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1.
J Gen Physiol ; 150(6): 835-850, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29743301

RESUMO

Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).


Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Potenciais da Membrana , Prótons , Animais , Cádmio/metabolismo , Células HEK293 , Humanos , Canais Iônicos/química , Caramujos , Zinco/metabolismo
2.
J Gen Physiol ; 149(4): 483-510, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28330839

RESUMO

Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel's cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure-function properties, ion selectivity, and cellular physiology.


Assuntos
Canais de Cálcio Tipo T/genética , Evolução Molecular , Animais , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/classificação , Canais de Cálcio Tipo T/metabolismo , Clonagem Molecular , Filogenia , Placozoa/química , Placozoa/metabolismo
3.
Dev Neurobiol ; 77(6): 677-690, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27513310

RESUMO

Carbon monoxide (CO) is physiologically produced via heme degradation by heme oxygenase enzymes. Whereas CO has been identified as an important physiological signaling molecule, the roles it plays in neuronal development and regeneration are poorly understood. During these events, growth cones guide axons through a rich cellular environment to locate target cells and establish synaptic connections. Previously, we have shown that another gaseous signaling molecule, nitric oxide (NO), has potent effects on growth cone motility. With NO and CO sharing similar cellular targets, we wanted to determine whether CO affected growth cone motility as well. We assessed how CO affected growth cone filopodial length and determined the signaling pathway by which this effect was mediated. Using two well-characterized neurons from the freshwater snail, Helisoma trivolvis, it was found that the CO donor, carbon monoxide releasing molecule-2 (CORM-2), increased filopodial length. CO utilized a signaling pathway that involved the activation of soluble guanylyl cyclase, protein kinase G, and ryanodine receptors. While increases in filopodial length often occur from robust increases in intracellular calcium levels, the timing in which CO increased filopodial length corresponded with low basal calcium levels in growth cones. Taken together with findings of a heme oxygenase-like protein in the Helisoma nervous system, these results provide evidence for CO as a modulator of growth cone motility and implicate CO as a neuromodulatory signal during neuronal development and/or regeneration. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 677-690, 2017.


Assuntos
Monóxido de Carbono/farmacologia , Cones de Crescimento/efeitos dos fármacos , Neurônios/citologia , Pseudópodes/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Carbazóis/farmacologia , Monóxido de Carbono/metabolismo , Inibidores Enzimáticos/farmacologia , Gânglios dos Invertebrados/citologia , Heme Oxigenase-1/metabolismo , Óxido Nítrico/metabolismo , Compostos Organometálicos/farmacologia , Oxidiazóis/farmacologia , Oxazinas/farmacologia , Técnicas de Patch-Clamp , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Caramujos , Fatores de Tempo
4.
J Biol Chem ; 290(41): 25151-63, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26306033

RESUMO

Brown adipocytes function to dissipate energy as heat through adaptive thermogenesis. Understanding the molecular mechanisms underlying the brown fat thermogenic program may provide insights for the development of therapeutic approaches in the treatment of obesity. Most studies investigating the mechanisms underlying brown fat development focus on genetic mechanisms; little is known about the epigenetic mechanisms in this process. We have discovered that ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase for di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), plays a potential role in regulating brown adipocyte thermogenic program. We found that UTX is up-regulated during brown adipocyte differentiation and by cold exposure in both brown adipose tissue (BAT) and white adipose tissue (WAT) of mice, suggesting a potential role in thermogenesis. Inactivation of UTX down-regulates brown fat specific gene expression, while overexpression of UTX does the opposite. Notably, activation of ß adrenergic signaling recruits UTX to the UCP1 and PGC1α promoters, leading to decreased H3K27me3, a histone transcriptional repressive mark. UTX demethylates H3K27me3 and subsequently interacts with the histone acetyltransferase (HAT) protein CBP, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulate brown adipocyte thermogenic program through coordinated control of demethylating H3K27me3 and acetylating H3K27, switching the transcriptional repressive state to the transcriptional active state at the promoters of UCP1 and PGC1α. We conclude that UTX may play a potential role in regulation of brown adipocyte gene expression and may mediate ß adrenergic activation of brown fat function.


Assuntos
Adipócitos Marrons/metabolismo , Histona Desmetilases/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Termogênese , Acetilação , Adipócitos Marrons/citologia , Animais , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular , Linhagem Celular , Temperatura Baixa , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Enzimológica da Expressão Gênica , Histona Desmetilases/genética , Canais Iônicos/genética , Metabolismo dos Lipídeos , Masculino , Potencial da Membrana Mitocondrial , Metilação , Camundongos , Proteínas Mitocondriais/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Proteína Desacopladora 1
5.
Eur J Neurosci ; 41(8): 1013-24, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25808424

RESUMO

Nitric oxide (NO) is a key regulator of neuronal excitability in the nervous system. While most studies have investigated its role as an intercellular messenger/modulator, less is known about potential physiological roles played by NO within NO-producing neurons. We showed previously that intrinsic production of NO within B5 neurons of the pond snail Helisoma trivolvis increased neuronal excitability by acting on three ionic conductances. Here we demonstrate that intrinsically produced NO affected two of the same conductances in another buccal neuron, B19, where it had the opposite, namely inhibitory, effect on neuronal activity. Using single-cell RT-PCR, we show that B19 neurons express NO synthase (NOS) mRNA. The inhibition of intrinsic NO production with NOS inhibitors caused membrane potential depolarization, transient spiking and an increase in input resistance. Inhibition of the main intracellular receptor of NO, soluble guanylyl cyclase, had similar effects on the parameters mentioned above. An investigation of the effects of NO on ion channels revealed that intrinsic NO mediated neuronal hyperpolarization by activating voltage-gated calcium channels that in turn caused the tonic opening of apamin-sensitive calcium-activated potassium channels. The analysis of action potentials in B5 and B19 neurons suggested that the opposite effects on neuronal excitability elicited by intrinsic NO were probably determined by differences in the ionic conductances that shape their action potentials. In summary, we describe a mechanism by which B19 neurons utilise intrinsically produced NO in a cell-type-specific fashion to decrease their neuronal activity, highlighting an important physiological role of NO within NO-producing neurons.


Assuntos
Potenciais da Membrana , Neurônios/fisiologia , Óxido Nítrico Sintase/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio/fisiologia , Células Cultivadas , Dados de Sequência Molecular , Neurônios/metabolismo , Canais de Potássio Cálcio-Ativados/fisiologia , RNA Mensageiro/metabolismo , Caramujos
6.
Dev Neurobiol ; 75(5): 435-51, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25251837

RESUMO

The electrical activity in developing and mature neurons determines the intracellular calcium concentration ([Ca(2+)]i), which in turn is translated into biochemical activities through various signaling cascades. Electrical activity is under control of neuromodulators, which can alter neuronal responses to incoming signals and increase the fidelity of neuronal communication. Conversely, the effects of neuromodulators can depend on the ongoing electrical activity within target neurons; however, these activity-dependent effects of neuromodulators are less well understood. Here, we present evidence that the neuronal firing frequency and intrinsic properties of the action potential (AP) waveform set the [Ca(2+)]i in growth cones and determine how neurons respond to the neuromodulator nitric oxide (NO). We used two well-characterized neurons from the freshwater snail Helisoma trivolvis that show different growth cone morphological responses to NO: B5 neurons elongate filopodia, while those of B19 neurons do not. Combining whole-cell patch clamp recordings with simultaneous calcium imaging, we show that the duration of an AP contributes to neuron-specific differences in [Ca(2+)]i, with shorter APs in B19 neurons yielding lower growth cone [Ca(2+)]i. Through the partial inhibition of voltage-gated K(+) channels, we increased the B19 AP duration resulting in a significant increase in [Ca(2+)]i that was then sufficient to cause filopodial elongation following NO treatment. Our results demonstrate a neuron-type specific correlation between AP shape, [Ca(2+)]i, and growth cone motility, providing an explanation to how growth cone responses to guidance cues depend on intrinsic electrical properties and helping explain the diverse effects of NO across neuronal populations.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Neurônios/fisiologia , Óxido Nítrico/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Técnicas de Patch-Clamp/métodos , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Caramujos
7.
PLoS One ; 8(11): e78727, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24236040

RESUMO

Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons.


Assuntos
Neurônios Motores/fisiologia , Óxido Nítrico/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , 4-Aminopiridina/farmacologia , Potenciais de Ação , Animais , Apamina/farmacologia , Canais de Cálcio/metabolismo , Células Cultivadas , Gânglios Autônomos/citologia , Cones de Crescimento/fisiologia , Caracois Helix , Doadores de Óxido Nítrico/farmacologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/agonistas , Tetraetilamônio/farmacologia
8.
Dev Neurobiol ; 73(7): 487-501, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23335470

RESUMO

In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]i ). Whole-cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP-induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation.


Assuntos
Acetilcolina/fisiologia , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Pseudópodes/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Células Cultivadas , Iodeto de Dimetilfenilpiperazina/farmacologia , Relação Dose-Resposta a Droga , Cones de Crescimento/efeitos dos fármacos , Caracois Helix , Neurônios/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Receptores Nicotínicos/fisiologia
9.
Eur J Neurosci ; 36(10): 3333-43, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22913584

RESUMO

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO-producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)-expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single-cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long-term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I(NaP) ), voltage-gated Ca currents (I(Ca) ) and small-conductance calcium-activated potassium (SK) channels. We show that NOS inhibitors 7-nitroindazole and S-methyl-l-thiocitrulline resulted in a decrease in I(NaP) , and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I(NaP) , riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I(Ca) , and blocked spontaneous and depolarization-induced spiking, suggesting that intrinsic NO controlled I(Ca) via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self-produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.


Assuntos
Potenciais de Ação , Neurônios/fisiologia , Óxido Nítrico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Apamina/farmacologia , Canais de Cálcio/fisiologia , Citrulina/análogos & derivados , Citrulina/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Indazóis/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Riluzol/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Caramujos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/fisiologia , Guanilil Ciclase Solúvel , Tioureia/análogos & derivados , Tioureia/farmacologia
10.
J Neurosci ; 30(5): 1699-711, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20130179

RESUMO

Nitric oxide (NO) is a radical and a gas, properties that allow NO to diffuse through membranes and potentially enable it to function as a "volume messenger." This study had two goals: first, to investigate the mechanisms by which NO functions as a modulator of neuronal excitability, and second, to compare NO effects produced by NO release from chemical NO donors with those elicited by physiological NO release from single neurons. We demonstrate that NO depolarizes the membrane potential of B5 neurons of the mollusk Helisoma trivolvis, initially increasing their firing rate and later causing neuronal silencing. Both effects of NO were mediated by inhibition of Ca-activated iberiotoxin- and apamin-sensitive K channels, but only inhibition of apamin-sensitive K channels fully mimicked all effects of NO on firing activity, suggesting that the majority of electrical effects of NO are mediated via inhibition of apamin-sensitive K channels. We further show that single neurons release sufficient amounts of NO to affect the electrical activity of B5 neurons located nearby. These effects are similar to NO release from the chemical NO donor NOC-7 [3-(2-hydroxy-1-methyl-2-nitrosohydazino)-N-methyl-1-propyanamine], validating the use of NO donors in studies of neuronal excitability. Together with previous findings demonstrating a role for NO in neurite outgrowth and growth cone motility, the results suggest that NO has the potential to shape the development of the nervous system by modulating both electrical activity and neurite outgrowth in neurons located in the vicinity of NO-producing cells, supporting the notion of NO functioning as a volume messenger.


Assuntos
Neurônios/metabolismo , Óxido Nítrico/metabolismo , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Apamina/farmacologia , Canais de Cálcio/metabolismo , Células Cultivadas , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Gânglios dos Invertebrados/metabolismo , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Hidrazinas/farmacologia , Dados de Sequência Molecular , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Caramujos
11.
Biochemistry ; 46(43): 12275-88, 2007 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17924653

RESUMO

Ca2+ regulates numerous biological processes through spatiotemporal changes in the cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store and plays an essential role in cytosolic Ca2+ homeostasis. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ concentration measurements in specific subcellular environments without using natural Ca2+ binding proteins such as calmodulin, which themselves participate as signaling molecules in cells. In this report, a strategy for creating such sensors by grafting a Ca2+-binding motif into chromophore sensitive locations in green fluorescence protein is described. The engineered Ca2+ sensors exhibit large ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentrations found in the ER (Kd values range from 0.4 to 2 mM). In addition to characterizing the optical and metal binding properties of the newly developed Ca2+ sensors with various spectroscopic methods, we also examined the kinetic properties using stopped-flow spectrofluorimetry to ensure accurate monitoring of dynamic Ca2+ changes. The developed Ca2+ sensor was successfully targeted to the ER of mammalian cell lines to monitor Ca2+ changes occurring in this compartment in response to stimulation with agonists. We envision that this class of Ca2+ sensors can be modified further to measure the Ca2+ concentration in other cellular compartments, providing tools for studying the contribution of these compartments to cellular Ca2+ signaling.


Assuntos
Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
12.
Dev Neurobiol ; 67(14): 1932-43, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17874460

RESUMO

Nitric oxide (NO), a gaseous messenger, has been reported to be involved in a variety of functions in the nervous system, ranging from neuronal pathfinding to learning and memory. We have shown previously that the application of NO via NO donors to growth cones of identified Helisoma buccal neurons B5 in vitro induces an increase in filopodial length, a decrease in filopodial number, and a slowing in neurite advance. It is unclear, however, whether NO released from a physiological source would affect growth cone dynamics. Here we used cell bodies of identified neurons known to express the NO synthesizing enzyme nitric oxide synthase (NOS) as a source of constitutive NO production and tested their effect on growth cones of other cells in a sender-receiver paradigm. We showed that B5 cell bodies induced a rapid increase in filopodial length in NO-responsive growth cones, and that this effect was blocked by the NOS inhibitor 7-NI, suggesting that the effect was mediated by NO. Inhibition of soluble guanylyl cyclase (sGC) with ODQ blocked filopodial elongation induced by B5 somata, confirming that NO acted via sGC. We also demonstrate that the effect of NO was reversible and that a cell releasing NO can affect growth cones over a distance of at least 100 microm. Our results suggest that NO released from a physiological source can affect the motility of nearby growth cones and thus should be considered a signaling molecule with the potential to affect the outcome of neuronal pathfinding in vivo.


Assuntos
Cones de Crescimento/ultraestrutura , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Pseudópodes/efeitos dos fármacos , Análise de Variância , Animais , Comunicação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Gânglios dos Invertebrados/citologia , Cones de Crescimento/efeitos dos fármacos , Caracois Helix , Indazóis/farmacologia , Neurônios/química , Oxidiazóis/farmacologia , Fatores de Tempo
13.
Eur J Neurosci ; 26(6): 1537-47, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17714493

RESUMO

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca(2+)](i)). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca(2+)](i) that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.


Assuntos
Cálcio/metabolismo , Cones de Crescimento/fisiologia , Óxido Nítrico/fisiologia , Pseudópodes/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , ADP-Ribose Cíclica/farmacologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/fisiologia , Gastrópodes , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/ultraestrutura , Guanilato Ciclase/metabolismo , Hidrazinas/farmacologia , Processamento de Imagem Assistida por Computador , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Neurônios/fisiologia , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos
14.
Cell Motil Cytoskeleton ; 63(4): 173-92, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16463277

RESUMO

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.


Assuntos
Movimento Celular/efeitos dos fármacos , Cones de Crescimento/fisiologia , Neuritos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Pseudópodes/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/fisiologia , Gânglios dos Invertebrados/metabolismo , Cones de Crescimento/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais , Caramujos , Fatores de Tempo
15.
Eur J Neurosci ; 22(12): 3006-16, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16367767

RESUMO

Nitric oxide (NO) is a gaseous messenger that has been shown to affect growth cone motility and neurite outgrowth in several model systems, but how NO brings about its effects is not understood. We have previously demonstrated that global and long-term application of NO to Helisoma trivolvis B5 neurons results in a transient increase in filopodial length, decrease in filopodial number and decrease in neurite outgrowth, all of which are mediated via soluble guanylyl cyclase (sGC) and involve an increase in the intracellular Ca2+ concentration [S. Van Wagenen & V. Rehder (1999)Journal of Neurobiology, 39, 168-185; K.R. Trimm & V. Rehder (2004) European Journal of Neuroscience, 19, 809-818]. The goal of the current study was twofold: to investigate the effects of short-term NO exposure on individual growth cones and to further elucidate the downstream pathway through which NO exerts its effects. Local application of the NO donor NOC-7 for 10-20 ms via puffer micropipette resulted in a transient increase in filopodial length and a small decrease in filopodial number. We show evidence that these effects of NO are mediated via sGC, protein kinase G and cyclic ADP ribose, resulting in the release of Ca2+ from intracellular stores, probably of the ryanodine-sensitive type. These results suggest that growth cones expressing sGC are highly sensitive to local and short-term exposure to NO, which they may experience during pathfinding, and that the stereotyped response of transient filopodial elongation seen in B5 neurons in response to NO requires intracellular Ca2+ release.


Assuntos
Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Cones de Crescimento/enzimologia , Guanosina Monofosfato/metabolismo , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismo , Pseudópodes/fisiologia , Animais , Células Cultivadas , Diagnóstico por Imagem/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Gânglios dos Invertebrados/citologia , Cones de Crescimento/efeitos dos fármacos , Caracois Helix , Hidrazinas/farmacologia , Modelos Biológicos , Neurônios/classificação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pseudópodes/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo
16.
J Biotechnol ; 119(4): 368-78, 2005 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-15935502

RESUMO

This study has investigated the expression of green fluorescent protein (GFP) variants in the cytosol and the endoplasmic reticulum (ER) of HeLa cells and evaluated the effects of the different cellular environments on the fluorescence properties of these GFP variants. Several GFP variants have been constructed by adding different N- or C-terminal signal sequences. These proteins were expressed and folded in distinct cellular compartments in HeLa cells. The localization of these GFP variants targeted to the endoplasmic recticulum was confirmed by the co-localization of DsRed2-ER as assessed by confocal microscopy. The addition of signal peptides targeting GFP variants to the ER or cytosol did not appear to alter the optical spectra of these GFP variants. However, the fluorescence intensity of these GFP variants in the ER was significantly less than that in the cytosol. Thus, the results clearly suggest that the cellular environment affects the formation and/or maturation of green fluorescence protein in vivo. These findings will be helpful in the future development and application of GFP technology aimed at investigating cellular functions performed in the ER and the cytosol.


Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mutação , Relação Estrutura-Atividade
17.
J Neurosci ; 24(13): 3421-35, 2004 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-15056722

RESUMO

The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT2betaPan, couples with the traditional G(q) pathway when expressed in HEK293 cells, but not to G(s) or G(i/o). Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.


Assuntos
Neurotransmissores/metabolismo , Palinuridae/fisiologia , Receptores de Neurotransmissores/metabolismo , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Aminas Biogênicas/farmacologia , Aminas Biogênicas/fisiologia , Linhagem Celular , Clonagem Molecular , Biologia Computacional/métodos , Sequência Conservada/fisiologia , Sistema Digestório/inervação , Drosophila/genética , Evolução Molecular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Rede Nervosa/metabolismo , Rede Nervosa/fisiologia , Palinuridae/genética , Proteína Quinase C/metabolismo , Receptores de Neurotransmissores/agonistas , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismo
18.
Eur J Neurosci ; 19(4): 809-18, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15009128

RESUMO

Nitric oxide (NO) has been demonstrated to act as a signaling molecule during neuronal development, but its precise function is unclear. Here we investigate whether NO might function at the neuronal growth cone to affect growth cone motility. We have previously demonstrated that growth cones of identified neurons from the snail Helisoma trivolvis show a rapid and transient increase in filopodial length in response to NO, which was regulated by soluble guanylyl cyclase (sGC) [S. Van Wagenen and V. Rehder (1999) J. Neurobiol., 39, 168-185]. Because in vivo studies have demonstrated that growth cones have longer filopodia and advance more slowly in regions where pathfinding decisions are being made, this study aimed to establish whether NO could function as a combined 'slow-down and search signal' for growth cones by decreasing neurite outgrowth. In the presence of the NO donor NOC-7, neurites of B5 neurons showed a concentration-dependent effect on neurite outgrowth, ranging from slowing at low, stopping at intermediate and collapsing at high concentrations. The effects of the NO donor were mimicked by directly activating sGC with YC-1, or by increasing its product with 8-bromo-cGMP. In addition, blocking sGC in the presence of NO with NS2028 blocked the effect of NO, suggesting that NO affected outgrowth via sGC. Ca2+ imaging of growth cones with Fura-2 indicated that [Ca2+]i increased transiently in the presence of NOC-7. These results support the hypothesis that NO can function as a potent slow/stop signal for developing neurites. When coupled with transient filopodia elongation, this phenomenon emulates growth cone searching behavior.


Assuntos
Neuritos/fisiologia , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/fisiologia , Caracois Helix , Fatores de Crescimento Neural/fisiologia , Neuritos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Transdução de Sinais/efeitos dos fármacos
19.
Cell Motil Cytoskeleton ; 57(1): 53-67, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14648557

RESUMO

The neuronal growth cone provides the sensory and motor structure that guides neuronal processes to their target. The ability of a growth cone to navigate correctly depends on its filopodia, which sample the environment by continually extending and retracting as the growth cone advances. Several second messengers systems that are activated upon contact with extracellular cues have been reported to affect growth cone morphology by changing the length and number of filopodia. Because recent studies have suggested that guidance cues can signal via G-protein coupled receptors to regulate phospholipases, we here investigated whether phospholipase A2 (PLA2) may control filopodial dynamics and could thereby affect neuronal pathfinding. Employing identified Helisoma neurons in vitro, we demonstrate that inhibition of PLA2 with 2 microM BPB caused a 40.3% increase in average filopodial length, as well as a 37.3% reduction in the number of filopodia on a growth cone. The effect of PLA2 inhibition on filopodial length was mimicked by the inhibition of G-proteins with 500 ng/ml pertussis toxin and was partially blocked by the simultaneous activation of PLA2 with 50 nM melittin. We provide evidence that PLA2 acts via production of arachidonic acid (AA), because (1) the effect of inhibition of PLA2 could be counteracted by supplying AA exogenously, and (2) the inhibition of cyclooxygenase, which metabolizes AA into prostaglandins, also increased filopodial length. We conclude that filopodial contact with extracellular signals that alter the activity of PLA2 can control growth cone morphology and may affect neuronal pathfinding by regulating the sensory radius of navigating growth cones.


Assuntos
Ácido Araquidônico/biossíntese , Gânglios dos Invertebrados/metabolismo , Cones de Crescimento/metabolismo , Caracois Helix/metabolismo , Fosfolipases A/metabolismo , Pseudópodes/metabolismo , Animais , Ácido Araquidônico/farmacologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sinais (Psicologia) , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/crescimento & desenvolvimento , Cones de Crescimento/efeitos dos fármacos , Caracois Helix/citologia , Caracois Helix/crescimento & desenvolvimento , Meliteno , Toxina Pertussis/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Receptores Acoplados a Proteínas-G/metabolismo
20.
J Neurosci Res ; 74(2): 210-20, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-14515350

RESUMO

Growth cones are essential for neuronal pathfinding during embryonic development and again after injury, when they aid in neuronal regeneration. This study was aimed at investigating the role of kinases in the earliest events in neuronal regeneration, namely, the formation of new growth cones from injured neuronal processes. Neurites of identified snail neurons grown in vitro were severed, and the formation of growth cones was observed from the ends of such transected processes. Under control conditions, all neurites formed a new growth cone within 45 min of transection. In contrast, growth cone formation in the presence of a general kinase inhibitor, K252a, was significantly inhibited. Moreover, decreasing the phosphorylation state of neurites by activating protein phosphatases with C2-ceramide also reduced growth cone formation. Pharmacological analysis with specific kinase inhibitors suggested that targets of protein kinase C (PKC) and tyrosine kinase (PTK) phosphorylation control growth cone formation. Inhibition of PKC with calphostin C and cerebroside completely blocked growth cone formation, whereas the inhibition of PTK with erbstatin analog significantly reduced growth cone formation. In contrast, inhibitors of protein kinase A, protein kinase G, CaM-kinase II, myosin light-chain kinase, Rho kinase, and PI-3 kinase had little or no effect 45 min after transection. These results suggest that the transformation underlying the formation of a growth cone from an injured (transected) neurite stump is highly sensitive to the phosphorylation state of key target proteins. Therefore, injury-induced signaling events will determine the outcome of neuronal regeneration through their action on kinase and phosphatase activities.


Assuntos
Gânglios dos Invertebrados/crescimento & desenvolvimento , Gânglios dos Invertebrados/lesões , Cones de Crescimento/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Gânglios dos Invertebrados/citologia , Cones de Crescimento/ultraestrutura , Regeneração Nervosa/fisiologia , Neurônios/citologia , Neurônios/ultraestrutura , Fosfoproteínas Fosfatases/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Caramujos
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