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1.
Toxicol Sci ; 159(1): 179-188, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28645175

RESUMO

Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (P7) mice. However, neonatal mice younger than 7 days-age remained unaffected by BoNT/A injection. Also, BoNT/A inhibited stimulus evoked acetylcholine release and stimulus-evoked twitch tension of diaphragm nerve muscle preparations (NMPs) of adult mouse and >P7 neonates but not that of P7. However, cholesterol depletion using methyl-ß-cyclodextrin (MßCD) sensitized

Assuntos
Toxinas Botulínicas Tipo A/toxicidade , Colesterol/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Toxinas Botulínicas Tipo A/metabolismo , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Endocitose , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/metabolismo
2.
Eur J Pharmacol ; 744: 211-9, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25446918

RESUMO

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.


Assuntos
Acetilcolina/metabolismo , Capsaicina/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Animais , Capsaicina/análogos & derivados , Exocitose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Canais de Cátion TRPV/metabolismo , Estimulação do Nervo Vago/métodos
3.
Am J Physiol Endocrinol Metab ; 303(4): E551-61, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22739110

RESUMO

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.


Assuntos
Acetilcolinesterase/deficiência , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 1/enzimologia , Neuropatias Diabéticas/enzimologia , Doenças da Junção Neuromuscular/enzimologia , Acetilcolinesterase/biossíntese , Animais , Butirilcolinesterase/biossíntese , Inibidores da Colinesterase/farmacologia , Neuropatias Diabéticas/fisiopatologia , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/deficiência , Hiperglicemia/enzimologia , Hiperglicemia/fisiopatologia , Masculino , Camundongos , Placa Motora/enzimologia , Placa Motora/fisiopatologia , Debilidade Muscular/enzimologia , Debilidade Muscular/fisiopatologia , Doenças da Junção Neuromuscular/fisiopatologia , Fisostigmina/análogos & derivados , Fisostigmina/farmacologia
4.
Neuropharmacology ; 58(8): 1189-98, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20211192

RESUMO

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K(i) for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 muM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 muM DCH or 5 muM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40-90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 degrees C compared to 37 degrees C. Unlike DAP, neither DCH nor ABS 130 increased Ca(2+) levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca(2+) levels within motor nerve endings.


Assuntos
Antídotos/farmacologia , Toxinas Botulínicas Tipo A/intoxicação , Cinamatos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Metaloexopeptidases/antagonistas & inibidores , Junção Neuromuscular/efeitos dos fármacos , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Acetilcolina/metabolismo , Amifampridina , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Técnicas In Vitro , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Reflexo/efeitos dos fármacos
5.
J Pharmacol Exp Ther ; 331(2): 361-71, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19654265

RESUMO

Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.


Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/toxicidade , Capsaicina/uso terapêutico , Fármacos Neuromusculares/antagonistas & inibidores , Fármacos Neuromusculares/toxicidade , Junção Neuromuscular/efeitos dos fármacos , Fármacos Neuroprotetores , Acetilcolina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Capsaicina/análogos & derivados , Capsaicina/antagonistas & inibidores , Capsaicina/farmacologia , Linhagem Celular , Eletrofisiologia , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Microscopia Confocal , Neurônios Motores/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Terminações Nervosas/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo
6.
Am J Physiol Endocrinol Metab ; 297(3): E602-8, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19602580

RESUMO

Peripheral neuropathy is a common complication of diabetes that leads to severe morbidity. In this study, we investigated the sensitivity of motor unit number estimate (MUNE) to detect early motor axon dysfunction in streptozotocin (STZ)-treated mice. We compared the findings with in vitro changes in the morphology and electrophysiology of the neuromuscular junction. Adult Thy1-YFP and Swiss Webster mice were made diabetic following three interdaily intraperitoneal STZ injections. Splay testing and rotarod performance assessed motor activity for 6 wk. Electromyography was carried out in the same time course, and compound muscle action potential (CMAP) amplitude, latency, and MUNE were estimated. Two-electrode voltage clamp was used to calculate quantal content (QC) of evoked transmitter release. We found that an early reduction in MUNE was evident before a detectable decline of motor activity. CMAP amplitude was not altered. MUNE decrease accompanied a drop of end-plate current amplitude and QC. We also observed small axonal loss, sprouting of nerve endings, and fragmentation of acetylcholine receptor clusters at the motor end plate. Our results suggest an early remodeling of motor units through the course of diabetic neuropathy, which can be readily detected by the MUNE technique. The early detection of MUNE anomalies is significant because it suggests that molecular changes associated with pathology and leading to neurodegeneration might already be occurring at this stage. Therefore, trials of interventions to prevent motor axon dysfunction in diabetic neuropathy should be administered at early stages of the disorder.


Assuntos
Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/diagnóstico , Doença dos Neurônios Motores/diagnóstico , Neurônios Motores/patologia , Animais , Glicemia/análise , Contagem de Células/métodos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/patologia , Neuropatias Diabéticas/sangue , Neuropatias Diabéticas/patologia , Neuropatias Diabéticas/fisiopatologia , Diagnóstico Precoce , Estimulação Elétrica , Eletrofisiologia/métodos , Hiperglicemia/induzido quimicamente , Hiperglicemia/complicações , Camundongos , Camundongos Transgênicos , Doença dos Neurônios Motores/sangue , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/patologia , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Prognóstico , Estreptozocina , Fatores de Tempo
7.
Ann N Y Acad Sci ; 1132: 61-70, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18567854

RESUMO

Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.


Assuntos
Envelhecimento/fisiologia , Conotoxinas/farmacologia , Venenos de Crotalídeos/farmacologia , Receptores Nicotínicos/classificação , Receptores Nicotínicos/metabolismo , Animais , Eletrofisiologia , Cinética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Subunidades Proteicas/classificação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Xenopus laevis
8.
Am J Physiol Regul Integr Comp Physiol ; 293(2): R592-600, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17537841

RESUMO

Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.


Assuntos
Glicemia/metabolismo , Hiperglicemia/metabolismo , Insulina/sangue , Óxido Nítrico/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo , Proteínas Quinases Ativadas por AMP , Doença Aguda , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Fluoresceínas , Masculino , Potenciais da Membrana/fisiologia , Complexos Multienzimáticos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR , Núcleo Hipotalâmico Ventromedial/citologia
9.
Am J Physiol Regul Integr Comp Physiol ; 292(4): R1418-28, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17170237

RESUMO

Glucose-sensing neurons in the ventromedial hypothalamus (VMH) are involved in the regulation of glucose homeostasis. Glucose-sensing neurons alter their action potential frequency in response to physiological changes in extracellular glucose, insulin, and leptin. Glucose-excited neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency when extracellular glucose is reduced. Central nitric oxide (NO) synthesis is regulated by changes in local fuel availability, as well as insulin and leptin. NO is involved in the regulation of food intake and is altered in obesity and diabetes. Thus this study tests the hypothesis that NO synthesis is a site of convergence for glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. With the use of the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein in conjunction with the membrane potential-sensitive dye fluorometric imaging plate reader, we found that glucose and leptin suppress, whereas insulin stimulates neuronal nitric oxide synthase (nNOS)-dependent NO production in cultured VMH GI neurons. The effects of glucose and leptin were mediated by suppression of AMP-activated protein kinase (AMPK). The AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) increased both NO production and neuronal activity in GI neurons. In contrast, the effects of insulin on NO production were blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Furthermore, decreased glucose, insulin, and AICAR increase the phosphorylation of VMH nNOS, whereas leptin decreases it. Finally, VMH neurons express soluble guanylyl cyclase, a downstream mediator of NO signaling. Thus NO may mediate, in part, glucose, leptin, and insulin signaling in VMH glucose-sensing neurons.


Assuntos
Glucose/metabolismo , Insulina/metabolismo , Leptina/metabolismo , Neurônios/metabolismo , Óxido Nítrico/biossíntese , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Androstadienos/farmacologia , Animais , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Glucose/antagonistas & inibidores , Masculino , Modelos Biológicos , Morfolinas/farmacologia , Neurônios/citologia , Óxido Nítrico/análise , Óxido Nítrico Sintase Tipo I/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/metabolismo , Ribonucleotídeos/farmacologia , Fatores de Tempo , Núcleo Hipotalâmico Ventromedial/citologia , Núcleo Hipotalâmico Ventromedial/enzimologia , Núcleo Hipotalâmico Ventromedial/metabolismo , Wortmanina
10.
Neuropharmacology ; 50(3): 345-53, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16288931

RESUMO

Effectiveness against chloroquine-resistant Plasmodia makes mefloquine a widely used antimalarial drug. However, mefloquine's neurologic effects offset this therapeutic advantage. Cellular actions which might contribute to the neurologic effects of mefloquine are not understood. Structural similarity to tacrine suggested that mefloquine might alter cholinergic synaptic transmission. Therefore, we examined mefloquine's effects at a model cholinergic synapse. Triangularis sterni nerve-muscle preparations were isolated from adult mice and examined with sharp electrode current clamp technique. Within 30 min of exposure to 10 microM mefloquine, miniature endplate potentials (mepps) occurred in summating bursts and their mean frequency increased 10-fold. The threshold concentration for the increase of mean mepp frequency was 0.6 microM mefloquine. Mefloquine continued to increase mean mepp frequency for preparations bathed in extracellular solution lacking Ca2+. In contrast, mefloquine no longer increased mean mepp frequency for preparations pre-treated with the intracellular Ca2+ buffer BAPTA-AM. Although mefloquine disrupts a thapsigargin-sensitive neuronal Ca2+ store, pre-treatment with thapsigargin did not alter the mefloquine-induced alterations of mepps. Since mefloquine, like oligomycin, inhibits mitochondrial FOF1H+ ATP synthase we tested the interaction between these two chemicals. Like mefloquine, oligomycin induced bursts and increased mean frequency of mepps. Furthermore, pre-treatment with oligomycin precluded the mefloquine-induced alterations of asynchronous transmsitter release. These data suggest that mefloquine inhibits ATP production which increases the concentration of Ca2+ within the cytosol of nerve terminals. This elevation of Ca2+ concentration selectively increases asynchronous transmitter release since 10 microM mefloquine did not alter stimulus-evoked transmsitter release.


Assuntos
Acetilcolina/metabolismo , Antimaláricos/farmacologia , Mefloquina/farmacologia , Junção Neuromuscular/citologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antimaláricos/química , Cálcio/metabolismo , Quelantes/farmacologia , Interações Medicamentosas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Mefloquina/química , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Oligomicinas/farmacologia , Tapsigargina/farmacologia
11.
Neuropharmacology ; 49(8): 1132-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16081111

RESUMO

Mefloquine is effective against drug-resistant Plasmodium falciparum. This property, along with its unique pharmacokinetic profile, makes mefloquine a widely prescribed antimalarial drug. However, mefloquine has neurologic effects which offset its therapeutic advantages. Cellular actions underlying mefloquine's neurologic effects are poorly understood. Here, we demonstrate that mefloquine inhibits human recombinant acetylcholinesterase. To explore the consequences of this action, we investigated mefloquine's actions at a model cholinergic synapse, the mouse neuromuscular junction. Sharp electrode recording was used to record miniature endplate potentials (mepps) in the Triangularis sterni muscle. Within 30 min of exposure to 10 microM mefloquine, mepps were altered in three ways: 10-90% rise time, 90-10% decay time and amplitude significantly increased. Mepp decay time increased linearly with mefloquine concentration. Pretreatment of muscles with the cholinesterase inhibitor physostigmine (3 microM) precluded the mefloquine-induced prolongation of mepp decay. Mefloquine also prolonged mepps at endplates of acetylcholinesterase knock-out mice. Since the selective butyrylcholinesterase inhibitor iso-OMPA (100 microM) also prolonged mepp decay at the neuromuscular junction of acetylcholinesterase knock-out mice, mefloquine inhibition of this enzyme is physiologically relevant. The non-selective anti-cholinesterase action can contribute to the neurologic effects of mefloquine.


Assuntos
Antimaláricos/farmacologia , Inibidores da Colinesterase , Mefloquina/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Acetilcolinesterase/genética , Acetilcolinesterase/fisiologia , Animais , Butirilcolinesterase/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Placa Motora/efeitos dos fármacos , Sistema Nervoso Parassimpático/efeitos dos fármacos , Fisostigmina/farmacologia , Sinapses/efeitos dos fármacos
12.
Cancer Res ; 64(17): 6327-36, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15342422

RESUMO

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-beta (TGF-beta) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-beta bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-beta in each other. In cocultures, PPT-I and TGF-beta expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-beta and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5' flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-beta, and transient tranfections all showed that c-myc is required for TGF-beta-mediated induction of PPT-I in BCCs. TGF-beta was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-beta is important to the regulation of PPT-I in the early events of bone invasion by BCCs.


Assuntos
Células da Medula Óssea/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes myc/fisiologia , Precursores de Proteínas/genética , Taquicininas/genética , Fator de Crescimento Transformador beta/biossíntese , Células da Medula Óssea/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Sequência Consenso , Citocinas/metabolismo , Genes myc/genética , Humanos , Precursores de Proteínas/biossíntese , Células Estromais/metabolismo , Células Estromais/patologia , Taquicininas/biossíntese , Transfecção , Fator de Crescimento Transformador beta/fisiologia
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