RESUMO
KEY POINTS: Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine and α-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity. ABSTRACT: Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Fusos Musculares/fisiologia , Receptores Colinérgicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Bungarotoxinas/farmacologia , Hemicolínio 3/farmacologia , Masculino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Células Receptoras Sensoriais , Tubocurarina/farmacologiaRESUMO
INTRODUCTION: The aim of this study was to compare the effects of adenosine-5'-triphosphate (ATP) and adenosine on the contractility of rodent extensor digitorum longus (EDL) muscle at normal and low temperatures. METHODS: Contractions of rat and mouse isolated EDL were induced by either electrical stimulation (ES) or exogenous carbachol and recorded in the presence of ATP or adenosine (both at 100 µM). RESULTS: ATP at all temperatures caused a decrease of the contractions induced by carbachol in rat and mouse EDL and ES-induced contractions in rat EDL, while it potentiated the ES-induced contractions of mouse EDL. Adenosine reduced the contractility of rat and mouse EDL evoked by ES and did not affect the carbachol-induced contractions of rat and mouse EDL at any temperature. DISCUSSION: Under various temperature conditions, ATP inhibits pre- but potentiates postsynaptic processes in the mouse EDL; in the rat EDL ATP causes only inhibition of neuromuscular conduction. Muscle Nerve 59:509-516, 2019.
Assuntos
Trifosfato de Adenosina/farmacologia , Contração Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Animais , Carbacol/farmacologia , Temperatura Baixa , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Camundongos , Agonistas Muscarínicos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fármacos Neuromusculares não Despolarizantes/farmacologia , Agonistas Purinérgicos/farmacologia , Ratos , Ratos Wistar , Tubocurarina/farmacologiaRESUMO
Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca2+-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.
Assuntos
Simulação por Computador , Células Ciliadas Auditivas/fisiologia , Modelos Biológicos , Estereocílios/fisiologia , Animais , Cálcio/metabolismo , Quelantes/farmacologia , Galinhas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Epitélio/ultraestrutura , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/ultraestrutura , Microscopia Eletrônica de Varredura , Fármacos Neuromusculares não Despolarizantes/farmacologia , Estereocílios/efeitos dos fármacos , Estereocílios/ultraestrutura , Processos Estocásticos , Técnicas de Cultura de Tecidos , Tubocurarina/farmacologiaRESUMO
INTRODUCTION: The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. METHODS: Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). RESULTS: At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. CONCLUSIONS: ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.
Assuntos
Trifosfato de Adenosina/farmacologia , Adenosina/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Temperatura , Análise de Variância , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Estimulação Elétrica , Hipotermia/induzido quimicamente , Masculino , Antagonistas Nicotínicos/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Wistar , Suramina/farmacologia , Teofilina/análogos & derivados , Teofilina/farmacologia , Tubocurarina/farmacologiaRESUMO
INTRODUCTION: In this study we examined the mechanisms of motor dysfunction in type 2 diabetes. METHODS: Contractile force was measured in isolated nerve-muscle preparations of db/db mice using various protocols for electrical stimulation. Sarcoplasmic reticulum Ca(2+) adenosine triphosphatase protein (SERCA) was quantified by comparing Ca(2+) -dependent and non-specific phosphorylation. RESULTS: Compared with controls, the muscle-nerve preparations of db/db mice displayed muscle atrophy, reduced axonal excitability, and force deficit when stimulated via the nerve. Muscle relaxation after contraction was slowed, and SERCA content was reduced. In contrast, the sensitivity of the neuromuscular junction to tubocurarine and muscle fiber excitability were not affected. CONCLUSIONS: The force deficit in db/db muscles was caused by atrophy and failure of neuromuscular signal transmission related to motor nerve axonal dysfunction. The slowed relaxation rate generally observed in diabetic muscles can, to a large extent, be explained by decreased SERCA pump content. Muscle Nerve 54: 460-468, 2016.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Músculo Esquelético/fisiopatologia , Doenças Musculares/etiologia , Doenças Musculares/patologia , Trifosfato de Adenosina/farmacocinética , Análise de Variância , Animais , Peso Corporal/genética , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica , Camundongos , Camundongos Mutantes , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Mutação/genética , Antagonistas Nicotínicos/farmacologia , Isótopos de Fósforo/farmacocinética , Receptores para Leptina/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tubocurarina/farmacologiaRESUMO
A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.
Assuntos
Alcaloides/isolamento & purificação , Curare/isolamento & purificação , Tubocurarina/isolamento & purificação , Alcaloides/farmacologia , Animais , Curare/química , Estrutura Molecular , Oócitos/efeitos dos fármacos , Venenos/farmacologia , Ratos , Tubocurarina/farmacologiaRESUMO
Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.
Assuntos
Alcaloides/química , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Strychnos/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Alcaloides/síntese química , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Tubocurarina/análogos & derivados , Tubocurarina/síntese química , Tubocurarina/química , Tubocurarina/farmacologia , Verapamil/farmacologiaRESUMO
Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite's vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule's position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K(+) channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components.
Assuntos
Caenorhabditis elegans/fisiologia , Receptores Colinérgicos/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biossíntese , Sinalização do Cálcio , Carbenoxolona/farmacologia , Copulação/fisiologia , Feminino , Junções Comunicantes/fisiologia , Levamisol/farmacologia , Masculino , Contração Muscular , Músculos/fisiologia , Rede Nervosa , Antagonistas Nicotínicos/farmacologia , Interferência de RNA , Receptores Colinérgicos/genética , Receptores Nicotínicos/biossíntese , Rodopsina/biossíntese , Transdução de Sinais , Tubocurarina/farmacologia , Vulva/fisiologiaRESUMO
Anurans (frogs and toads) are important models for comparative studies of communication, auditory physiology, and neuroethology, but to date, most of our knowledge comes from in-depth studies of a relatively small number of model species. Using the well-studied green treefrog (Hyla cinerea), this study sought to develop and evaluate the use of auditory evoked potentials (AEPs) as a minimally invasive tool for investigating auditory sensitivity in a larger diversity of anuran species. The goals of the study were to assess the effects of frequency, signal level, sex, and body size on auditory brainstem response (ABR) amplitudes and latencies, characterize gross ABR morphology, and generate an audiogram that could be compared to several previously published audiograms for green treefrogs. Increasing signal level resulted in larger ABR amplitudes and shorter latencies, and these effects were frequency dependent. There was little evidence for an effect of sex or size on ABRs. Analyses consistently distinguished between responses to stimuli in the frequency ranges of the three previously-described populations of afferents that innervate the two auditory end organs in anurans. The overall shape of the audiogram shared prominent features with previously published audiograms. This study highlights the utility of AEPs as a valuable tool for the study of anuran auditory sensitivity.
Assuntos
Anuros/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico , Retroalimentação Fisiológica , Modelos Biológicos , Vocalização Animal , Animais , Anuros/anatomia & histologia , Anuros/crescimento & desenvolvimento , Limiar Auditivo , Tronco Encefálico/anatomia & histologia , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/fisiologia , Feminino , Imobilização , Masculino , Fármacos Neuromusculares não Despolarizantes/farmacologia , Neurônios/fisiologia , Reprodutibilidade dos Testes , Caracteres Sexuais , Especificidade da Espécie , Texas , Tubocurarina/farmacologia , Aumento de PesoRESUMO
Tomopteris helgolandica Greeff 1879 (Tomopteridae) is a transparent holoplanktonic polychaete that can emit a bright light. In this study, we investigated the emission pattern and control of this deep-sea worm's luminescence. Potassium chloride depolarisation applied on anaesthetised specimens triggered a maximal yellow light emission from specific parapodial sites, suggesting that a nervous control pathway was involved. Pharmacological screening revealed a sensitivity to carbachol, which was confirmed by a dose-light response associated with a change in the light emission pattern, where physiological carbachol concentrations induced flashes and higher concentrations induced glows. The light response induced by its hydrolysable agonist, acetylcholine, was significantly weaker but was facilitated by eserine pretreatment. In addition, a specific inhibitory effect of tubocurarine was observed on carbachol-induced emission. Lastly, KCl- and carbachol-induced light responses were significantly reduced when preparations were pre-incubated in Ca(2+)-free artificial seawater or in different calcium channel blockers (verapamil, diltiazem) and calmodulin inhibitor (trifluoperazine) solutions. All of these results strongly suggest that T. helgolandica produces its light flashes via activation of nicotinic cholinergic receptors and a calcium-dependent intracellular mechanism involving L-type calcium channels.
Assuntos
Carbacol/farmacologia , Medições Luminescentes , Poliquetos/fisiologia , Cloreto de Potássio/metabolismo , Receptores Nicotínicos/metabolismo , Zooplâncton/fisiologia , Acetilcolina/farmacologia , Análise de Variância , Animais , Oceano Atlântico , Carbacol/agonistas , Carbacol/antagonistas & inibidores , Relação Dose-Resposta a Droga , Noruega , Água do Mar/química , Tubocurarina/farmacologiaRESUMO
Acetylcholine (ACh) is well known to be involved in the control of breathing. However, no information is available on the role of ACh receptors (AChRs) within the lamprey respiratory network. The present study was performed on in vitro brainstem preparations of adult lampreys to investigate whether ACh affects respiratory activity possibly through an action on the paratrigeminal respiratory group (pTRG) that has been identified as an essential component of the respiratory network. Respiratory activity was monitored as vagal motor output. Bath application of 100 µM physostigmine or 1 µM nicotine increased respiratory frequency, while bath application of 100 µM D-tubocurarine or 0.25 µM α-bungarotoxin reduced respiratory frequency and increased the duration of vagal bursts. Since these effects were mimicked by microinjections of the same drugs into the pTRG, ACh proved to influence respiratory activity by acting on α7 nicotinic AChRs located within the pTRG. During apnea caused by partial blockade of ionotropic glutamate receptors at the level of the pTRG, bath application of bicuculline and strychnine restored the respiratory rhythm, although at reduced frequency. Similar results were obtained by the concurrent removal of both fast synaptic excitatory and inhibitory transmission. Blockade of pTRG α7 nicotinic AChRs suppressed this respiratory activity, thus indicating that pTRG neurons expressing these receptors contribute to respiratory rhythm generation. Together, these findings identify a novel cholinergic modulatory and possibly subsidiary rhythmogenic mechanism within the respiratory network of the adult lamprey and encourage further studies on the respiratory role of cholinergic receptors in different animal species.
Assuntos
Tronco Encefálico/fisiologia , Receptores Nicotínicos/fisiologia , Centro Respiratório/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Tronco Encefálico/efeitos dos fármacos , Bungarotoxinas/farmacologia , Lampreias , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nicotina/farmacologia , Fisostigmina/farmacologia , Respiração/efeitos dos fármacos , Centro Respiratório/efeitos dos fármacos , Estricnina/farmacologia , Tubocurarina/farmacologia , Nervo Vago/fisiologia , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.
Assuntos
Cálcio/metabolismo , Junções Comunicantes/metabolismo , Potenciais de Ação , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Eletrofisiologia/métodos , Modelos Biológicos , Músculos/metabolismo , Fármacos Neuromusculares não Despolarizantes/farmacologia , Piridazinas/farmacologia , Interferência de RNA , Transmissão Sináptica , Tubocurarina/farmacologiaRESUMO
Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating neonicotinoid efficiency and open novel strategies for optimizing insect pest control.
Assuntos
Cálcio/metabolismo , Imidazóis/farmacologia , Inseticidas/farmacologia , Neurônios/metabolismo , Nitrocompostos/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animais , Bungarotoxinas/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Agonistas Colinérgicos/farmacologia , Baratas , AMP Cíclico/metabolismo , Guanidinas/farmacologia , Insetos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Neonicotinoides , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Tubocurarina/farmacologiaRESUMO
We examined the effects of nicotine on differentiation and function of monocyte-derived human dendritic cells (DCs). NiDCs, which were the DCs differentiated in the presence of nicotine, showed lower levels of CD1a. Secretion of IL-12 and TNF-α by lipopolysaccharide (LPS)-stimulated NiDCs was significantly suppressed compared to monocyte-derived DCs grown without nicotine. NiDCs displayed a diminished capacity to induce allogeneic T cell proliferation with a reduced production of IFN-γ, and maintained/enhanced LPS-mediated expression of coinhibitory molecules. Interestingly, NiDCs enhanced the expression of nuclear receptor peroxisome proliferator-activated receptors γ (PPAR γ), which has immunomodulatory properties. Expression of PPAR γ and PPAR γ-target genes was significantly inhibited by pretreatment with d-tubocurarine, antagonist of non-selective nicotinic acetylcholine receptors (nAChR). In addition, reduction of Th1 responses was inhibited after blocking nAChR-mediated signal. These data suggest the effect of nicotine on altering DC immunogenicity by impeding Th1 immunity is partially mediated by upregulation of PPAR γ.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Nicotina/farmacologia , PPAR gama/biossíntese , Receptores Nicotínicos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-12/metabolismo , Lipopolissacarídeos/imunologia , Monócitos/metabolismo , Antagonistas Nicotínicos/farmacologia , PPAR gama/genética , Fenótipo , Fumar/efeitos adversos , Fumar/imunologia , Células Th1/imunologia , Células Th2/imunologia , Tubocurarina/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
The nature of the potassium channels involved in determining endothelium-derived hyperpolarizing factor-mediated relaxation was investigated in first-order small mesenteric arteries from male endothelial nitric oxide synthase (eNOS-/-)-knockout and control (+/+) mice. Acetylcholine-induced endothelium-dependent relaxation of small mesenteric arteries of eNOS-/- was resistant to N-nitro-L-arginine and indomethacin and the guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one. Apamin and the combination of apamin and iberiotoxin or apamin and charybdotoxin induced a transient endothelium-dependent contraction of small mesenteric arteries from both eNOS-/- and +/+ mice. Acetylcholine-induced relaxation in eNOS-/- mice was unaffected by charybdotoxin or apamin alone but significantly inhibited by the combination of these agents. However, the combination of scyllatoxin and iberiotoxin did not mimic the inhibitory effect of the apamin/charybdotoxin combination. Tubocurarine alone completely blocked acetylcholine-induced relaxation in eNOS-/- mice. Single channel analysis of myocytes from small mesenteric arterioles revealed a large conductance calcium-activated potassium channel that was sensitive to iberiotoxin, charybdotoxin, and tetraethylammonium. Tubocurarine blocked this channel from the cytosolic side but not when applied extracellularly. Solutions of nitric oxide (NO) gas also relaxed small mesenteric arteries that had been contracted with cirazoline in a concentration-dependent manner, and the sensitivity to NO was reduced by iberiotoxin and the combination of apamin, scyllatoxin, or tubocurarine with charybdotoxin but not by apamin, charybdotoxin, scyllatoxin, or tubocurarine alone. These data indicate that acetylcholine-induced endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries from eNOS-/- involved the activation of tubocurarine and apamin-/charybdotoxin-sensitive K-channels. In eNOS+/+ mice, the acetylcholine-induced response was primarily mediated by NO and was sensitive to iberiotoxin and the combination of apamin and charybdotoxin.
Assuntos
Acetilcolina/farmacologia , Fatores Biológicos/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Canais de Potássio/metabolismo , Animais , Apamina/administração & dosagem , Apamina/farmacologia , Charibdotoxina/administração & dosagem , Charibdotoxina/farmacologia , Relação Dose-Resposta a Droga , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Knockout , Óxido Nítrico/farmacologia , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Canais de Potássio/efeitos dos fármacos , Tubocurarina/administração & dosagem , Tubocurarina/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
1. The 2 Hz train-of-four ratio (TOF(ratio)) is used to monitor the degree of patient curarization. Using a rat phrenic nerve-hemidiaphragm preparation, we showed that antinicotinic agents, such as hexamethonium, d-tubocurarine and pancuronium, but not cisatracurium, decreased contractions produced by physiological nerve activity patterns (50 Hz) more efficiently than those caused by 2 Hz trains. Uncertainty about the usefulness of the TOF(ratio) to control safe recovery from curarization prompted us to investigate the muscarinic and adenosine neuromodulation of tetanic (50 Hz) fade induced by antinicotinic agents at concentrations that cause a 25% reduction in the TOF(ratio) (TOF(fade)). 2. Tetanic fade caused by d-tubocurarine (1.1 µmol/L), pancuronium (3 µmol/L) and hexamethonium (5.47 mmol/L) was attenuated by blocking presynaptic inhibitory muscarinic M(2) and adenosine A(1) receptors with methoctramine (1 µmol/L) and 1,3-dipropyl-8-cyclopentylxanthine (2.5 nmol/L), respectively. These compounds enhanced rather than decreased tetanic fade induced by cisatracurium (2.2 µmol/L), but they consistently attenuated cisatracurium-induced TOF(fade). The effect of the M(1) receptor antagonist pirenzepine (10 nmol/L) on fade produced by antinicotinic agents at 50 Hz was opposite to that observed with TOF stimulation. Blockade of adenosine A(2A) receptors with ZM 241385 (10 nmol/L) attenuated TOF(fade) caused by all antinicotinic drugs tested, with the exception of the 'pure' presynaptic nicotinic antagonist hexamethonium. ZM 241385 was the only compound tested in this series that facilitated recovery from tetanic fade produced by cisatracurium. 3. The data suggest that distinct antinicotinic relaxants interfere with fine-tuning neuromuscular adaptations to motor nerve stimulation patterns via activation of presynaptic muscarinic and adenosine receptors. These results support the use of A(2A) receptor antagonists together with atropine to facilitate recovery from antinicotinic neuromuscular blockade.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Bloqueadores Neuromusculares/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Animais , Diafragma/efeitos dos fármacos , Diafragma/fisiologia , Sinergismo Farmacológico , Estimulação Elétrica/métodos , Hexametônio/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Junção Neuromuscular/fisiologia , Pancurônio/farmacologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/fisiologia , Ratos , Ratos Wistar , Receptor A2A de Adenosina/metabolismo , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Período Refratário Eletrofisiológico/efeitos dos fármacos , Tubocurarina/farmacologiaRESUMO
Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction. PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the ß2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect. In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.
Assuntos
Broncoconstrição , Agentes Neurotóxicos , Acetilcolinesterase , Animais , Atropina/farmacologia , Cromakalim/farmacologia , Estimulação Elétrica , Fumarato de Formoterol/farmacologia , Sulfato de Magnésio/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular , Agentes Neurotóxicos/farmacologia , Nicotina/farmacologia , Ratos , Tubocurarina/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Tubocurarine (d-TC), a non-depolarizing competitive blocker of nicotinic acetylcholine receptors is extensively utilized for the relaxation of skeletal muscles. Drug repositioning is a forthright approach to reduce the cost and speed up drug development process. Herein, we have attempted to evaluate the analgesic and anti-inflammatory activity of d-TC for its possible repurposing in pain and inflammation-related issues. EXPERIMENTAL APPROACH: We examined the soluble epoxide hydrolase inhibitory (sEHI) activity of d-TC employing in silico high throughput screening protocols, in vitro cell-free sEH inhibitory assay, and in in vivo rodent models for its repositioning in pain and inflammation-related disorders. KEY RESULTS: In molecular docking study, d-TC displayed impressive hydrogen bonding interactions within the cavity of sEH enzyme with good docking score. d-TC also exhibited notable sEH inhibitory activity (IC50 3.72 nm) at the in vitro assay. Oral absorption capability of d-TC (0.1 and 0.2 mg/mL) was determined using an in vitro everted intestinal sac model employing rat ileum tissue that revealed significant oral absorption of d-TC. Besides, in vivo studies revealed that oral administration of d-TC (0.1 and 0.2 mg/kg) in rodents significantly attenuated hyperalgesia (cold plate test, tail immersion test and formalin test) and inflammation (estimation of rectal temperature, acetic acid induced pleurisy test and cotton pellet-induced granuloma test) induced in robust preclinical models. Conclusion and implications These findings are novel and warrant immediate efforts to reposition d-TC as a new therapeutic candidate in the management of hyperalgesia, inflammation, and associated conditions.
Assuntos
Receptores Nicotínicos , Tubocurarina , Ratos , Animais , Tubocurarina/farmacologia , Tubocurarina/uso terapêutico , Epóxido Hidrolases , Reposicionamento de Medicamentos , Simulação de Acoplamento Molecular , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Inibidores Enzimáticos/farmacologiaRESUMO
It is widely recognized that neuronal network activity can be modulated via activation of nicotinic and muscarinic acetylcholine receptors located pre- and postsynaptically. It was established in our earlier study that the activation of presynaptic nicotinic receptors greatly facilitates the retinotectal glutamatergic transmission. In the present study, we have determined a transmitter of tectal recurrent excitation and explored the effects of muscarinic acetylcholine receptor activation on the recurrent excitation and the activity of frog tectum column in vivo. Discharge of a single retinal ganglion cell was elicited by a minimal electrical stimulation of the retina. Evoked activity of the tectum column was recorded using the carbon-fiber microelectrode inserted into the tectum layer F. We found the following: 1. The recurrent excitation in the tectum column was not affected by d-tubocurarine (10 µM) and was greatly depressed by the kynurenic acid (500 µM), demonstrating glutamatergic nature of the recurrent excitation. 2. The glutamatergic recurrent excitation was largely reduced by carbamylcholine (100 µM) and oxotremorine-M (10 µM), demonstrating that the activation of muscarinic receptors, located, presumably, on the presynaptic terminals of recurrent pear-shaped neurons, inhibits the recurrent excitation in the tectum column. 3. The muscarinic inhibition of glutamatergic recurrent transmission had critical influence on the activity of the tectum column, preventing the generation of an output signal through suppression of the NMDA receptor activation and establishing necessary conditions for returning of the network to its resting state.
Assuntos
Ácido Glutâmico/fisiologia , Neurônios Eferentes/fisiologia , Receptores Muscarínicos/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais Sinápticos/fisiologia , Teto do Mesencéfalo/fisiologia , Animais , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios Eferentes/efeitos dos fármacos , Oxotremorina/farmacologia , Rana temporaria , Potenciais Sinápticos/efeitos dos fármacos , Teto do Mesencéfalo/efeitos dos fármacos , Tubocurarina/farmacologiaRESUMO
1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 µmol/L)-, cisatracurium (0.32 µmol/L)- and vecuronium (0.36 µmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 µmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.