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1.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638858

RESUMO

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.


Assuntos
Membrana Celular/metabolismo , Hidrazonas/farmacologia , Lisossomos/metabolismo , Naftóis/farmacologia , Canais de Potássio/metabolismo , 4-Aminopiridina/farmacologia , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Microscopia Confocal/métodos , Oócitos/citologia , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Transporte Proteico/efeitos dos fármacos , Xenopus laevis
2.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356653

RESUMO

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinamatos/química , Depsídeos/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinaptossomos/metabolismo
3.
Am J Physiol Cell Physiol ; 321(4): C684-C693, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34432539

RESUMO

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.


Assuntos
Fundo Gástrico/metabolismo , Motilidade Gastrointestinal , Contração Muscular , Músculo Liso/metabolismo , Potássio/metabolismo , Canais de Potássio Shal/metabolismo , 4-Aminopiridina/farmacologia , Animais , Fundo Gástrico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Cinética , Masculino , Potenciais da Membrana , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/genética , Venenos de Aranha/metabolismo
4.
Exp Brain Res ; 239(9): 2841-2849, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34283252

RESUMO

Phoenixin-14 (PNX-14) has a wide bioactivity in the central nervous system. Its role in the hypothalamus has been investigated, and it has been reported that it is involved in the regulation of excitability in hypothalamic neurons. However, its role in the regulation of excitability in entorhinal cortex and the hippocampus is unknown. In this study, we investigated whether i. PNX-14 induces any synchronous discharges or epileptiform activity and ii. PNX-14 has any effect on already initiated epileptiform discharges. We used 350 µm thick acute horizontal hippocampal-entorhinal cortex slices obtained from 30- to 35-day-old mice. Extracellular field potential recordings were evaluated in the entorhinal cortex and hippocampus CA1 region. Bath application of PNX-14 did not initiate any epileptiform activity or abnormal discharges. 4-Aminopyridine was applied to induce epileptiform activity in the slices. We found that 200 nM PNX-14 reduced the frequency of interictal-like events in both the entorhinal cortex and hippocampus CA1 region which was induced by 4-aminopyridine. Furthermore, PNX-14 led to a similar suppression in the total power of local field potentials of 1-120 Hz. The frequency or the duration of the ictal events was not affected. These results exhibited for the first time that PNX-14 has a modulatory effect on synchronized neuronal discharges which should be considered in future therapeutic approaches.


Assuntos
Córtex Entorrinal , Hipocampo , 4-Aminopiridina/farmacologia , Animais , Camundongos , Neurônios
5.
World Neurosurg ; 153: e168-e178, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34166824

RESUMO

PURPOSE: To study the 24-hour expression of long noncoding RNAs (lncRNAs) in synaptic and extrasynaptic neurons expressing N-methyl-D-aspartate receptor (NMDAR), and normal neuronal cultures, via microarray analysis. MATERIALS AND METHODS: Cortical neurons from embryonic (day E18) Sprague-Dawley rats were used for primary neuronal culture. NMDAR activation was blocked and the cells were then incubated for 6 hours. Total RNA was extracted, quantified, and analyzed for purity and integrity. Double-stranded cDNA was synthesized, followed by quantile normalization, quantitative polymerase chain reaction validation, and data analysis. The interactions between transcription factors and lncRNAs were analyzed by Pearson correlation. RESULTS: The lncRNA profiles were obtained after synaptic and extrasynaptic NMDAR activation of rat cortical neuron cultures for 24 hours. In total, 251 lncRNAs were consistently upregulated, and 335 were downregulated, after extrasynaptic NMDAR activation compared with normal neurons. After synaptic NMDAR activation, only 9 lncRNAs were upregulated and 2 were downregulated. CONCLUSIONS: Differential expression of lncRNAs after synaptic and extrasynaptic NMDAR activation suggests that lncRNAs may be responsible for extrasynaptic NMDAR-induced neurodegeneration.


Assuntos
Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , 4-Aminopiridina/farmacologia , Animais , Bicuculina/farmacologia , Córtex Cerebral/citologia , Regulação para Baixo , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Glicina/farmacologia , Glicinérgicos/farmacologia , Análise em Microsséries , N-Metilaspartato/farmacologia , Reação em Cadeia da Polimerase , Bloqueadores dos Canais de Potássio/farmacologia , Cultura Primária de Células , Ratos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Regulação para Cima
6.
J Physiol ; 599(12): 3195-3220, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33942325

RESUMO

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3 - cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3 - cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.


Assuntos
Epilepsia , Espaço Extracelular , 4-Aminopiridina/farmacologia , Animais , Encéfalo/metabolismo , Epilepsia/tratamento farmacológico , Espaço Extracelular/metabolismo , Humanos , Camundongos , Simportadores de Sódio-Bicarbonato/metabolismo
7.
Clin Biomech (Bristol, Avon) ; 86: 105382, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34000628

RESUMO

BACKGROUND: People with multiple sclerosis have reduced walking speed and impaired gait pattern. Prolonged release-fampridine is a potassium channel blocker that improves nerve conduction in patients with multiple sclerosis, leading to walking benefits. Whether fampridine alters gait pattern is unknown. METHODS: In this crossover, randomized controlled trial, patients with multiple sclerosis were tested for responder status during a 4-week run-in period. Patients were considered responders if they improved their 25-ft walk test by 10% and improved their perceived walking capacity. Responders were randomized to prolonged release-fampridine (10 mg b.i.d.) or placebo for a 6-week period. After a 2-week wash-out period, they were allocated to the other treatment for 6 weeks. Participants were assessed before and after both conditions. Three-dimensional gait analysis assessed kinematic, kinetic, mechanic and energetic variables while walking on a treadmill at comfortable speed. Six-minute walk test and 25-ft walk test were used to assess walking speed on middle and short-distances, respectively. Patient-reported outcome measures were also used. Repeated measures ANCOVAs were applied to assess the treatment effects. FINDINGS: Out of 39 included patients, 24 responders (12 women; Expanded Disability Status Scale:4.25[4-5]; age:46 ± 10 years; maximal speed:0.93 ± 0.38 m·s-1) were identified. Among them, prolonged release-fampridine reduced the external mechanical work (-0.039 J·kg-1·m-1;p = 0.02), and improved knee flexion during swing phase (+5.3°; p = 0.02). No differences were found in other walking tests and patient-reported outcomes, at group-level. INTERPRETATION: Prolonged release-fampridine increases knee flexion during swing phase and lowers mechanical external work. Whether these changes are related to clinically meaningful improvements in walking capacity and other functional variables should be further investigated.


Assuntos
Esclerose Múltipla , 4-Aminopiridina/farmacologia , 4-Aminopiridina/uso terapêutico , Adulto , Feminino , Marcha , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/uso terapêutico , Resultado do Tratamento , Caminhada
8.
Artigo em Inglês | MEDLINE | ID: mdl-33653963

RESUMO

As an antagonist of voltage-gated potassium (Kv) channels, 4-aminopyridine (4-AP) is used as symptomatic therapy in several neurologic disorders. The improvement of visual function and motor skills and relieve of fatigue in patients with MS have been attributed to 4-AP. Its prolonged release formulation (fampridine) has been approved for the symptomatic treatment of walking disability in MS. The beneficial effects were explained by the blockade of axonal Kv channels, thereby enhancing conduction along demyelinated axons. However, an increasing body of evidence suggests that 4-AP may have additional properties beyond the symptomatic mode of action. In this review, we summarize preclinical and clinical data on possible neuroprotective features of 4-AP.


Assuntos
4-Aminopiridina/farmacologia , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , 4-Aminopiridina/uso terapêutico , Animais , Humanos , Inflamação/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Transdução de Sinais
9.
Mil Med ; 186(Suppl 1): 479-485, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33499448

RESUMO

INTRODUCTION: Traumatic peripheral nerve injuries (TPNIs) are increasingly prevalent in battlefield trauma, and the functional recovery with TPNIs depends on axonal continuity. Although the physical examination is the main tool for clinical diagnosis with diagnostic work up, there is no diagnostic tool available to differentiate nerve injuries based on axonal continuity. Therefore, treatment often relies on "watchful waiting," and this leads to muscle weakness and further reduces the chances of functional recovery. 4-aminopyridine (4-AP) is clinically used in multiple sclerosis patients for walking performance improvement. Preliminary results in conscious mice suggested a diagnostic role of 4-AP in distinguishing axonal continuity. In this study, we thought to evaluate the diagnostic potential of 4-AP on the axonal continuity in unawake/sedated animals. MATERIALS AND METHODS: Rat sciatic nerve crush and transection injuries were used in this study. Briefly, rats were anesthetized with isoflurane and mechanically ventilated with oxygen-balanced vaporized isoflurane. Sciatic nerve and triceps surae muscles were exposed by blunt dissection, and a stimulating electrode was placed under a sciatic nerve proximal to the crush injury. A force transducer measured muscle tension response to electrical stimulation of sciatic nerve. Muscle response was measured before crush, after crush, and 30 minutes after systemic 4-AP (150 µg/kg) or local (4-AP)-poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG) treatment. RESULTS: We found that both crush and transection injuries in sciatic nerve completely abolished muscle response to electrical stimulation. Single dose of systemic 4-AP and local (4-AP)-PLGA-PEG treatment with crush injury significantly restored muscle responses to electrical stimulation after 30 minutes of administration. However, systemic 4-AP treatment had no effect on muscle response after nerve transection. These results clearly demonstrate that 4-AP can restore nerve conduction and produce muscle response within minutes of administration only when there is a nerve continuity, even in the sedated animal. CONCLUSIONS: We conclude that 4-AP could be a promising diagnostic agent in differentiating TPNI based on axonal continuity.


Assuntos
Axônios , 4-Aminopiridina/farmacologia , 4-Aminopiridina/uso terapêutico , Animais , Masculino , Camundongos , Traumatismos dos Nervos Periféricos/diagnóstico , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Nervo Isquiático
10.
Cell Biochem Biophys ; 79(1): 57-71, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33095400

RESUMO

Endothelin-1 (ET-1) is a potent endogenously derived vasoconstrictor, which increases pulmonary hypertension via stimulation of [Ca2+]i level in pulmonary artery smooth muscle cells (PASMCs). In this communication, we sought to investigate the mechanism by which ET-1 causes stimulation of Ca2+ concentration in caveolae vesicles of bovine PASMCs (BPASMCs). ET-1 activates PKC-α in the caveolae vesicles by O2.- derived from PKCζ-NADPH oxidase dependent pathway. PKC-α phosphorylates Kv1.5 channels leading to a marked stimulation of Na+ and Ca2+ concentration in the caveolae vesicles. The stimulation of Ca2+ concentration in the caveolae vesicles by ET-1 occurs predominantly via Cav1.2 channels. Additionally, an increase in Na+ concentration by ET-1 due to stimulation of Nav1.5 channels marginally increases Ca2+ level in the caveolae vesicles via reverse-mode Na+/Ca2+ exchanger (NCX-1) and also through "slip-mode conductance" Nav1.5 channels. 4-AP, a well-known inhibitor of Kv channels, also increases Ca2+ concentration in the caveolae vesicles via Cav1.2 channels, reverse-mode NCX-1 and Nav1.5 channels by phosphorylation independent modulation of Kv1.5 channels without the involvement of PKCζ-NADPH oxidase-PKCα signaling axis. Overall, PKCζ-NADPH oxidase-PKCα dependent phosphorylation of Kv1.5 by ET-1 modulates Nav1.5-NCX1-Cav1.2 axis for stimulation of Ca2+ concentration in caveolae vesicles of BPASMCs, which provides a crucial mechanism for better understanding of ET-1-mediated modulation of pulmonary vascular tone.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Endotelina-1/metabolismo , Músculo Liso Vascular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína Quinase C/metabolismo , Trocador de Sódio e Cálcio/metabolismo , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Cavéolas/metabolismo , Membrana Celular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Fosforilação , Isoformas de Proteínas , Proteína Quinase C-alfa/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais , Sódio/metabolismo
11.
Eur Arch Otorhinolaryngol ; 278(8): 3057-3063, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33226461

RESUMO

OBJECTIVE: 4-Aminopyridine (4-AP) is a potassium channel blocker that enhances nerve excitability. In this study, rat models that have facial nerve crush injury (FNCI) were grouped and treated with methylprednisolone (MP), 4-AP, and a combination of these two drugs. Electrophysiologic and histopathologic outcomes of these groups will be compared with a control group. MATERIALS AND METHODS: Thirty healthy male Wistar rats (mean weight of 265 g) were used in this study. The rats were randomly divided into five groups with six subjects in each: Group 1 (sham group), Group 2 (control group), Group 3 (MP group), Group 4 (4-aminopyridine group), and Group 5 (4-AP + MP group). All groups except the sham group underwent crush injury to the right facial nerve. Electrophysiologic and histologic recovery was recorded three weeks postoperatively. RESULTS: The 4-AP group and the combined group had a more significant recovery at Nerve Excitability Thresholds (NET) at the end of three weeks. The methylprednisolone group and the control group had a minimal recovery of NET. Histologically, when compared with the control group, the combined group was the only group that had significant recovery at all three of axonal degeneration, axon diameter, and myelin thickness. CONCLUSION: In this experimental study, we demonstrated that a combination treatment of 4-AP and MP is more effective in the recovery of peripheric FNCI than in the no-treatment control group and in the 4-AP- or MP-alone groups. Moreover, our results suggested that 4-AP can be a potent alternative to MP in the treatment of the FNCI. LEVEL OF EVIDENCE: N/A.


Assuntos
Lesões por Esmagamento , Traumatismos do Nervo Facial , 4-Aminopiridina/farmacologia , Animais , Modelos Animais de Doenças , Nervo Facial , Traumatismos do Nervo Facial/tratamento farmacológico , Masculino , Metilprednisolona/farmacologia , Regeneração Nervosa , Ratos , Ratos Wistar , Recuperação de Função Fisiológica
12.
PLoS Comput Biol ; 16(12): e1008463, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315892

RESUMO

Cerebellar stellate cells (CSCs) are spontaneously active, tonically firing (5-30 Hz), inhibitory interneurons that synapse onto Purkinje cells. We previously analyzed the excitability properties of CSCs, focusing on four key features: type I excitability, non-monotonic first-spike latency, switching in responsiveness and runup (i.e., temporal increase in excitability during whole-cell configuration). In this study, we extend this analysis by using whole-cell configuration to show that these neurons can also burst when treated with certain pharmacological agents separately or jointly. Indeed, treatment with 4-Aminopyridine (4-AP), a partial blocker of delayed rectifier and A-type K+ channels, at low doses induces a bursting profile in CSCs significantly different than that produced at high doses or when it is applied at low doses but with cadmium (Cd2+), a blocker of high voltage-activated (HVA) Ca2+ channels. By expanding a previously revised Hodgkin-Huxley type model, through the inclusion of Ca2+-activated K+ (K(Ca)) and HVA currents, we explain how these bursts are generated and what their underlying dynamics are. Specifically, we demonstrate that the expanded model preserves the four excitability features of CSCs, as well as captures their bursting patterns induced by 4-AP and Cd2+. Model investigation reveals that 4-AP is potentiating HVA, inducing square-wave bursting at low doses and pseudo-plateau bursting at high doses, whereas Cd2+ is potentiating K(Ca), inducing pseudo-plateau bursting when applied in combination with low doses of 4-AP. Using bifurcation analysis, we show that spike adding in square-wave bursts is non-sequential when gradually changing HVA and K(Ca) maximum conductances, delayed Hopf is responsible for generating the plateau segment within the active phase of pseudo-plateau bursts, and bursting can become "chaotic" when HVA and K(Ca) maximum conductances are made low and high, respectively. These results highlight the secondary effects of the drugs applied and suggest that CSCs have all the ingredients needed for bursting.


Assuntos
4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cerebelo/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Células de Purkinje/efeitos dos fármacos , 4-Aminopiridina/administração & dosagem , Animais , Cádmio/administração & dosagem , Cerebelo/citologia , Cerebelo/fisiologia , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Técnicas de Patch-Clamp , Células de Purkinje/fisiologia
13.
Oncol Rep ; 44(5): 2152-2164, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32901871

RESUMO

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA­mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK­8, and comet assay. The results demonstrated that shRNA­induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Dano ao DNA , Reparo do DNA , Etoposídeo/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , 4-Aminopiridina/administração & dosagem , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Ensaio Cometa/métodos , Sinergismo Farmacológico , Etoposídeo/administração & dosagem , Recombinação Homóloga , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Inibidores da Topoisomerase II/administração & dosagem , Inibidores da Topoisomerase II/farmacologia
14.
Hum Mutat ; 41(11): 1999-2011, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32906212

RESUMO

Clinical and genetic features of five unrelated patients with de novo pathogenic variants in the synaptic vesicle-associated membrane protein 2 (VAMP2) reveal common features of global developmental delay, autistic tendencies, behavioral disturbances, and a higher propensity to develop epilepsy. For one patient, a cognitively impaired adolescent with a de novo stop-gain VAMP2 mutation, we tested a potential treatment strategy, enhancing neurotransmission by prolonging action potentials with the aminopyridine family of potassium channel blockers, 4-aminopyridine and 3,4-diaminopyridine, in vitro and in vivo. Synaptic vesicle recycling and neurotransmission were assayed in neurons expressing three VAMP2 variants by live-cell imaging and electrophysiology. In cellular models, two variants decrease both the rate of exocytosis and the number of synaptic vesicles released from the recycling pool, compared with wild-type. Aminopyridine treatment increases the rate and extent of exocytosis and total synaptic charge transfer and desynchronizes GABA release. The clinical response of the patient to 2 years of off-label aminopyridine treatment includes improved emotional and behavioral regulation by parental report, and objective improvement in standardized cognitive measures. Aminopyridine treatment may extend to patients with pathogenic variants in VAMP2 and other genes influencing presynaptic function or GABAergic tone, and tested in vitro before treatment.


Assuntos
4-Aminopiridina/farmacologia , Mutação/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Adulto , Eletrofisiologia , Exocitose/efeitos dos fármacos , Feminino , Humanos , Masculino , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
15.
Am J Physiol Regul Integr Comp Physiol ; 319(4): R439-R447, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32847398

RESUMO

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K+ concentration ([K+]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K+ permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na+-K+ pump blocker) or 4-aminopyridine (4-AP; voltage-gated K+ channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K+ permeability, facilitated by opening of 4-AP-sensitive K+ channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K+ channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.


Assuntos
Aclimatação/fisiologia , Temperatura Baixa , Locusta migratoria/fisiologia , Fibras Musculares Esqueléticas/fisiologia , 4-Aminopiridina/farmacologia , Aclimatação/efeitos dos fármacos , Animais , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Ouabaína/farmacologia
16.
Cell Rep ; 32(1): 107869, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32640234

RESUMO

Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K+ conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K+ conductances mediated by Kv1.8, Kv7.4, Kv11.1, Kv12.1, and BKCa channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials.


Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Canais de Potássio/metabolismo , Som , 4-Aminopiridina/farmacologia , Animais , Células CHO , Cricetulus , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/metabolismo
17.
Restor Neurol Neurosci ; 38(4): 301-309, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32651338

RESUMO

BACKGROUND: Stroke-induced ischemia affects both cortex and underlying white matter. Dalfampridine extended release tablets (D-ER) enhance action potential conduction in demyelinated axons, which may positively affect post-stroke recovery. OBJECTIVE: Based on promising preliminary data, we compared efficacy of D-ER administered at 7.5 mg or 10 mg with placebo on post-stroke ambulation. Primary study outcome (response) was a ≥20% increase on the 2-minute walk test (2 MinWT) at 12 weeks after first drug administration. METHODS: This was a multicenter, randomized, placebo-controlled, 3-arm, parallel-group, safety and efficacy trial. After obtaining baseline measures of 2 MinWT, Walk-12, and Timed Up and Go, subjects entered a 2-week, single-blind placebo run-in period and were randomized 1:1:1 to receive 7.5 mg D-ER, 10 mg D-ER, or placebo, dosed twice-daily for 12 weeks. Follow-up evaluations occurred at weeks 14 and 16 when subjects were off study drug. RESULTS: The study was terminated early with 377 of planned 540 patients enrolled, due to no treatment effect. At week 12, mean increase in distances walked in 2 minutes were similar among the 3 study groups (14.9±40.0 feet; 19.4±39.6 feet; and 20.4±38.3 feet for placebo, 7.5 mg D-ER, and 10 mg D-ER, respectively). The proportion of subjects who showed ≥20% improvement on 2 MinWT at week 12 was 13.5%, 14.0%, and 19.0%, for placebo, 7.5 mg D-ER, and 10 mg D-ER, respectively; these were nonsignificant changes from baseline for all groups. CONCLUSIONS: D-ER at either a 7.5-mg or 10-mg dose did not significantly increase performance on the 2 MinWT in stroke survivors with gait impairment, although this study was terminated early before full enrollment. (Clinical Trial # NCT02271217).


Assuntos
4-Aminopiridina/farmacologia , Isquemia Encefálica/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Caminhada/fisiologia , 4-Aminopiridina/administração & dosagem , Adulto , Preparações de Ação Retardada/farmacologia , Método Duplo-Cego , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Avaliação de Resultados em Cuidados de Saúde
18.
Biol Pharm Bull ; 43(7): 1123-1127, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612075

RESUMO

Nitric oxide (NO) is an important regulator of the retinal blood flow. The present study aimed to determine the role of voltage-gated K+ (KV) channels and ATP-sensitive K+ (KATP) channels in NO-mediated vasodilation of retinal arterioles in rats. In vivo, the retinal vasodilator responses were assessed by measuring changes in the diameter of retinal arterioles from ocular fundus images. Intravitreal injection of 4-aminopyridine (a KV channel inhibitor), but not glibenclamide (a KATP channel blocker), significantly attenuated the retinal vasodilator response to the NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Intravitreal injection of indomethacin (a non-selective cyclooxygenase inhibitor) also reduced the NOR3-induced retinal vasodilator response. The combination of 4-aminopyridine and indomethacin produced a greater reduction in the NOR3-induced response than either agent alone. 4-Aminopyridine had no significant effect on pinacidil (a KATP channel opener)-induced response. These results suggest that the vasodilatory effects of NO are mediated, at least in part, through the activation of 4-aminopyridine-sensitive KV channels in the retinal arterioles of rats. NO exerts its dilatory effect on the retinal vasculature of rats through at least two mechanisms, activation of the KV channels and enhancement of prostaglandin production.


Assuntos
4-Aminopiridina/farmacologia , Arteríolas/efeitos dos fármacos , Óxido Nítrico/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Vasos Retinianos/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Arteríolas/fisiologia , Indometacina/farmacologia , Masculino , Ratos Wistar , Vasos Retinianos/fisiologia , Vasodilatação/fisiologia
19.
Neuron ; 107(1): 52-64.e7, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32362337

RESUMO

At neuronal synapses, synaptotagmin-1 (syt1) acts as a Ca2+ sensor that synchronizes neurotransmitter release with Ca2+ influx during action potential firing. Heterozygous missense mutations in syt1 have recently been associated with a severe but heterogeneous developmental syndrome, termed syt1-associated neurodevelopmental disorder. Well-defined pathogenic mechanisms, and the basis for phenotypic heterogeneity in this disorder, remain unknown. Here, we report the clinical, physiological, and biophysical characterization of three syt1 mutations from human patients. Synaptic transmission was impaired in neurons expressing mutant variants, which demonstrated potent, graded dominant-negative effects. Biophysical interrogation of the mutant variants revealed novel mechanistic features concerning the cooperative action, and functional specialization, of the tandem Ca2+-sensing domains of syt1. These mechanistic studies led to the discovery that a clinically approved K+ channel antagonist is able to rescue the dominant-negative heterozygous phenotype. Our results establish a molecular cause, basis for phenotypic heterogeneity, and potential treatment approach for syt1-associated neurodevelopmental disorder.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Neurônios/fisiologia , Transmissão Sináptica/genética , Sinaptotagmina I/genética , 4-Aminopiridina/farmacologia , Animais , Células Cultivadas , Humanos , Camundongos , Transtornos do Neurodesenvolvimento/fisiopatologia , Neurônios/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Sinaptotagmina I/química
20.
Brain ; 143(4): 1127-1142, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32293668

RESUMO

Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.


Assuntos
4-Aminopiridina/farmacologia , Esclerose Múltipla/patologia , Fármacos Neuroprotetores/farmacologia , Neurite Óptica/patologia , Degeneração Retiniana/patologia , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células-Tronco Neurais/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar
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