ABSTRACT
Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.
Subject(s)
Etomidate/pharmacology , Evoked Potentials/drug effects , Exocytosis/drug effects , Hypnotics and Sedatives/pharmacology , Motor Endplate/drug effects , Neuromuscular Junction/drug effects , Synaptic Vesicles/drug effects , Animals , Calcium Channels, P-Type/drug effects , Calcium Channels, P-Type/metabolism , Calcium Channels, Q-Type/drug effects , Calcium Channels, Q-Type/metabolism , Diaphragm/drug effects , Diaphragm/innervation , Dose-Response Relationship, Drug , Female , Mice , Receptors, Nicotinic/drug effectsABSTRACT
Em um delineamento experimental usando o fatorial 3x2, três crioprotetores internos, glicerol (GLI), etilenoglicol (EG) e dimetilformamida (DMF), e dois externos, gema de ovo (GEMA) e lipoproteína de baixa densidade (LDL), avaliaram-se a motilidade ao descongelamento de GLI-GEMA 53,9±1,96, sendo superior aos demais tratamentos (P<0,05). Na avaliação de morfologia ao descongelamento, não houve diferença (P>0,05) entre os tratamentos EG-GEMA 68,3±1,58, EG-LDL 72,2±2,39 e DMF-GEMA 68,7±1,67 que foram mais altos que os demais (P<0,05). A avaliação de integridade de membrana por fluorescência ao descongelamento GLI-GEMA 34,2±2,28 e EG-GEMA 30,9±1,32 não diferiram entre si (P>0,05), mas foram mais elevados que os demais (P<0,05), enquanto que a HOST dos tratamentos DMF-GEMA 13,6±1,30 e DMF-LDL 9,8±0,78 diferirem entre si (P<0,05) e foram mais baixas que as demais (P<0,05). O uso de etilenoglicol associado à gema de ovo pode ser uma alternativa ao uso de glicerol nos protocolos de congelamento de sêmen de touros.
The experiment was designed as 3 x 2 factorial design, with three internal cryoprotectants, glycerol (GLY), etileneglycol (EG) and dymethilformamide (DMF) and two external, egg yolk (YOLK) and density low lipoproteina (LDL). The motility at thawing for GLY-YOLK (53.9±1.96) was higher than other treatments (P<0.05). The percentage of cells with normal morphology at thawing was not different between EG-YOLK (68.3±1.58), EG-LDL (72.2±2.39) and DMF-YOLK (68.7±1.67), but they were higher than the others (P<0.05). The evaluation of membrane integrity through fluorescent probes at thawing indicate that GLY-YOLK (34.2±2.28) and EG-YOLK (30.9±1.32) were not different (P>0.05), but were higher than the others (P<0.05). The evaluation of membrane integrity through hypoosmotic swelling test (HOST) indicate that DMF-YOLK (13.6±1.30) and DMF-LDL (9.8±0.78) were different (P<0.05) and lower than the others (P<0.05). The use of ethylene glycol associated to egg yolk can be a viable alternative to the use of glycerol in bull semen freezing protocols.
Subject(s)
Animals , Cattle , Cryoprotective Agents , Glycerol/analysis , Semen/cytology , Cattle/classification , CryopreservationABSTRACT
Well-defined hybrids of linear poly(ethylene glycol)s (PEGs) and dendritic polyesters were prepared via the dendronization of the alcohol end groups of the mono and difunctional linear PEGs. Though useful for rudimentary product characterization, GPC and NMR could not verify the overall structural purity of these linear-dendritic hybrids. On the other hand, the detailed data provided by MALDI-ToF mass spectrometry enabled confirmation of the high structural purity of the dendronized PEGs at each step of the dendronization procedure. The well-defined number of functionalities on these dendronized PEGs, renders them particularly useful for research in the biomedical sphere where functionality and purity are of the utmost importance. The MALDI-ToF mass spectrometric approach described herein represents a valuable technique for detailed monitoring of these dendronization reactions, as well as a variety of other polymer end group modifications.
Híbridos bem definidos de poli(etilenoglicol) lineares (PEGs) e poliésteres dendriméricos foram preparados via "dendronização" de álcool e grupos de PEGs lineares mono e bifuncionais. Embora úteis para a caracterização rudimentar de produtos, Cromatografia por Permeação em Gel e RMN podem não demonstrar a pureza estrutural global desses híbridos lineares dendríticos. Por outro lado, informações detalhadas provenientes de espectrometria de massas MALDI-ToF permitiram a confirmação de elevada pureza estrutural de PEGs "dendronizados" em cada passo do processo de "dendronização". O número de funcionalidades bem definidas destes PEGs "dendronizados", torna-os particularmente úteis para pesquisa na área biomédica, na qual funcionalidade e pureza são de grande importância. A abordagem de espectrometria de massas MALDI-ToF descrita aqui representa uma técnica valiosa para o monitoramento detalhado destas reações de "dendronização", bem como diversas modificações de outros polímeros e grupos.
