a probe concentration of 10 microM the relative variation of photoincorporation was more pronounced for the gamma subunit (three- to fourfold) than for the alpha subunit (about twofold). By contrast, the relative distribution of radioactivity among alpha-subunit labeled residues (alphaTyr190, alphaCys192, alphaCysC193, and alphaTyr198) did not change. Altogether, these results reveal that during the course of agonist-induced receptor desensitization, the site-lining peptide loops, which belong to adjacent alpha and gamma subunits, move closer to each other.


Assuntos
Acetilcolina/metabolismo , Receptores Nicotínicos/metabolismo , Torpedo/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Sítios de Ligação , Compostos de Diazônio/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Receptores Nicotínicos/efeitos da radiação , Trítio , Raios Ultravioleta
7.
FEBS Lett ; 309(2): 213-7, 1992 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-1505686

RESUMO

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically released carbamoylcholine ('caged' carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N-[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N-(alpha-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm-1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.


Assuntos
Carbacol/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Análise de Fourier , Conformação Proteica , Receptores Nicotínicos/química , Receptores Nicotínicos/efeitos da radiação , Espectrofotometria Infravermelho , Torpedo
8.
Mol Pharmacol ; 36(2): 327-32, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2549388

RESUMO

In low ionic strength buffer (5 mM Tris.HCl), the binding of [3H] nitrendipine to dihydropyridine calcium antagonist binding sites of mouse forebrain membranes is increased by both Na+ and Ca2+. Radiation inactivation was used to determine the target size of [3H]nitrendipine binding sites in 5 mM Tris.HCl buffer, in the presence and absence of these cations. After irradiation, [3H] nitrendipine binding in buffer with or without Na+ was diminished, due to a loss of binding sites and also to an increase in Kd. After accounting for radiation effects on the dissociation constant, the target size for the nitrendipine binding site in buffer was 160-170 kDa and was 170-180 kDa in the presence of sodium. In the presence of calcium ions, [3H]nitrendipine binding showed no radiation effects on Kd and yielded a target size of 150-170 kDa. These findings suggest, as in the case of opioid receptors, the presence of high molecular weight membrane components that modulate cation-induced alterations in radioligand binding to dihydropyridine binding sites.


Assuntos
Encéfalo/metabolismo , Cátions/metabolismo , Receptores Nicotínicos/efeitos da radiação , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/efeitos da radiação , Encéfalo/efeitos da radiação , Cálcio/farmacologia , Canais de Cálcio , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Técnicas In Vitro , Masculino , Camundongos , Nitrendipino/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Receptores Nicotínicos/metabolismo , Sódio/farmacologia
9.
Neurosci Lett ; 87(1-2): 145-50, 1988 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-3380333

RESUMO

Radiation inactivation and simple target theory were employed to determine the molecular weight of an insect CNS alpha-bungarotoxin binding component in the presence and absence of a cross-linking reagent, dimethyl suberimate. In the presence of the cross-linker, the number of binding sites decreased, and the apparent molecular weight (236,000) was approximately double the control value (112,000). This, together with sedimentation data, suggests that the lower value represents only a portion of the insect nicotinic receptor molecule. A model is presented to account for the increase in target size and reduction in the number of alpha-[3H]bungarotoxin binding sites in the presence of dimethyl suberimate.


Assuntos
Baratas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Dimetil Suberimidato/farmacologia , Gânglios/metabolismo , Imidoésteres/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Baratas/efeitos dos fármacos , Gânglios/efeitos dos fármacos , Gânglios/efeitos da radiação , Peso Molecular , Receptores Nicotínicos/efeitos da radiação
10.
J Biol Chem ; 259(9): 5384-7, 1984 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-6325434

RESUMO

[3H]Nitrendipine and high intensity ultraviolet irradiation have been used to photoaffinity label the protein component of the high affinity nitrendipine-binding site in subcellular membrane fractions from canine cardiac muscle. Irradiation of isolated cardiac membranes in the presence of [3H]nitrendipine resulted in the covalent labeling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 32,000. Incorporation of [3H]nitrendipine did not occur in the absence of irradiation. The photoaffinity labeling of the 32,000-Da protein by [3H]nitrendipine was inhibited by excess unlabeled nitrendipine, nifedipine, or verapamil. EDTA, ATP, and La3+, which are known to reduce high affinity nitrendipine binding, also inhibited the photoaffinity labeling of this membrane protein by [3H]nitrendipine. The 32,000-Da [3H]nitrendipine-labeled protein was found to be enriched in the ryanodine-sensitive fraction of cardiac sarcoplasmic reticulum and absent from the ryanodine-insensitive fraction of cardiac sarcoplasmic reticulum which is known to lack high affinity nitrendipine binding. Therefore, the 32,000-Da photoaffinity-labeled [3H]nitrendipine-binding protein exhibits properties identical to those expected for the protein component of the high affinity nitrendipine-binding site in isolated cardiac membranes.


Assuntos
Marcadores de Afinidade , Bloqueadores dos Canais de Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Miocárdio/metabolismo , Nifedipino/análogos & derivados , Fluoreto de Fenilmetilsulfonil/farmacologia , Receptores Nicotínicos/metabolismo , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Sulfonas/farmacologia , Animais , Canais de Cálcio , Cães , Luz , Nifedipino/metabolismo , Nifedipino/farmacologia , Nitrendipino , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/efeitos da radiação , Raios Ultravioleta , Verapamil/farmacologia
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